8th European A A V Conference

Association of Avian Veterinarians

6th Scientic E C A M S Meeting

European College of Avian Medicine and Surgery

In collaboration with AFVAC Genac

Association Franaise des Vtrinaires pour Animaux de Compagnie Groupe dEtude des Nouveaux Animaux de Compagnie
Arles, France, April 24-30, 2005

AAV

AFVAC

ISBN : 2-9508885-9-3
AFVAC - 40 rue de Berri - 75008 PARIS - FRANCE Phone : + 33 (0) 1 53 83 91 60 - Fax : + 33 (0) 1 53 83 91 69 - Email : contact@afvac.com

Association of Avian Veterinarians European committee (EAAV) www.eaav.org, www.aav.org European College of Avian Medicine and Surgery (ECAMS) www.ecams-online.org Association Franaise des Vtrinaires pour Animaux de Compagnie (AFVAC) www.afvac.com Groupe dEtude des Nouveaux Animaux de Compagnie (GENAC) www.afvac.com

8th European A A V Conference 6th Scientic E C A M S Meeting

Arles, April 24-30, 2005

In collaboration with AFVAC Genac EAAV CONFERENCE CHAIRMAN

Jean-Marie Pricard, DVM, Certied in Avian Diseases, Certied in Epidemiology - Clinique vtrinaire, Sigean, France

EAAV SCIENTIFIC COMMITTEE CHAIRMAN

Jaime Samour, MVZ, PhD, Dip ECAMS Fahad bin Sultan Falcon Center, Riyadh - Kingdom of Saudi Arabia

ECAMS SCIENTIFIC COMMITTEE CHAIRMAN

Nico J. Schoemaker, DVM, PhD, Diplomate ECAMS, Diplomate ABVP (Avian) Division of Avian and Exotic Animal Practice, Utrecht University, The Netherlands

AAV EUROPEAN CONFERENCE 2005 COMMITTEES 26-30 APRIL 2005 ARLES PROVENCE FRANCE
ORGANISING COMMITTEE Jean-Marie PERICARD (France), Chairman of the organizing committee Lorenzo CROSTA (Italy, Spain), EAAV President Peter SANDMEIER (Switzerland), Chairman, 2007 EAAV Conference Helga GERLACH (Germany), Treasurer Niel FORBES (United Kingdom), EAAV-ECAMS delegate SCIENTIFIC COMMITTEE Jaime SAMOUR (Saudi Arabia), Chairman of the scientic committee Jean-Pierre ANDRE (France) Tom BAILEY (Dubai, UAE) John CHITTY (United Kingdom) Gerry DORRESTEIN (Netherlands) Helga GERLACH (Germany) Nigel HARCOURT-BROWN (United Kingdom) Jean-Michel HATT (Switzerland) Rdiger KORBEL (Germany) (and website coordinator) Suzan OROSZ (USA) Jean-Marie PERICARD (France) LOCAL ORGANISING COMMITTEE Jean-Marie PERICARD Michel BELLANGEON Franck RIVAL (President of GENAC) Didier BOUSSARIE Emmanuel RISI Guillaume LELOCH Grard GELLY Jean-Franois QUINTON Sylvie DUFOUR (Administration director AFVAC) Didier-Nol CARLOTTI (President of AFVAC)

ECAMS ORGANISING COMMITTEE M. Krautwald-Junghanns (Germany) Nico J. Schoemaker (Netherlands) EDITORS EAAV Tom BAILEY John CHITTY Nigel HARCOURT-BROWN Jaime SAMOUR AUDITORS Peter COUTTEEL (Belgium) Nigel HARCOURT-BROWN (United Kingdom)

SPONSORING
We greatly appreciate the support by our exhibitors and sponsors :

For this conference :

BAYER HEALTHCARE VETOQUINOL

&
ELLMAN International Inc. HARRISONS BIRD FOODS HEALTH AND HYGIENE JANSSEN SANTE ANIMALE KARL STORZ LYON ELECTRONIC CO. INC. MEDLAB MELET SCHLOESING Laboratoires OPTOMED EVITEC OXBOW PET PRODUCTS SCANELIS SCIL ANIMAL CARE COMPANY VERSELE-LAGA

For the pre-conference scientic tours :

LPO (Ligue pour la protection des oiseaux) BIRDLIFE INTERNATIONAL RESERVE NATURELLE DE CRAU STATION BIOLOGIQUE DE LA TOUR DU VALAT

For the practical laboratories :

ALCYON BIRD-BITS CENTRAVET ELLMAN International Inc. ESCULAPE GENIA GER KARL STORZ MEDICAL SOLUTION NIKON SCIL ANIMAL CARE COMPANY VETOQUINOL 3M

Welcome to Arles 2005 !

The French Companion Animal Veterinary Association (AFVAC) and its exotics study group (GENAC) are happy and proud to contribute to Arles 2005, a joint event with AAV and ECAMS. The GENAC is a very active group of our Association. They took up the challenge of such a meeting with enthusiasm and competence. Bravo! Many thanks also to AAV and its Board for their effective collaboration with our national group. Bird medicine and surgery has become a well recognized discipline. This is due to the incommensurable involvement of dedicated veterinarians. This has justied the creation and development of ECAMS, a species-oriented European College of Veterinary Specialists, third partner of the adventure. Thank you as well When a eld expands, congresses are the indispensable stages of its development. This one will be remembered, I am sure, because of the outstanding and sophisticated programme set up by the organizers. Plenary Sessions, Master Classes and Practical Laboratories are the corner-stones of a well-balanced scientic meeting like this one. I am also convinced that the remarkable environment of Provence will contribute to the success of this gathering of highly motivated colleagues. Please take at least a few hours to enjoy the place I thank deeply all the organizing team and particularly Jean-Marie Pricard who spared no effort and time to make this event possible and, I am already convinced, successful. I wish you all a pleasant and fruitful meeting.

Didier-Nol Carlotti, President of AFVAC

A conference mirror of our activity

It wasnt the aim, but it is the result : This conference reects the role played by the avian veterinarian within our profession and society. The main role of the avian veterinarian is to treat and prevent the diseases of privately owned pet birds, pet shop birds, breeding centre birds, wild birds as well as those birds found in zoological parks, rehabilitation centres and reintroduction programs. By doing it, this veterinarian participates in the protection of the sanitary state of the poultry industry, and in the prevention and epidemiological survey of the zoonosis. But the veterinarian has also, in these different situations, a role of zootechnician, nutritionist and behaviourist. As a matter of fact, all these aspects are dealt with during this conference ! The participation of more than 160 colleagues from 21 countries in the 2 pre conference scientic tours, 71 oral presentations, 2 round table discussions, 6 master classes, 26 posters and 9 practical labs, displays, beyond our diversity, the similarity of our care to improve a scientic eld in rapid progression. I wish everybody to make use of this conference, but also to enjoy Provence and the exceptional birds living in these top places of biodiversity in Europe that are the Camargue, the Crau and the Alpilles.

Jean-Marie Pricard, Chairman of the organising committee

Table of Contents
Scientic meeting of the European College of Avian Medicine and Surgery
S.I.C.O. Santos & J.T. Lumeij. Concealed avian sexual dichromatism and multiple angle reectance spectrometry ......................................... J.-M. Hatt & J.T. Lumeij. Evidence-based avian medicine How to ask questions and nd answers ............................................................. R. Korbel & K. Sturm. Review on lightsources for birdhousing under articial light circumstances .................................................................. U. Benheim & I. Davidson. Molecular Identication and Prevalence of Psittacine Circovirus in Israel ............................................................ M. Hochleitner et al. Fungal cultures in feather probes of psittacine birds ...................................................................................................... S.E. Orosz. The senses of birds : their unique qualities ...................... J. van Engelen, I. Akkerdaas & N.J. Schoemaker. A study into the analgesic efcacy of Buprenorphine and Butorphanol in pigeons (Columba livia domestica) .................................................................... E.S. Story, D. Carboni & T.N. Tully. Establishing normal tear production values in hispaniolan Amazon parrots (Amazona ventralis) .................... J. Samour. Haemopathological responses in falcons to Newcastle disease ................................................................................................. M.-E. Krautwald, G. Stelzer & V. Schmidt. Food - intoxication in nestling psittacines ............................................................................... I. Westerhof, N.J. Schoemaker & P.Y. Barthez. Spiral Computed Tomography in Respiratory diseased Birds ....................................... T.A. Bailey & O. Combreau. Domestication and Disease : Challenges for Houbara Bustard Captive Breeding Projects in the Middle East ...... B.W. Ritchie et al. Epizootiology of Proventricular Dilatation Disease in Breeding Cockatiels ..........................................................................

3 5 7 9 11 13

19 21 23 25 27 29 31

Main European Association of Avian Veterinarians Conference

J. Chitty. Veterinary aspects of the great bustard (Otis tarda) Reintroduction project in the UK. ......................................................... M. Barrows, M. Hartley, J.M. Pittman. Diseases and management of captive crested screamers (Chauna torquata) ....................................... G. Cousquer. Clinical approach to the downer swan ........................... U. He, J.M. Blanco, R. Valboa, D. Villanua, C. Gortazar. Trichomonas gallinae in free-living birds of prey, passerines birds and woodpigeons (Columba palumbus) ......................................................................... E. Jourdain, M. Gauthier-Clerc, D. Bicout, Y. Kayser, P. Sabatier. A study of free-ranging wild birds to better understand their role in the circulation of west nile virus in Camargue, Southern France .................. O. Lambert, H. Pouliquen, B. Philippe, N. de la Cotte, M. LHostis. Wild bird exposure to anticoagulant rodenticides in Loire-atlantique (France), a 2 year study on waterbirds and raptors (2002-2003) .......... G. Riggs Mycobacterial infection in waterfowl collections : a conservation perspective ............................................................................................. M.G. Muller, A. George, A.T. Mannil. Haematological values of gyr hybrid falcons ......................................................................................... J. Samour, J.L. Naldo. Causes of morbidity and mortality in captive falcons in Saudi Arabia ......................................................................... T.A. Bailey, C. Silvanose, E. Pesci, A. Di Somma. Histoplasma sp. incidental nding, underdiagnosed, or occasional opportunistic pathogen of falcons in the Middle East ? ............................................................ U. Wernery, F.A. Hassan, T.A. Bailey, B. Johnston, J. Kinne. Mycobacteriosis in hunting falcons in the Middle East ................. S. Le Dran-Quenechdu, L. Marion, M. Popoff Epidemiology of avium botulism in France : example of Grand Lieu Lake ....................... A. Y Gancz, I.K Barker, L. Robbin, A. Dibernardo, K. McKeever, B. Hunter. A West Nile virus outbreak in North American owls .................

37 41 47

56

60

66 70 77 85

92 99 106 112

J. Hars, P. Auge, J. Pradel, M. Mortamais, D. Chavernac, G. Balanca, S. Zientara, I. Schuffenecker, H. Zeller. Surveillance of West Nile virus in the avifauna of Southern France ........................................................ P. Zsivanovits, D. Monks, N. Forbes. Adenovirus infection in raptors ... D. N. Phalen, G. Olsen, K. Russell. Diagnosis, prevention, and treatment of iron storage disease using the European starling as a model G. Rossi, S. Pesaro, C. Peccati, R. Ceccherelli, P. Petroselli. Biomolecular study of a progression of haemocromatosis in hill mynahs (Gracula religiosa) ................................................................................. J. Grfols, F. Bargall, D. Perpin, A. Ramis, J. Ballester. Circovirus infection in a canary breeding ock ....................................................... M. Carnarius, H.A Hafez, A. Henning, H.J Henning, M. Lierz. Central nervous symptoms caused by thiamine deciency in juvenile goshawks (Accipiter gentilis) .................................................................................. V. Schmidt, M-E. Krautwald-Junghanns. A comprehensive study on a disease complex associated with pigeon circovirus infection, young pigeon disease. ...................................................................................... D. Gelli, V. Ferrari, F. Franceschini, M Bedin, D. Bernardini, S. Romagnoli. Serum biochemical and electrophoretic patterns in the Eurasian buzzard (Buteo buteo) Reference values .......................... G. Werquin Hepatic haemosiderosis in birds : not only total dietary iron level, but several dietary factors and bird-specic defense mechanisms contribute to the development of the disease ........................................ O. Amann, M.J.M Visschers, G.M. Dorrestein, N.J Schoemaker, I. Westerhof, J.T Lumeij. Exocrine pancreatic insufciency in racing pigeons (Columba livia domestica) ........................................................ M. Lierz, H.M. Hafez. Endoscopic guided multiple entry surgery in birds ... E. Risi, D. Ordonneau, O. Gauthier. Two cases of femoral head luxations on cormorants (Phalacrocorax carbo) treated with a modied Meij-Hazewinkel-Nap technique. ......................................................... S. Villaverde, J. Benito de la Vibora, R. Gonzalez, M. Freire, M. Lopez Pea, F. Muoz Guzon, J. Rodriguez-Quiros. Management of the femorotibial luxation by coaptation splinting, intramedullary pins, external skeletal xator and tension bands : a pilot study in domestic pigeons (Columba livia domestica) .......................................................

120 130 136

141 148

151

159

166

171

179 184

190

196

T.N. Tully,A. Stevens, O. Diaz - Figaroa, T. Zachariah, M.A. Mitchell. Use of a dental composite to correct beak deviation in Psittacine species ..... A.M. Lennox, L. Crosta, M. Buerkle. The effects of isourane anaesthesia on gastrointestinal transit time ............................................. M. Desmarchelier, Y. Rondenay, G. Fitzgerald, S. Lair. Monitoring of end-expired partial pressures of carbon dioxide in anaesthetised birds of prey .................................................................................................... A. Scope, J. Burhenne, W.E. Haefeli, M. Hess. Pharmacokinetics and pharmacodynamics of the new antifungal agent voriconazole in birds ....... J.M. Pricard. Clinical assessment on the use of uconazole per os in 24 African grey parrots (Psittacus Erithacus) : acceptance, side effects and efciency ......................................................................................... G.H. Wilson, S. Hernandez-Divers, S.C. Budsberg, K.S. Latimer, M. Pethel. Pharmacokinetics and use of meloxicam in psittacine birds .... M. Pees, K. Kuhring, M-E. Krautwald-Junghanns. The use of enalapril in birds. Indications, clinical experiences, pharmacokinetics and possible side effects ......................................................................... E.M. Weilacher, F.R. Ungemach, M-E. Krautwald-Junghanns. Pharmacokinetics, bioavailability and compatibility of doxycycline in birds after oral administration ................................................................ M-E. Krautwald-Junghanns, V. Schmidt, N. Reitz. Oral furazolidone and chloramphenicol for treatment of gastro-intestinal bacterial infections ............................................................................................... C.H. Grund, B. Khler, R.T. Korbel. Evaluation of various tissues for diagnosis of psittacine beak and feather disease (PBFD) ..................... U. Bendheim, A. Lublin, N. Edery. Reliability of crop biopsy as a diagnostic tool for proventricular dilatation disease in psittacines birds . D. Perpin, H. Fernndez-Bellon, C. Lpez, A. Ramis. Myenteric ganglioneuritis in four non-psittacine birds ............................................ G. Le Loch, M. Deville, E. Risi, S. Bretagne, J. Guillot. Evaluation of the serological test Platelia Aspergillus for the diagnosis of aspergillosis ..........................................................................................

203 207

211 217

222 230

233

238

243 249 254 257

260

D. Monks, P. Zsivanovits, J. Chitty, C. Fisk, N.A. Forbes. Assessment of disc fusion tests to evaluate the effects of antibacterial nebulisation agents against bacteria isolated from the avian trachea ......................... A.Lublin, S. Mechani, V. Bumbarov. Diagnoses of rotavirus in pet birds vs Poultry ...................................................................................... R. Raue, H. Gerlach, H. Mller. Phylogenetic analysis of the hexon loop 1 region of adenovirus isolated from psittacine birds indicates the existence of a new psittacine adenovirus .............................................. D. Ordonneau, Y. Roman, D. Chaste-Duvernoy. Plasma electrophoresis reference ranges in various bird species .......................................................... Y. Roman, D. Ordonneau, D. Chaste-Duvernoy. Early detection of an acute inammatory condition by plasma protein electrophoresis and haematology in peafowls ....................................................................... R.T. Korbel. Digital scanning ophthalmoscopy in birds ........................ L. Crosta, L. Timossi. Imaging techniques in avian obstetrics ............ I. Fischer, A. Liesegang, J-M. Hatt. Quantitative computed tomography for the assessment of bone density in budgerigars : a pilot study ............ N.A. Forbes, P. Zsivanovits, D. Monks, S. Smith. Biosecurity of the avian hospital facility by design, protocols and procedures ................... G.M. Dorrestein. Consequence of severe dyspnoea for organ function ..... D. Reavill. A review of avian endoventricular mycosis submitted during 1998-2004 .............................................................................................. S. Pesaro, R. Ceccherelli, C. Tarantino, S. Scoccianti, E. Bert, G. Rossi. The rst Italian outbreak of polyomavirus infection in macaws ................... T.K. Ritzman. Cloacal disease and disorders in the avian patient ....... F. Rival. Auricular diseases in birds ...................................................... B. Stockdale. The nutritional implications of producing the optimal egg in captive birds .............................................................................. M.D. Stanford. The effect of UV-B radiation on calcium metabolism in grey parrots ............................................................................................

267 274

277 283

290 298 301 307 310 314 318 321 327 333 340 348

M. Lichtenberger, W. Chavez, C. Cray, C. Orcutt, D. DeBehnke, E. Stumpp, S. Diehl, B. Feldman, C. Page, L. Mull, L. Inman, R. Kirby. Response to uid resuscitation subsequent to acute blood loss in mallard ducks (Anas platyrhynchos) .............................................. J. Hooimeijer. Parrots dont bite children ............................................. Concurrent Master Classes sessions P. Zwart. The avian respiratory system : normal and pathological aspects .................................................................................................. M. Lichtenberger. Shock, uid therapy and CPCR for the avian patient ... S. Echols, S. Orosz. A master class of the urinary system : from anatomy to treatment options ................................................................ G.M. Dorrestein. Passerine and softbill medecine and surgery .......... P. Macwhirter. Avian clinical anatomy from an evolutionary perspective ............................................................................. M-E. Krautwald-Junghanns, M. Pees. Imaging non-invasive diagnostic techniques of the urogenital tract ............................................................ Posters European Association of Avian Veterinarians Conference Fischer, P. Keller, J-M. Hatt . Inuence of a seed diet versus a formulated diet on bodyweight and intestinal ora in budgerigar (Melopsittacus undulatus) over a six month period ............................... M. Gauthier-Clerc, J-P. Gendner, C. Le Bohec, Y. Le Maho. Effects of ipper bands on free-living penguins ..................................................... M. Gauthier-Clerc, C. Le Bohec, Y. Le Maho. Infestation by ticks Ixodes uriae in penguin colonies ......................................................................... S. Lafon, J-L. Pingret, C. Boucraut-Baralon. Real-Time PCR for diagnosis of three common infectious diseases in caged birds : Chlamydiophylosis, beak and feather disease and avian polyomavirosis

354 359

365 374 388 409 425 434

447 451

452

453

S. Le Dran-Quenechdu, M. Kammerer, H. Pouliquen, M. Larhantec . Vanadium content of oiled birds : is it a good marker for oil exposure ? ...... S. Le Dran-Quenechdu, E. Risi, O. Lambert. Cause of mortality of wild birds collected in a wildlife center in France : example of Nantes Wildlife Center ....................................................................................... A. Martel, A. Van Caelenberg, I. Gielen, F. Pasmans, H. van Bree. Computed tomographic anatomy of the pigeon (Columba livia) ............ H. Pouliquen, O. Lambert, N. de la Cotte, M. LHostis. Determination of the possible exposure of raptors and waterbirds to ve anticoagulant rodenticides in Loire Atlantique (France 44) .......................................... C. Remple, C. Nurse. Use of a composite silicone dental impression material to create a form-tting, exible, support cushion to facilitate wound healing in bumblefoot ................................................................. E. Risi, P. Costiou. Congenital abnormalities in a wild young Tawny owl (Strix aluco) ............................................................................................ Risi E, NGuyen F, Albaric O, Abadie J. Successful treatment of a cervical rhabdomyosarcoma on an Indian Fantail pigeon (Colomba livia) by surgical resection ...................................................................... R. Schuster, J. Kinne, U. Wenery, P. Mckinney. Endo parasites of the Asian houbara bustard (Chlamydotis undulata macqueenii) ........... C, Silvanose, T.A. Bailey, A. Di Somma. Antifungal susceptibility testing of fungi isolated from the respiratory tract of falcons ..................... J. Tena. Health control of various capercaillies (Tetrao urogallus) captured in the Principality of Andorra ............................................... S. Villaverde, U. He, J.M. Blanco. Treatment of crop stulas due to severe trichomoniasis in two booted eagles (Hieraaetus pennatus) .... J-P. Andr, F. Degorce-Rubiales. Mucocele in a lesser sulphur-crested cockatoo (Cacatua sulphurea) ........................................................... M. Barrows, M. Hartley, J.M. Pittman. Zinc toxicosis in a wattled crane (Bugeranus carunculatus) ...................................................................... J. Chitty. A new method for the cytological sampling of feather pulp ...

456

459 461

464

467

470

473 476 479 482 484 488 491 495

A. Horvatek, Z. Gottstein, C. Grozdanic, H. Mazija, E. PruknerRadovcic. Klebsiella oxytoca infection in monk parakeet .................. T.K. Ritzman. Chronic polychondritis in an African grey parrot (Psittacus erithacus) ............................................................................................ H. Wilson, R. Roberts, N. Northrup, S. Hernandez-Divers, K. Latimer. Radiation tolerance doses of the cutaneous and mucosal tissues in psittacine birds using cobalt-60 teletherapy ....................................... M-E. Terrrier, J. Barrat, J-R. Gaillet. Diseases found in French birds according to SAGIR database ............................................................... F. Rival. Clinical approach to ophthalmologic disorders in birds .......... A.M. Lennox, M. Stanford. Post-intubation stenosis in psittaccine birds ................................................................................................ D. Monks, N.A. Forbes, M. Fisher. Ixodes frontalis as a cause of avian disease in the UK ................................................................................... E. Bert, L. Tomassone, C.J. Soto Pieiro, I. Acosta Guevara, M. Gonzalez Navarrete, X. Galvez Aguilera. The role of avian medicine in a parrot conservation project of Amazona leucocephala in Cuba : preliminary results ..................................................................................

498 501

504 507 509 512 515

518

Practical Laboratories European Association of Avian Veterinarians Conference List of the practical laboratories ........................................................

523

Scientic meeting of the European College of Avian Medecine and Surgery ABSTRACTS

Division of Avian and Exotic Animal Medicine, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University

CONCEALED AVIAN SEXUAL DICHROMATISM AND MULTIPLE ANGLE REFLECTANCE SPECTROMETRY

S.I.C.O.Santos, J.T.Lumeij, DVM, PhD

ABSTRACT
Numerous avian species have plumage that reects both in the human visible range and in the ultraviolet (UV) part of the spectrum.It has been shown that birds employ their ability to detect UV light, apart from foraging and navigation, in behavioural interactions and mate-choice. Reectance spectrometry is considered an accurate and objective way to assess plumage colour and its results are independent of the colour perception system, either from the researcher or the studied subject. Most studies have only used one angle geometry (illumination/observation) to assess noniridescent plumage colours. In iridescent feathers, since their colour changes with the observation/illumination angles, more than one angle geometry has been used. However, the inuence of angle geometry in the colour assessment of non-iridescent feathers has been proved to be signicant as well. All avian species studied thus far by spectrometry, with the exception of the Long-tailed nch (Poephila acuticauda) (LANGMORE and BENNETT, 1999), have been proven to be sexually dichromatic when the UV part of the spectrum was considered. The Long-tailed nch has been classied by single angle spectrometry as monochromatic, even when the UV part of the spectrum was considered. Considering that angle geometry does have an inuence on spectrometric results and that birds exhibit different positions towards the opposite sex during mating displays, we hypothesize that all diurnal avian species are sexually dimorphic and that this trait can be visualized by using multiple angle spectrometry. Different body regions, actively involved in sexual displays, from the Blue fronted Amazon parrot (Amazona aestiva), Long-tailed nch (Poephila acuticauda), Java nch (Padda oryzivora), and Magpie (Pica pica), were assessed with a spectrometer using different illumination (45 and 90) and observation angles (30, 45, 60, 75, 90, 135). Resulting reectance data were summarized in several parameters (brightness, colour intensity, hue, contrast and UV chroma), both in the UV and in the total range, and were analysed by logistic regression. All studied species, including the Long-tailed nch previously classied as monomorphic by Langmore & Bennett (1999), proved to be sexually dichromatic. One hundred percent correct sex prediction could be achieved by combining different colour parameters from different body regions and angle geometries. The results of this study will not only have impact on avian morphology studies but also all subsequent theories in different elds of biology such as evolution and behaviour.

REFERENCES
1. LANGMORE N E and BENNETT A T D. Strategic concealment of sexual identity in an estrildid nch. Proc R Soc Lond Ser B - Biol Sci 1999; 266: 543 550.

AUTHORS ADRESS
S.I.C.O. Santos Division of Avian and Exotic Animal Medicine Department of Clinical Sciences of Companion Animals University of Utrecht Yalelaan 8 3584 CM Utrecht / The Netherlands e-mail: s.i.c.o.santos@vet.uu.nl

Division of Zoo Animals and Exotic Pets, Department of Small Animals Vetsuisse Faculty University of Zurich, Switzerland

EVIDENCE-BASED AVIAN MEDICINE HOW TO ASK QUESTIONS AND FIND ANSWERS

J.-M. Hatt, Prof Dr med vet; J.T. Lumeij, DVM, PhD

ABSTRACT
This paper presents current denitions of evidence-based medicine (EBM) and proposes to adopt for veterinary medicine the following modied denition by Cockroft & Holmes (2003): Evidence-based veterinary medicine is the use of current best evidence in making clinical decisions. A special challenge in applying EBM is the translation of a clinical problem as encountered in practice, into an answerable clinical question. The process of developing a well-formed clinical question is explained and the key elements (Patient or Problem, Intervention, Comparison and Outcome) introduced. Patient: By categorising e.g. species, age, management (captive or free), a search in databases will be more focused. Example: Lead intoxication in free ranging vultures. Intervention: Denition of the diagnostic or therapeutic intervention and the exposure or prognostic factor that the patient has been or will be subjected to. Example: Will an iron level of <150 ppm in mynah diets signicantly reduce the incidence of iron storage disease? Comparison: Searching for best evidence often implies a choice between two interventions or an intervention and no treatment at all. Example: How does PCR compare to ELISA in the diagnosis of Chlamydophila psittaci? Outcome: The outcome that is desired may have to be included in the question, in relation to aspects such as time-frame, cost-benet, welfare. Example: Is there an enhancement of ventriculotomy healing in Amazon parrots after inclusion of a collagen patch during surgery? Subsequently sources of evidence will be discussed and the hierarchy of evidence is introduced, which is a spectrum of potential sources with the source most likely to provide the best evidence at the top. Previously given examples will be used to demonstrate retrieval of evidence. Of special interest is the Medline database (Pubmed; www.pubmedcentral.nih.gov) and the Veterinary Science Database (formerly VetCD; www.cabi-publishing.org), the approach to answer a specic clinical question and to retrieve general information quickly, using these databases will be presented.

Further reading COCKROFT P and HOLMES M. Handbook of evidence-based veterinary medicine. Blackwell Publishing, Oxford. 2003

AUTHORS ADDRESS
Division of Zoo Animals and Exotic Pets Department of Small Animals Vetsuisse Faculty University of Zurich Winterthurerstrasse 260 8057 Zurich Switzerland Jean-Michel.Hatt@access.unizh.ch

Klinik fr Vgel, Ludwig-Maximilians-University Munich, Sonnenstrasse, Oberschleissheim, Germany

REVIEW ON LIGHT SOURCES FOR BIRD HOUSING UNDER ARTIFICIAL LIGHT CIRCUMSTANCES
R. Korbel, Prof Dr med vet; K. Sturm, Dr med vet

ABSTRACT
Unlike man most of the diurnal birds are capable of penta- / tetrachromatic vision including ultraviolet perception. Furthermore birds are able to resolve motion pictures at a higher icker frequency (approximately 180 frames / sec) rather than man (15 80 frames / sec). These circumstances have to be considered when keeping birds - pet birds as well as poultry, pigeons and raptors - under articial light circumstances, as detection of sex via UV-reection of feathers, assessment of food sources and prey patches, feeding of chicks are based on UV-vision and thus are of importance not only regarding to animal welfare aspects. Following a review on avian vision capacities known so far the proposed paper will give a technical orientated review of commercially available light sources including uorescent lamps, energy saving light sources, LED (light emitting diodes)-light sources, incandescent light as well as discharge lamps. These data will be compared and discussed regarding their suitability providing birds, which are kept under articial light sources, with light that fulls their needs regarding to vision aspects. The table on the following page gives a quick orientation of light sources available on the market, which to a certain extent meet avian requirements:

AUTHORS ADRESS Ruediger Korbel, Prof Dr med vet, Dr med vet habil, Cert Vet Ophthalmol Klinik fr Vgel Ludwig-Maximilians-University Munich Sonnenstrasse 18 D 85764 Oberschleissheim / Germany e-mail: korbel@lmu.de

Light sources according to their suitability for birds Light source Brandname Incandescent Conventional Spectral range Flickerfree* UVA portion 400 -1100nm, max at 950nm yes none not described

ESU Birdlife Brightlight Spot full-spectrum Incandescent Lamp Fluorescent + CF Conventional True-light-Solux Vita-lite (Duro-Test) Activa (Sylvania/Osram) Arcadia Birdlamp ESU Avian Birdlamp Halogen Conventional DECOSTAR IRC cold-light reector lamp (Osram) HID Conventional SoLux LED Conventional Golden Dragon (Osram) 3-bandspectrum full-spectrum full-spectrum full-5-bandspectrum full-spectrum full-spectrum

yes

according to none ballast according to described ballast according to described ballast according to described ballast according to 12% UVA, ballast 2.4% UVB according to 10% UVA, ballast 3% UVB none none (absorbs radiation below 390nm)

80% in infrared yes full-spectrum nearUV to infrared yes

according to according to lter ballast

natural daylight according to practically none (lter) spectrum ballast narrow-band spectrum 380-700nm, peak at 410nm yes (DC) yes (DC) none none

* Flicker frequency over 180 Hz no UVA according to Thrush 2000 CCG: operates at twice the mains supply current: 100 - 120 Hz, ECG: operates at 30-50 megahertz frequencies DC: operates under direct current

Koret School of Veterinary Medicine, Hebrew University, Jerusalem. Division of Avian and Fish Diseases, Kimron Veterinary Institute, Bet Dagan, Israel

MOLECULAR IDENTIFICATION AND PREVALENCE OF PSITTACINE CIRCOVIRUS IN ISRAEL

U. Bendheim, DVM; I. Davidson, PhD

ABSTRACT
Psittacine Beak and Feather Disease (PBFD) was rst detected in Israel in two Sulphur-Crested Cockatoos, imported from Singapore, who showed typical clinical signs: feather deformation, feather loss and necrosis of the upper beak (BENDHEIM and HOCHLEITHNER, 1991). The diagnosis was conrmed by histopathology and electron microscopy. Since then, most birds suspected for PBFD infection have shown typical clinical signs. The diagnosis was conrmed by PCR in different laboratories. During the last 4 years PBFD is being monitored routinely in 4 breeding farms and some Psittacines collections in order to detect apparently healthy PBFD carriers and are assessed by the bird types and years. This was done from blood samples taken from the brachial vein. The birds tested for PBFD by PCR in the years 2001-2003 showed the following results: All Psittacines Cockatoos Grey Parrots Other Psittacines 13,9 % positive 28,0 % positive 4,9 % positive 6,7 % positive n=417 n=126 n=142 n=149

During the years 2001-2003 the infectivity rate increased from 6.3% in the year 2001 to 18.9% and 19.7% in the following 2 years. Until December 2003 the PBFD infectivity was assessed in 417 birds by PCR performed only by the Molecular Diagnostic Services in Westville, South Africa (MDS), while afterwards it was performed also in the Kimron Veterinary Institute for research purposes, where the Ypelaar PCR (YPELAAR et al, 1999) was applied and validated by sequencing. The virus was detected in both laboratories with a similar efcacy. Most positive cases were detected in clinically-affected birds, including cockatoos and other psittacines. In several cases PBFDV sequences were detected in apparently healthy birds.

To broaden the molecular detection capability and the environmental monitoring of circoviruses, we analyzed feathers that were shed in the proximity of clinicallyaffected birds and were able detect viral-sequences in several cases.

REFERENCES
1. BENDHEIM U. and HOCHLEITHNER M. Unpublished case 1991. 2. YPELAAR, I., BASSAMI, M.R., WILCOX, G.E. and S.R. RAIDAL. A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus. Vet Microbiol 1999; 68: 141 8.

AUTHORS ADDRESS
U. Bendheim, DVM P.O. Box 196 Shawe Zion 25227 Israel

10

Tierklinik Strebersdorf, Vienna, Austria

FUNGAL CULTURES IN FEATHER PROBES OF PSITTACINE BIRDS

M. Hochleithner, Dr med vet; C. Hochleithner, Dr med vet; E. Leidinger; N. Ziegler; G. Harrison, DVM

ABSTRACT
Microbiological testing in cases of feather picking in psittacine birds is often recommended as standard procedure. Positive fungal cultures of Aspergillus sp. are considered to be controversal. In this study feather probes (feathers plugged 1cm lateral of christa sternalis both sides) of feather picking and non-feather picking birds of different psittacine species were collected and incubated on sabourauds glucose 2% agar. The study included birds with the history and signs of feather picking (African grey parrots (n=52), cockatoos (n=16), Amazon parrots (n=10), macaws (n=17) and budgerigars (n=5)) as well as birds with no history or signs of any feather or skin problem, brought to the clinic for other reasons (African Grey Parrots (n=48), cockatoos (n=19), Amazon parrots (n=32), macaws (n=11) and budgerigars (n=15)). Differences between frequency distributions of culture results of feather-picking and non feather picking birds were tested for signicance with Fischer Exact test (Table 1). With the exception of Amazon parrots the results demonstrate a signicant increase in prevalence of Aspergillus sp growth (within 72 hours) on feather probes from feather picking birds compared with those from non picking birds. Species Feather picking Non feather picking P value of difference Fisher Exact test Aspergillus pos neg pos neg African Grey Parrot 46 6 9 39 0.000 Cockatoos Amazon Macaws Budgerigar Total 15 2 14 5 82 1 8 3 0 18 0 2 1 1 13 19 30 10 14 194 0.000 0.236 0.000 0.000 0.000

Tab. 1 Aspergillus sp culture results from feather samples collected from feather plucking psittacines and psittacines without feather disorders.

11

The clinical signicance of positive culture results is not clear. First of all positive test results can be observed in non-feather picking birds. Furthermore, the fact that feather picking birds show a higher frequency of positive cultures might be secondary to poor feather structure caused by feather picking, making the feathers vulnerable to secondary infections. In a second study feathers of 40 African grey parrots were collected and incubated on sabourauds glucose 2% agar which had been prepared with the content of the uropygeal gland of each bird as well as without this agar modication. Signicant higher numbers of positive cultures were found using the secretion of the uropygeal gland which seems to be in contrast to literature were this gland secretion should prevent fungal growth on feathers in birds. Finally the secretion of the uropygeal gland was examined histologically. Staining was performed by a cytological standard staining (Hemacolor, VWR International). A few slides were additionally stained by Gram-stain (VWR International).

AUTHORS ADRESS
M. Hochleitner, Dr med vet Tierklinik Strebersdorf Muhlweg 5 Vienna, AT 1210 / Austria e-mail: hochleithner@aon.at

12

Perrysburg Animal Care Perrysburg, Ohio USA

THE SENSES OF BIRDS : THEIR UNIQUE QUALITIES

Susan E. Orosz, DVM, PhD This paper will review the special senses of birds with particular emphasis on aspects with clinical applications. Birds are unique and wonderful creatures and their senses are an integral part of their makeup. Of all the senses of birds, vision is by far the most important. Birds are exquisitely visual animals. The cross-sectional diameter of the optic nerves of birds are larger than the diameter of their cervical spinal cord (KING and McCLELLAND,1984; PORTMAN and STINGELIN, 1961; BREAZILE and KVENZEL, 1993). This large collection of myelinated axons is from the ganglion cell layer of the retina. In comparison, humans are highly visual primates but have only 40% of the numbers of retinal axons per optic nerve when compared to pigeons or chicks (BINGGELI, and PAULE, 1969; RAGER and RAGER, 1978). Raptors have even larger numbers of neurons per optic nerve as their acuity surpasses that of other living beings (FOX and WESTENDORF, 1976). The eyes of birds comprise a considerable volume of the head and are very large in relation to brain size. The basic components of the eye are similar to mammals except for the scleral ossicles to help maintain the shape of the globe. Light has to pass through the cornea, anterior chamber, lens, and the vitreous body before is reaches the retina. It is in the retina where light energy is converted to electrical impulses by bleaching of the photoreceptors. These components of the globe or optic media are very transparent and are able to transmit light down into the near ultraviolet spectrum (at least 310 nm) (EMMERTON, 1980). This ability to see into the near ultraviolet allows them to discern ripeness of food items, identify birds within their own species as individuals, and to determine the sex within a species (KORBEL and GROPP, 1999) that, to us humans, appears visually as sexually monomorphic. These factors need to be taken into account clinically as humans, for example, determine ripeness largely by taste and smell rather than by sight. The visual impression of objects and colors are important to birds. The shape of the globe of a particular species of birds is the result of its ecological requirements (GNTRKN, 2000). Acuity is maximized by increasing the anterior focal length of the eye, thereby allowing the optic image to be spread out over a larger retinal surface. The eye of the ostrich has an axial length of 50 mm, which is the largest of any land vertebrate and twice that of a human (WALLS, 1942). Another technique that achieves the same goal is represented in the eyes of raptors. Their tubular-shaped globes result in high visual acuity. Part of this results from the shape

13

and the remainder is from the high ganglion cell to receptor hookup ratio that increases visual resolution. However, this non-pooled system requires high light intensity to function adequately. Consequently, these daylight raptors have reduced resolution at dusk (RAYMOND, 1985). Clinically, this may make it difcult for the bird in a low-light hospital cage to locate its food, but it may also allow the bird to be calmer than at higher light levels. Information from the retina is sent to the area that is represented as the superior collicus in mammals. This is a relay station for visual information. This area is so large in birds that it is described as the optic lobe. It sits dorsally over the midbrain. This reex pathway is thought to relay information concerning moving objects (ROGERS and MILES, 1972), as occurs in mammals. It is also involved in enhancing contrast under dim light conditions (HELLMANN et al, 1995). This area may also be involved in pecking and food selection. Studies have shown that birds such as the Clarks nutcracker have visual memory for over 6,600 caches of stores containing 33,000 seeds (Vander WALL and BALDA, 1977). The lowly pigeon has not been considered the smartest bird but they have been found to understand visual concepts of animals (ROBERTS and MAZMANIAN, 1988) as well as same versus different (WRIGHT, 1988). They are able to rank optic patterns by using transitive inference logic (von FERSEN et al, 1992; GNTRKN, 2000). We know clinically that parrots know the difference between individual dogs, as they love to call them by name, and they know humans as individuals as well. Memory of visual images and working memory are also observed in birds. It was supposed that working memory--the ability to temporarily store and manipulate currently relevant information-- was the consequence of a prefrontal cortex and a neocortex containing grey matter. However, birds are able to handle working memory without a laminated neocortex. Instead, they appear to use their neostriatum caudolaterale. Data demonstrates that even though they do not possess the same anatomic components as mammals, they evolved neuronal mechanisms to master equivalent cognitive demands (DIEKAMP et al, 2002). We are just beginning to unravel the vast complexities of their visual system and its relationship to learning and memory. We need to think about this level of complexity when developing toys and enrichment items as they are very color oriented. From the enrichment perspective, they are also great discriminators of thousands of images. When we approach their needs, we must bear in mind that they have very active visual brains that need stimulating. Sensitive hearing and vocal communication are important in the behavioral repertoire of birds. Birds have the most highly evolved auditory system of non-mammalian species. While the mammalian ear is considered to be more specialized than birds, that does not mean that the avian ear is inferior (NECKER, 2000). The normal range for hearing often does not exceed 10 kHz but it does have a restriction to an upper limit of 20kHz (SCHWARTZKOPFF, 1973). As with some mammalian species, like the elephant, there are some birds that are able to hear infrasound (NECKER, 2000).

14

Hearing involves complex interactions between the sense organ, the ear, and the processing of sound in the central nervous system. Airborne sound waves enter through the external acoustic meatus and cause the tympanic membrane to vibrate. The tympanic membrane is attached to its ossicle, the columella, and motion in the membrane sets up a wave motion in the membranous labyrinth of the inner ear. The wave motion is converted to mechanical energy by the discharge of sensory receptors located on the papilla, the hair cells. The electrical energy and its discharges are transmitted to the CNS by the eighth cranial nerve (VIII). In the CNS, further processing occurs and is interpreted by higher centers. Owls have the ability to discriminate changes in the location of sound sources that are as small as 3 degrees apart and can aim their heads within 2 degrees of the source. They have a spatial map in their midbrains that is much larger than their behavioral perception. For example, a typical neuron has a spatial receptive eld that spans 40 degrees many times wider than the behavioral threshold (BALA et al, 2003). The ability to discriminate frequency differences in birds is similar to that of humans (KUHN et al, 1980), as is their ability to discriminate 2 sounds of different intensity (KLUMP and BAUR, 1990). Time resolution of hearing in birds is also similar to humans (DOOLING, 1992). Two sounds separated by a gap are recognized as separate if the gap exceeds 210 msec (WILKINSON and HOSE, 1975). Echolocation occurs in only two families of birds, the Steatornidae, which are represented by the oilbird and by swiftlets. These birds use this sense in dark caves for orientation (NECKER, 2000). Hearing represents the sensory component to the motor response, vocalization. Studies of vocalization and vocal learning are providing further information about the avian brain and its processing. For example, the mammalian basal ganglia and its connections with the thalamus and cortex are important for motor control and cognitive functions. It appears that the avian brain has a closed loop for vocal learning that is similar to that of mammals (LUO et al, 2001). This suggests that birds, like mammals, have a comparable ability to learn and should not be relegated to second class citizen status in the animal world. This has important implications from a lab animal perspective and a humane one as well. The chemical senses of bird include their ability to taste and their ability to smell. Chemesthesis is the perception of chemically induced pain. Sensitivity to chemical irritants is adaptive so that they avoid actual physical damage by avoiding noxious stimuli. The sensory input for this chemical avoidance sense in birds appears to be the components of the trigeminal nerve (CN V). It seems that birds rarely avoid mammalian irritants even though the trigeminal nerve is responsive to the chemical stimuli (MASON et al, 1989; MASON and OTIS, 1990). Many aromatic chemicals are aversive to birds. These chemicals are aversive on a purely chemical basis. This aversive quality is unlearned, it appears on the initial contact, and there is no evidence that aversion is altered by GI feedback. Further, birds do not seem to associate aversion of the stimulus with other chemosensory cues, suggesting that conditioned avor avoidance learning does not occur (CLARK, 1995). It has also been determined

15

that birds do not habituate to the stimulus so that the avoidance persists without reinforcement (CLARK and SHAH, 1994). As a result, oral medications may continue to be refused despite frequent attempts to habituate a bird to their taste. Birds have relatively few taste buds when compared to other vertebrates. Taste buds are distributed throughout the oropharynx, often in close association with salivary gland openings (BERKHOUDT, 1985). The avian taste bud does not open directly onto the epithelial surface, suggesting that saliva would be important to the transference of the chemical sense to a mechanical nerve ending associated with the taste bud. Although most of the chemical sense of birds is toward avoidance of a noxious compound, their taste preferences are not well documented. While we humans may enjoy something or project that a bird may like it, our taste preferences may serve the bird poorly. Many species including parrots, budgerigars and nectar feeders have a preference for sweetness using natural sugars mixed with drinking water (KARE and ROGERS, 1976; STROMBERG and JOHNSEN, 1990). Finches in the family of Carduelidae have a great preference for salt (MASON and ESPAILLAT, 1990). Birds without a nasal salt gland tend to refuse foods with a concentration that is hypertonic to their body uids (BARTOLOMEW and MacMILLAN, 1960). Birds may be tolerant of sour but this tolerance is species dependant. Cockatiels are more sensitive to organic acids than to inorganic acids (MATSON et al, 2001). This data suggest to the clinician that food preferences are individualized and that they tend to avoid inorganic acids while sweetness may help to hide drugs and other medications. The somatosensory system allows for the animal to perceive its body surface with the external environment. The information received by the exteroreceptive cutaneous receptors is taken into the spinal cord to be sent up to the level of the telencephalon for interpretation. The mechanoreceptors, the thermoreceptor, and the nociceptors (pain) serve different but important functions. It is important for the clinician to note that the pain pathways appear to be similar in birds when compared to mammals (OROSZ, 1996), indicating that issues involving pain and its management should be addressed in a manner similar to other companion animals. The Herbst corpuscles allow ducks billing in the water to discern the texture of the items in the murky water. They also provide information to a macaw, for example, that you are dremeling its beak, both by the texture and pressure and by the heat generated. Birds have unique anatomic features including their lissencephalic brain. While in the past, that feature suggested bird brains were not endowed with the mental capacity of mammals and humans, recent data shows that these preconceived notions are not true. Birds have a greater capacity visually than humans and a similar capacity with hearing. They are not taste or smell oriented but their somatosensory system is similar neuroanatomically to humans. Bird brains and their sensory systems are not that simple! Birds cognitive and learning abilities, along with their pain perception, need to be considered both in their husbandry and medical treatment. Taste preferences, or perhaps, taste aversion, will require us to be remain creative about medicating birds. We should also consider their visual perception (eg, ability or inability to discern food in ambient light and calming effect of low levels of light) when treating or convalescing birds.

16

REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. BALA AD, SPITZER MW, TAKAHASHI TT. Prediction of auditory spatial acuity from neural images on the owls auditory space map. Nature 2003; 424: 771 4. BARTOLOMEW GA, MACMILLAN RE. The water requirements of mourning doves and their use of sea water and NaCl solutions. Physiol Zool 1960; 33; 171. BERKHOUDT H. (1985.) Structure and function of avian taste receptors. In: LEVY AS, MCLELLAND (eds): Form and Function in Birds III. New York, NY, Academic Press 1985; 463 96. BINGGELI RL, PAULE WJ. The pigeon retina: quantitative aspects of the optic nerve and ganglion cell layer. J Comp Neurol 1969; 137: 1 18. BREAZILE JE, KVENZEL WJ. Systema nervosum centrale. In: BAUMEL JJ (ed): Handbook of Avian Anatomy: Nomina Anatomica Avium. Cambridge, MA, Nuttall Ornithological Club No. 23, 1993; 493 554. CLARK L. Modulation of avian responsiveness to chemical irritants: effects of prostaglandin E1 and analgesics. J Exp Zool 1995; 271: 432 40. CLARK L, SHAH PS. Tests and renements of a general structure-activity model for avian repellents. J Chem Ecol 1994; 20: 321 39. DIEKAMP B, KALT T, GNTRKN O. Working memory neurons in pigeons. J Neurosci 2002; 22(4): RC210. DOOLING R. Hearing in birds. In: WEBSTER DB, FAY RR, POPPER AN (eds): The Evolutionary Biology of Hearing. New York, NY: Springer-Verlag 1992; 545 59. EMMERTON J, SCHWEMER J, MUTH I, SCHLECHT P. Spectral transmission of the ocular media of the pigeon (Columbia livia). Invest Opthalmol Visual Sci 1980; 19: 1382 7. FOX R, LEHMKUHLE SW, WESTENDORF DH. Falcon visual acuity. Science 1976; 192: 263 5. GNTRKN O. Sensory physiology: Vision. In: WHITTOW GC (ed): Sturkies Avian Physiology. 5th ed. San Diego, CA: Academic Press 2000 HELLMANN B, WALDMANN C, GNTRKN O. Cytochrome oxidase activity revelas percolations of the pigeons ectostriatum. Neuroreport 1995; 6: 881 5. KARE MR, ROGERS JG. Sense organs: taste. In: STURKIE PD (ed) Avian Physiology. Berlin, Springer Verlag 1976 KING AS, MCCLELLAND J. Birds, Their Structure and Function. 2nd ed. Bath, United Kingdom, Bailliere Tindall 1984: 237 314. KLUMP GM, BAUR A. Intensity discrimination in the European starling (Sturnus vulgaris). Naturwissenschaften 1990; 77: 545 7. KORBEL RT, GROPP U. (1999). Ultraviolet perception in birds. Proc Annu Conf Assoc Avian Vet. 77-81. KUHN A, LEPPELSACK HJ, SCHWARTZKOPFF J. Measurement of frequency discrimination in the starling (Sturnus vulgaris) by conditioning of heart rate. Naturwissenschaften 1980; 67: 102. LUO M, DING L, PERKEL DJ. An avian basal ganglia pathway essential for vocal learning forms a closed topographic loop. J Neurosci 2001; 21(17): 6836 45. MASON JR, ESPAILLAT JE. Differences in taste preference between redwinged blackbirds and European starlings. Wilson Bull 1990; 102: 292 9.

17

21. MASON JR, OTIS DL. Aversiveness of six potential irritants on consumption by red-winged blackbirds (Agelaius phoeniceus) and European starlings (Sturnus vulgaris). Wilson Bull 1990; 102: 292 9. 22. MASON JR, ADAMS MA, CLARK L. Anthranilate repellency to starlings: chemical correlates and sensory perception. J Wildl Manage 1989; 53: 55 64. 23. MATSON KD, MILLAM JR, KLASING KC. Thresholds for sweet, salt, and sour taste stimuli in cockatiels (Nymphicus hollandicus). Zoo Biol 2001; 20: 1 13. 24. NECKER R. The avian ear and hearing. In: WHITTOW GC (ed): Sturkies Avian Physiology. 5th ed. San Diego, CA, Academic Press 2000. 25. OROSZ SE. Principles of avian clinical neuroanatomy. Semin. Avian Exotic Pet Med. 1996; 5: 127 39. 26. PORTMAN A, STINGELIN W. The central nervous system, In: MARSHAL AJ (ed): Biology and Comparative Physiology of Birds, vol II. New York, Academic Press 1961: 1 36. 27. RAGER G, RAGER U. Systems-matching by degeneration. I. A quantitative electronic microscopic study of the generation and degeneration of retinal ganglion cells in the chicken. Exp Brain Res 1978; 33: 65 78. 28. RAYMOND L. Spatial visual acuity of the eagle, Aquila audax: A behavioral, optical and anatomical investigation. Vision Res 1985; 25: 1477 91. 29. ROBERTS WA, MAZMANIAN DS. Concept learning at different levels of abstration by pigeons, monkeys, and people. J Exp Pyschol Anim Behav Process 1988; 14: 247 60. 30. ROGERS JJ, MILES FA. Centrifugal control of the avian retina. V. Effects of lesions of the isthmo-optic nucleus on visual behaviour. Brain Res 1972; 48: 147 56. 31. SCHWARTZKOPFF J. Mechanoreception. In: FARNER DS, KING JR, PARKES KC (eds) Avian Biology. New York, NY, Academic Press 1973; 417 77. 32. STROMBERG MR, JOHNSEN PB. Hummingbird sweetness preferences: taste or viscosity. Condor 1990; 32: 606 12. 33. Vander WALL SB, BALDA RP. Coadaptation of the Clarks nutcracker and the pinyon pine for efcient seed harvest and dispersal. Ecol Mon 1977; 47: 89 111. 34. von FERSEN L, WYNNE CDL, DELIUS JD, STADDON JER. Transitive inference formation in pigeons. J Exp Psychol Anim Behav Process 1992; 17: 334 41. 35. WALLS GL. The Vertebrate Eye and Its Adaptive Radiations. Cranbook Institute of Science, Bloomeld Hills, MI 1942. 36. WILKINSON R, HOSE PE. Time resolution of acoustic signals by birds. Nature 1975; 258: 320 1. 37. WRIGHT AA, COOK RG, RIVERA JJ, SANDS SF, DELIUS JED. Concept learning by pigeons: matching-to-sample with trial-unique video picture stimuli. Anim Learn Behav 1988; 16: 436 44. AUTHORS ADRESS S. Orosz, DVM, PhD, Dipl. ABVP-certied in Avian Practice Perrysburg Animal Care Perrysburg, Ohio USA e-mail: DrSusanOrosz@aol.com

18

Division of Avian and Exotic Animal Medicine, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, The Netherlands

A STUDY INTO THE ANALGESIC EFFICACY OF BUPRENORPHINE AND BUTORPHANOL IN PIGEONS (Columba livia domestica)
J. van Engelen, DVM; I. Akkerdaas, DVM; N.J. Schoemaker, DVM, PhD

ABSTRACT
In the last decade opioid drugs have been studied for their efcacy in providing analgesia in avian species. Most studies have been performed in the United States of America were it was found that butorphanol has an isourane-sparing effect (CURRO et al, 1994), and was found to be more effective in providing analgesia than buprenorphine in conscious African Grey Parrots (PAUL-MURPHY et al, 1999). In Germany, however, it was found that buprenorphine in a dose of 0.5 mg/kg had an analgetic effect in racing pigeons (GAGGERMEIER et al, 2000) as well. Since butorphanol was not readily available in The Netherlands the authors wanted to compare the analgesic efcacy between butorphanol and buprenorphine in pigeons anaesthetized with isourane. In this study, change of heart rate was used as the measure for analgesia, since in mammals there is evidence that cardiovascular and pain regulatory pathways are functionally linked (RADICH and MAIXNER, 1984; ZAMIR and MAIXNER, 1986). The study was performed in 9 pigeons. Each pigeon was studied 3 times, every time with a different form of premedication, the latter being determined randomly according to the Latin Square. At least one week was scheduled between each study to allow the birds to fully recover. Premedication comprised either buprenorphine (0.5 mg/kg), butorphanol (2 mg/kg) or saline (control) with a xed volume of 0.7 ml IM. The total volume was divided over both pectoral muscles. The investigator was unaware of the premedication the pigeon had received prior to measurements. Fourty-ve minutes after premedication, anaesthesia was induced by mask with isourane 4% in 100% oxygen (1l/min), after which the pigeons were intubated and ventilated with intermittent possitive pressure (IPPV). Anaesthesia was maintained at an end-tidal concentration of 1.8 % isourane. One hour after premedication, a noxious stimulus (toe pinch) was administered for a period of 10 seconds, 3 times with three minute intervals. During the experiment heart rate, body temperature and end-tidal concentrations of CO2, O2 and isourane were monitored. Reactions to the stimuli were also registered. Differences between the heart frequencies in the three groups were compared by means of an ANOVA for repeated measures. Although the heart rate in pigeons receiving butorphanol was signicantly lower compared to those receiving saline or buprenorphine, there was no signicant difference in heart frequency in any of the pigeons during the noxious stimuli.

19

We therefore conclude that no analgesic effect could be measured in pigeons receiving either buprenorphine or butorphanol (at the trial dose rates), as assessed by an increased heart rate response to a standard noxious stimulation.

REFERENCES
1. CURRO TG, BRUNSON DB, PAUL-MURPHY J. Determination of the ED50 of isourane and evaluation of the isourane-sparing effect of butorphanol in cockatoos (Cacatua spp.). Vet Surg 1994; 23: 429 33. 2. GAGGERMEIER B, HENKE J, SCHATZMANN U, ERHARDT W, KORBEL R. Untersuchungen zur Schmerzlinderung mit buprenorphin bei Haustauben (Columba livia Gmel., 1789, var. domestica). Deutsche Vet Gesellsch, Mnchen 2000: 75 6. 3. PAUL-MURPHY J, BRUNSON DB, MILETIC V. Analgesic effect of butorphanol and buprenorphine in conscious African grey parrots (Psittacus erithacus erithacus and Psittacus erithacus timneh). Am J Vet Res 1999; 60: 1218 21. 4. RADICH A and MAIXNER W. The role of sinoaortic and cardiopulmonary baroreceptor reex arcs in nociceptive and stress-induced analgesia. In: KELLY DD (ed): Stress-Induced Analgesia. New York Academy of Science 1984; 385 401. 5. ZAMIR M, MAIXNER W. The relationship between cardiovascular and pain regulatory systems. In: KELLY DD (ed): Stress-induced Analgesia. New York Academy of Science 1986; 371 384.

AUTHORS ADRESS
N.J. Schoemaker, DVM, PhD, Dipl. ABVP-certied in Avian Practice Division of Avian and Exotic Animal Medicine Department of Clinical Sciences of Companion Animals University of Utrecht Yalelaan 8 3584 CM Utrecht / The Netherlands e-mail: N.J. Schoemaker@vet.uu.nl

20

Louisiana State University School of Veterinary Medicine Baton Rouge, Louisiana, United States

ESTABLISHING NORMAL TEAR PRODUCTION VALUES IN HISPANOILAN AMAZON PARROTS (Amazona ventralis)
Eric S. Story, DVM, Debra Carboni, MS, DVM, Thomas N. Tully, Jr., DVM, MS

ABSTRACT
There are currently 2 methods used to measure tear production in animals, the Schirmer Tear Test (STT) and the Phenol Red Thread Test (PRTT). There have been 2 reports of values obtained when performing STT in birds (WILLIAMS, 1994; KORBEL and LEITENSTORFER, 1996). No investigations have been performed to conrm the viability of the PRTT in avian species. The PRTT has been investigated in dogs and cats (BROWN et al, 1996, 1997). The PRTT is faster and causes less reexive tearing than the STT due to the signicantly less irritating contact with the cornea in species where it has been evaluated. The purpose of this study was to determine if the PRTT is a feasible measurement of tear production in birds, to determine the mean and normal range of PRTT values in normal Amazon Parrots prior to topical anesthesia (PRTT1) and after application of topical anesthesia (PRTT2), and to compare PRTT and STT values before and after topical anesthesia in Amazon parrots. Based on prior research reports, two primary hypotheses were proposed for this investigation: 1) STT1 (without anesthesia) and STT2 (with topical corneal anesthesia) will not be signicantly different from previous reports, 2) PRTT measurements of tear production will be easier to perform in birds, but will have a wider standard deviation than the STT. Twenty six (26) Hispanoilan Amazon parrots maintained at the LSU School of Veterinary Medicine in a controlled environment allowed for the rst study of this type using a statistically signicant population of a similar psittacine species with a known life and medical history. Corneal anesthesia was accomplished using topical proparacaine after the initial STT and PRTT and prior to a second STT and second PRTT. To perform the STT, 5mm lter paper strips were used (Schirmer Tear Test Strips, Alcon Canada, Mississauga, Ontario, Canada) and maintained behind the ventral eyelid margin for 1 minute. To perform the PRTT, Phenol Red Thread Test Zone Quick (Menicon America, San Mateo, California, USA) was used, placing a 3mm bend in the thread then inserting into the lateral canthus for 15 seconds. Tear absorption and a resulting red color change on the thread was measured using Jameson calipers. After all tests were performed a sodium uorescein dye was used to identify any corneal ulceration or trauma. For statistical analysis of tear production measurements paired t-test were used to compare PRTT and STT values prior to and following topical anesthesia. To compare

21

the individual birds from day to day the proc GLM test for repeated measures was used. P< 0.05 was considered signicant for our statistical results. Pearsons correlations between PRTT & STT were also determined during this investigation.

RESULTS
PRTT1 PRTT2 STT1 STT2 15.5 +/- 3.3 mm 15.5 +/- 3.6 mm 8.0 +/- 2.3 mm 5.1 +/- 2.5 mm (8-22) (7-23) (3-12) (1-12) [9-22] [8-23] [3-13] [0-10]

PRTT1 phenol red thread test without topical corneal anesthesia PRTT2 phenol red thread test with topical corneal anesthesia STT1 Shirmer tear test without topical corneal anesthesia STT2 Shirmer tear test with topical corneal anesthesia Statistical results of this study indicate the PRTT is a feasible measurement of tear production in the Amazon parrot and can be used to identify Amazon parrots with low tear production. Corneal anesthesia did not signicantly effect PRTT values (p = 0.97) but did signicantly effect the STT values (p < 0.000). Based on our ndings the PRTT test is a feasible option to measure tear production in Amazon parrots and most likely other avian species. We also determined that the 5mm commercial STT test strips could be used to measure tear production in Amazon parrots with results almost identical to previous studies using modied STT test strips.

REFERENCES
1. 2. 3. 4. BROWN MH, GALLAND JC, DAVIDSON HJ, BRIGHTMAN AH. The phenol red thread tear test in dogs. Vet Comp Ophth 1996; 6: 274 7. BROWN MH, GALLAND JC, DAVIDSON HJ, BRIGHTMAN AH. The phenol red thread tear test in healthy cats. Vet Comp Ophth 1997; 7: 249 52. KORBEL R, LEITENSTORFER D. Clinical estimation of lacrimal function in various bird species using a modied Schirmer tear test. Israel J Vet Med 1996; 51: 171 5. WILLIAMS D. Avian ophthalmology. In: RITCHIE B, HARRISON GJ, HARRISON LR (eds): Avian Medicine: Principles and Application. Lake Worth, Florida: Wingers 1994; 673 94.

AUTHORS ADDRESS Eric Storey, DVM LSU School of Veterinary Medicine Dept. VCS Skip Bertman Dr. Baton Rouge, 70803 LA / United States e-mail: storeye@vetmed.lsu.edu

22

Falcon Specialist Hospital and Research Institute, Fahad bin Sultan Falcon Center, Riyadh, Kingdom of Saudi Arabia

HAEMOPATHOLOGICAL RESPONSES IN FALCONS TO NEWCASLE DISEASE

J. Samour MVZ, PhD

ABSTRACT
Newcastle disease (ND) is an infectious and highly contagious disease which commonly affects falcons in Saudi Arabia and the Middle East as a whole. ND is produced by Paramyxovirus 1 (PMV 1). The disease is thought to be highly prevalent subsequent to the extensive feeding of pigeons throughout the region. Haematology analysis was carried out in 34 falcons suspected of undergoing Newcastle disease based on the anamnesis, clinical signs, gastroscopy and radiology examinations. All falcons have been fed on freshly killed or live pigeons in the past 7 to 14days. Clinical symptoms varied accordingly to the clinical presentation. The clinical signs of falcons affected with the neurotrophic form varied in type and severity but could include initially, reduced to total absence of appetite, shredding and icking of food, regurgitation and vomiting, metallic-green coloured urates leading to dysphagia and salivation due to tongue paresia, paresia or paralysis of the nictitant membrane, amaurosis, progressive paralysis of the legs, hyperesthesia, clonic spasm, ataxia, head tremors and tics or sudden death. Clinical signs associated with the viscerotrophic form vary also in type and severity but commonly include initially reduce to total absence of appetite, shredding and icking of food, regurgitation and vomiting, metallic-green coloured urates, leading to severe depression, mucoid diarrhoea, constant distress vocalisation, the vomiting of partially digested blood or sudden death. All 34 falcons died 3 to 8 days after the clinical diagnosis was made. Post-mortem examination of cases affected with the neurotrophic form revealed only a slightly enlarged liver. No other gross pathological nding could be made. Falcons affected with the viscerotrophic form showed a slightly enlarged liver and a grossly distended ventriculus. The walls of the ventriculus were thickened and petechial haemorrhages were observed in most cases in the isthmus and in the ventriculus. The pathological appearance of the ventriculus of affected individuals could be observed during gastroscopy and at radiology in 65% of the cases. A selection of tissues including brain, heart, lung, liver, kidney and spleen were submitted for virus isolation. Newcastle disease virus was isolated in all 34 falcons. Isolates were sent to the World Reference Laboratory in Weybridge, UK, for further studies. Laboratory results from 30 falcons revealed moderate leucocytosis (12.43 1.39 x 109/l) (normal 3.8-11.5 x 109/l) with absolute

23

heterophilia (84.33 1.1 %, normal 60-68%). In most cases the heterophils showed signs of toxicity (grades 3 and 4) including basophilic cytoplasm, loss of granulation and loss of lobulation. Four falcons showed moderate to severe leucopenia (1.8 0.45 x 109/l) with moderate heteropenia (50 4.05 %) and severe monocytosis (31.33 8.3 %, normal 0-4 %). The haematology ndings highlighted the importance of expressing the results of the differential white cell count in both absolute and percentage values. In all cases examined the absolute heterophil count was within established normal ranges. However, absolute heterophilia was only detected when the percentage of heterophils in the blood lms was taken into consideration. The toxic changes observed in the heterophils were considered typical of the acute phase of the ND infection.

REFERENCES
1. 2. 3. 4. 5. ALEXANDER DJ. Newcastle disease. State Vet J 1995; 5(3): 21 24. RITCHIE BW. Avian Viruses, Function and Control, Lake Worth, Fl: Wingers Publishing Inc 1995; 253 - 311. KALETA EF and BALDAUF C. Newcastle disease in free-living and pet birds. In: ALEXANDER DJ (ed): Newcastle Disease. Norwell: Kluwer Academic Publishers 1988; 197 - 246. WERNERY U. Newcastle disease. In: Samour J (ed) Avian Medicine. London: Mosby 2000; 264 - 266. CAMPBELL TW. Avian Hematology and Cytology, 2nd edition, Ames: Iowa State University Press 1998; 3 - 19.

AUTHOR ADDRESS
Jaime Samour, MVZ, PhD Falcon Specialist Hospital and Research Institute Fahad bin Sultan Falcon center, P.O. Box 55, Riyadh 11322, Kingdom of Saudi Arabia e-mail: falcon@shabakah.com

24

Clinic for Birds and Reptiles University Leipzig, Germany

FOOD - INTOXICATION IN NESTLING PSITTACINES

M.-E. Krautwald-Junghanns, Prof Dr Med vet; G. Stelzer; V. Schmidt

ABSTRACT
Food intoxication is often suspected in a case of abnormal mortality in bird ocks. However, it is difcult to prove this suspicion without a thorough veterinary investigation at quite an early stage. In the following noteworthy case, a psittacine breeder took legal proceedings against a food company. After having used their psittacine diet 26 nestling birds of different species died within 5 months. After changing the food no further mortality was noticed. Consequently there was a strong suspicion of food related intoxication. A time consuming extensive and costly examination followed, in which different specialized veterinary institutions as well as 5 different veterinarians were involved. The investigations contained a gross necropsy of all dead psittacine nestlings. In addition histological, cytological, parasitological, microbiological and virological examinations were performed. The food as well as the birds livers and kidneys were examined for their vitamin and mineral-content. Additionally the food was examined for mycotoxins and for the composition of amino-acids. Further to this food trials with budgerigars were undertaken, which resulted in the death within a few days of the positive group (fed with the food under trial). It was noted that the food (compared to the recommendations by the National Research Council and the AAVs Expert group) contained an increased Vit A, Vit D3 and Vit E - content, which corresponded partially to an increased content of these vitamins in the liver /kidneys of the dead birds. The pathological ndings were different in the various birds without any apparent common theme. A variety of changes were recorded including (visceral?) gout, crop stasis and liver alterations. However, these ndings could not be directly connected to hypervitaminosis A/ D3 or E. Some birds which showed a 5 fold increased content of Vitamin A in the liver (levels about 7200 I.E./ g liver) for example did not exhibit any typical pathological ndings. On the other hand some birds fed with the same food showed a decreased Vit. A level in the liver.

25

In relation to mineral content, copper, zinc, - as well as selenium - levels were elevated when compared to the published recommendations. This corresponded again to partially increased contents of the minerals mentioned in liver and kidneys of the nestling birds. The relationship between these ndings in the food, the content of the different vitamins and minerals in the organs and their interaction between each other as well as the pathological ndings are discussed taking into account of the different species and age of the birds. The decision of the court will also be presented and discussed.

AUTHORS ADRESS
Prof. Dr. M.-E. Krautwald-Junghanns Clinic for Birds and Reptiles Department of Small Animals University Leipzig An den Tierkliniken 17 04103 Leipzig Germany e-mail: krautwald@vmf.uni-leipzig.de

26

Division of Avian and Exotic Animal Medicine, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, The Netherlands

SPIRAL COMPUTED TOMOGRAPHY IN RESPIRATORY DISEASED BIRDS

I.Westerhof DVM, PhD; N.J. Schoemaker, DVM, PhD; P.Y. Barthez, DVM, PhD

ABSTRACT
Respiratory problems occur frequently in birds. Diagnosis of the exact cause of the respiratory distress is difcult. Conventional radiography is usually performed in order to identify abnormalities in the respiratory tract. However, using radiography, small lesions may be missed or misinterpreted. In the literature computed tomography has been described as a non-invasive diagnostic tool (KRAUTWALD-JUNGHANNS et al, 1998[1]; KRAUTWALD-JUNGHANNS et al, 1998 [2]; KRAUTWALD-JUNGHANNS et al, 1998[3]; ROMAGNANO and KRAUTWALD-JUNGHANNS, 1997). In these studies sagital cross sections were used as the standard view. This study describes spiral CT in 20 birds with respiratory problems. Spiral CT, i.e., helical or in volume acquisition, CT is an imaging technique which has, thus far not been described in birds. Spiral CT enables scanning of a complete organ during the time of maximal enhancement without artefacts caused by respiratory movements (SOUCEK et al, 1990). The number of scan slides used in the birds were at least 250, with a total scanning time of less than 1 minute per bird. In this study birds were scanned during inhalation anaesthesia with isourane to facilitate precise positioning of the birds. Birds were xed in sternal recumbency. Transverse total body cross sections of the whole bird from cranial to caudal, starting at the beak and ending at the tail, were included in the standard scanning protocol. CT scans of respiratory diseased birds are compared with those of healthy birds. CT ndings are described and compared with conventional radiographic ndings. The CT images provided superior information of the respiratory tract compared to the radiographs. The individual results will be discussed during the presentation. In 10 birds the ndings are also compared with post mortem ndings. It is concluded that spiral CT provides detailed information about alterations of the respiratory tract in birds. Spiral CT requires less examination time under anaesthesia than conventional radiography or conventional computed tomography. On the other hand, spiral CT generates many detailed CT images that requires more interpretation time and experience from the interpreter of the CT images.

27

REFERENCES
1. KRAUTWALD-JUNGHANNS ME, SCHUMACHER F, SOHN HG. Examination of the lower respiratory tract of Psittacines and Amazoniae varieties by means of reconstructed computer x ray tomography. 1: Examination of healthy parrots. Tierarztl Prax Ausg K Kleintiere Heimtiere 1998; 26(1): 61 70. KRAUTWALD-JUNGHANNS ME, SCHUMACHER F, SOHN HG. Examination of the lower respiratory tract of Psittacines and Amazoniae varieties by means of reconstructive transmission computed tomography. 2: Examination of parrots with respiratory symptoms. Tierarztl Prax Ausg K Kleintiere Heimtiere. 1998; 26(2): 139 49. KRAUTWALD-JUNGHANNS ME, VALERIUS KP, DUNCKER HR, SOHN HG. CT-assisted versus silicone rubber cast morphometry of the lower respiratory tract in healthy amazons (genus Amazona) and grey parrots (genus Psittacus). Res Vet Sci. 1998; 65(1): 17 22. ROMAGNANO A, KRAUTWALD-JUNGHANNS ME. Respiratory Radiology and Imaging. Proc Avian Spec Adv Prog & Small Mamm and Reptile Med and Surg. 1997; 17 21. SOUCEK M, VOCK P, DAEPP M, KALENDER W. Spiral CT: a new volume scanning technique. II. Clinical applications. Rntgenpraxis 1990; 43: 365 75.

2.

3.

4. 5.

AUTHORS ADRESS
I. Westerhof, DVM, PhD Division of Avian and Exotic Animal Medicine Department of Clinical Sciences of Companion Animals University of Utrecht Yalelaan 8 3584 CM Utrecht / The Netherlands e-mail: I. Westerhof@vet.uu.nl

28

Dubai Falcon Hospital, PO Box 23919, Dubai, United Arab Emirates

DOMESTICATION AND DISEASE : CHALLENGES FOR HOUBARA BUSTARD CAPTIVE BREEDING PROJECTS IN THE MIDDLE EAST
T.A. Bailey, MRCVS, PhD; O. Combreau

ABSTRACT
In recent years, there has been a surge of interest in the propagation of bustards in captivity. By managing bustards under intensive captive breeding conditions man has started the process of domestication, exposing the birds to new environments and diseases. As a consequence captive bustards are vulnerable to a wide range of disorders. Houbara captive breeding programmes involve managed movements of animals between captive and free-living populations. Conservationists are concerned that novel diseases or genetic conditions may be transferred between captive and wild populations by these movements, endangering captive-breeding populations, released animals and free-living populations. The larger projects have started to address these issues by initiating veterinary programmes that include; 1) investigating the health of wild populations, 2) considering health when founder stock are selected, 3) managing the health of stock during the breeding programme and 4) health screening during the release stages. This presentation reviews health surveys conducted on free-living, rehabilitated and captive houbara in the region. In the context of this information the health challenges posed by domestication are assessed and veterinary recommendations for what may become a new regional industry, houbara bustard farming, are presented. Bustards maintained in captivity in the Middle East are derived from four sources: 1) 2) 3) 4) Wild caught birds smuggled directly into collections in the region. Conscated smuggled wild-caught birds that have come through an ofcial rehabilitation programme and are placed in captivity. Ofcially sanctioned projects collecting eggs from wild populations. Exchanges of breeding stock between the larger captive breeding projects.

Health issues vary according to the source of the bird. Consequently, we conducted health surveys on wild and rehabilitated bustards including: Free-living bustards - samples collected from three populations (China, Pakistan and the UAE) of live and dead (hunted) free-living bustards were examined from 1993 to 2001. Rehabilitated bustards - the health status of conscated bustards in a survey between 1992 and 1999.

29

Experience has shown that the productivity of rehabilitated birds is poor and an ever present risk of disease, in spite of expensive quarantine and health screening, exists. Viral diseases (e.g. PMV-1, avipox, reovirus, adenovirus, avian leucosis) represent the clearest health threat from rehabilitated houbara incorporated into breeding programmes, because of inability to treat infections, the lack of knowledge concerning recovery from infection and the potential for recovered, but latently infected birds, to shed virus and to infect other birds by either vertical or horizontal transmission. Consequently, the integration of any birds that have passed through the illegal trade into large breeding projects cannot be recommended. The best sources of healthy breeding stock for captive projects are from sources 3 and 4. The founder breeding stock of the major breeding projects were all derived from eggs collected from the wild and were hatched in captivity and hand-reared. It is getting harder to obtain birds from source 3, because wild populations are declining and obtaining permission to conduct responsible egg collection in range states (e.g. central Asia) is both logistically complicated and can be controversial. Projects therefore need to initiate genetic management to manage their founders and to investigate new ways of collecting new genetic material. Health surveys show that wild houbara bustards are not naturally exposed to the viral diseases that are common in captivity. This may explain the apparently high susceptibility of smuggled birds to infectious diseases. Appropriate protocols will need to be developed to screen captive bred birds before they are released back into the wild. Projects will also need to develop health screening standards for birds that are exchanged between projects. The health issues surrounding the production, utilisation and ultimately domestication of houbara bustards in the Middle East are complex and challenging. The research requirements of the young houbara bustard industry are large, considering the low level of productivity to high level of investment that has taken place. Ultimately, the larger breeding centres will need to improve co-operation and develop common research goals, so that research funding is prioritised and existing and new problems are solved.

AUTHORS ADRESS
T. Bailey, BSc, BVSc, MRCVS, Cert Zoo Med, MSc, PhD Dubai Falcon Hospital PO Box 23919 Dubai / United Arab Emirates e-mail: tom.bailey@dfh.ae

30

Emerging Diseases Research Group, University of Georgia, College of Veterinary Medicine, Athens, USA

EPIZOOTIOLOGY OF PROVENTRICULAR DILATATION DISEASE IN BREEDING COCKATIELS

B. W. Ritchie, DVM, PhD; C. R. Gregory, DVM, PhD; K. S. Latimer, DVM, PhD; D. Pesti, MS; M. Ard, BS

ABSTRACT
Proventricular dilatation disease (PDD) continues to cause morbidity and mortality among companion and aviary birds. Field observations have been used to suggest that the etiologic agent of PDD can be spread through horizontally and vertically. To evaluate issues related to the natural transmission of the PDD agent, breeding pairs of cockatiels in which at least one individual of each pair was histologically positive for PDD were placed in enclosures to facilitate breeding and both the adults and their offspring were followed to determine the rate of transmission in the population.

INTRODUCTION
Proventricular dilatation disease (PDD) is used to describe an inammatory response characterized by the accumulation of lymphocytes and plasma cells in the central and peripheral nervous systems, especially the nerves that supply the muscles in the proventriculus and other digestive organs including crop, ventriculus and small intestine. Central nervous system signs associated with PDD, which may occur in addition to or independent of gastrointestinal signs, may include ataxia, abnormal head movements, seizures and proprioceptive or motor decits (GREGORY, 1995). The most common clinical signs of PDD include depression, weight loss (with or without decreased appetite), constant or intermittent regurgitation, and/or passage of undigested food in the feces indicating a malabsorptive or maldigestive disorder (GREGORY, 1995). This disease was rst discussed in the late 1970s in birds imported into the United States and Germany (GREGORY, 1995; GERLACH, 1991). Subsequently, a PDD epornitic has been occurring in psittacine birds in North America and Europe, probably as a result of the widespread importation and shipment of birds to satisfy the demands of the pet trade. There is no reference to spontaneous disease in free-ranging psittacine birds; however, they should be considered psittacine or nonpsittacine birds susceptible. Given the severe nature of PDD and its potential to affect a wide range of bird species, the importation of psittacine birds or their eggs into any region with indigenous Psittaciformes must be considered extremely risky.

31

ETIOLOGY of PDD
Since its initial description in the late 1970s, multiple etiologies have been proposed for PDD; however, none have been proven to be the etiologic agent (GREGORY, 1995; GERLACH, 1991; MANNL et al, 1987; GASKIN et al, 1991; GREGORY et al, 1997; GRUND et al, 1999). Adenovirus-like particles were demonstrated within intranuclear inclusion bodies in one affected bird. Paramyxovirus-like viral particles were demonstrated within inclusion bodies located in the neural cells of the spinal cord and in visceral nerve ganglia of another bird. Similar inclusion bodies have been described in the nerves of pigeons with paramyxovirus infections (GERLACH, 1991). Birds with PDD have been shown to lack detectable levels of antibodies to paramyxovirus (serotypes 1, 2, 3, 4, 6 and 7), Pachecos disease virus (an avian herpesvirus), avian polyomavirus and avian encephalitis virus (GREGORY, 1995). An eastern equine encephalomyelitis (EEE) virus was recovered from neonates with abdominal distention from an aviary with a history of PDD. The disease in these neonates was termed avian viral serositis. This nding was used to suggest that PDD may be caused by EEE virus (GASKIN et al, 1991), even though EEE virus occurs primarily in the eastern portion of the United States, and PDD has been shown to occur throughout the United States, Canada and Europe. Experimental and epizootiologic ndings suggests that EEE virus is not the cause of PDD (GREGORY et al, 1997). A paramyxovirus related to Hitchner B1 was recovered from birds with PDD. Antibodies to this virus could be detected using a virus specic ELISA, however, antibodies were not detected using standard hemagglutination-inhibition assays available for paramyxoviruses (GRUND et al, 1999; GRUND et al, 2002). Experimentally infected African grey parrots either died soon after inoculation or seroconverted and shed virus with morphologic characteristics suggestive of paramyxovirus in their excrement (GRUND et al, 1999). Using electron microscopy, viruses with morphologic characteristics suggestive of paramyxovirus, enterovirus, coronavirus and reovirus have been detected in tissues, secretions or excretions from birds that have been either histologically positive for PDD or with gross distention of the proventriculus. None of these viruses have been consistently demonstrated in all birds with conrmed PDD.

EXPERIMENTAL and NATURAL INDUCTION of PDD

Exposing susceptible psittacine birds to a tissue homogenate derived from affected birds can experimentally induce the lymphoplasmacytic ganglioneuritis that characterizes PDD (Gregory, et al., unpublished data, 2004). Clinical changes in experimentally infected birds vary from acute onset of a combination of central nervous system and gastrointestinal signs, followed by death within 11 days of inoculation, to induction of only gastrointestinal signs that were rst noted 3 months after inoculation. The absence of disease in contact controls suggests that the etiologic agent of PDD is not readily transmissible or requires a specic route of inoculation that was not favored by the experimental conditions. Alternatively, it could be speculated that the unaffected contact controls were already immune to infection.

32

However, all birds, both those that received the virus containing inoculum and the birds that received the control inoculum were derived from the same initial group of research birds. All of the experimentally infected birds were susceptible and none of the contact control birds developed disease. These ndings suggest that a lack of successful transfer of the virus from an infected to an uninfected bird was more likely than some specic immunity in all of the contact control birds. To evaluate the epizootiologic characteristics associated with naturally and experimentally induced disease, breeding pairs of cockatiels in which at least one individual of each pair was histologically positive for PDD were placed in a closed indoor room and provided nesting boxes. Most offspring were allowed to mature without interference while some were experimentally inoculated with tissue homogenates known to induce PDD. Adults and their offspring were monitored for clinical changes suggestive of PDD and birds that developed morbidity were euthanized and tissues were histologically evaluated. Results of this study conrm that some birds can be in direct contact with PDD positive birds for prolonged periods without developing disease, diseased adults can produce clinically normal offspring, chicks produced by positive parents are susceptible to disease, any immunity passed from a hen to her chicks is only transient, the severity of histologic lesions do not correlate with chronicity or death and the period from exposure to the suspect PDD agent to development of overt clinical signs can be more than a year.

ACKNOWLEDGMENTS
In memory of Dr. W.L. Buddy Steffens, a gifted and cherished scientist, colleague and friend. Major sustained contributions that have made this work possible have been provided by the UGARF Animal Health Fund, Veterinary Medical Experiment Station, Zoo Atlanta, Riverbanks Zoological Park, the Stop PDD Challenge and the Kenosha Exotic Bird Club. Hundreds of aviculturists, bird clubs and veterinarians have also made important contributions.

REFERENCES
1. GREGORY CR (1995). Proventricular dilatation disease. In: Ritchie BW (ed): Avian Viruses: Function and Control. Lake Worth, FL, Wingers Publishing; 1995:439-448. 2. GERLACH S. Macaw wasting disease - a 4 year study on clinical case history, epizootiology, analysis of species, diagnosis, microbiological and virological results. Proc Europ Chap Assoc Avian Vet 1991; 273 81. 3. MANNL A, GERLACH H, LEIPOLD R. Neuropathic gastric dilatation in Psittaciformes. Avian Dis 1987; 31: 214 21. 4. GASKIN JM, HOMER B, ESKELUND K. Preliminary ndings in avian viral serositis. A newly recognized syndrome of psittacine birds. J Assoc Avian Vet 1991; 5: 27 34. 5. GREGORY CR, LATIMER KS, NIAGRO FD, et al. Investigations of eastern equine encephalomyelitis virus as the causative agent of psittacine proventricular dilatation syndrome. J Avian Med Sur 1997; 11: 187 93.

33

6. GRUND C, GRIMM F, KOSTERS J, et al. Serological studies on persistent PMV-1 infection associated with PDD. Proc Assoc Avian Vet 1999; 19 23. 7. GRUND CH, WERNER O, GELDERBLOM HR, et al. Avian paramyxovirus serotype 1 isolates from the spinal cord of parrots display a very low virulence. J Vet Med B Infect Dis Vet Public Health 2002; 49: 445 51.

AUTHORS ADRESS
B. W. Ritchie, DVM, PhD Emerging Diseases Research Group College of Veterinary Medicine University of Georgia Athens, GA 30602 USA e-mail: britchie@vet.uga.edu

34

Main European Association of Avian Veterinarians Conference TEXTS

Strathmore Veterinary Clinic, Andover, Hants, United Kingdom

VETERINARY ASPECTS OF THE GREAT BUSTARD (Otis tarda) REINTRODUCTION PROJECT IN THE UK
J.R. Chitty BVetMed, CertZooMed, MRCVS, P. Osborne BSc DPhil, A. Fraser BSc, D.Waters, T. Kapranova, A. Khrustov

KEYWORDS
Great bustard - Otis tarda - Disease surveillance Reintroduction - Mortality

ABSTRACT
Great bustard eggs were taken from disturbed nests in Russia. These were hatched in captivity and chicks transported to the UK for reintroduction to the wild. This paper will discuss the disease screening protocol devised for this project and the results gained. It will also discuss the disease syndromes seen and health problems encountered from incubation to release. A set of haematological normal values for captive juvenile great bustards will be proposed.

1 INTRODUCTION
The great bustard (Otis tarda) is a globally threatened species needing conservation action across Europe. They became extinct as a breeding species in the United Kingdom (UK) in 1832 probably as a result of hunting, agricultural change and inclement weather. The factors that caused the loss are no longer thought to operate. Suitable habitat exists in pockets across England and especially on the Salisbury Plain where a large area is protected for military training and conservation purposes. The Plain combines short grass areas for lekking, long grassland for feeding and adjacent arable land for nesting. Pilot studies on arthropods in long grassland suggest that their density is sufcient for chick-rearing but the precautionary creation of additional food-rich areas among arable crops is recommended. Genetic studies indicate that Britains bustards probably belonged to the central European group and that restocking should not use birds from Iberia. Only Russia has sufcient birds to supply a reintroduction project and losses there through nest devastation are high. By rescuing eggs, articially incubating them and transporting chicks to Britain, the project should have zero detriment to the donor population (OSBORNE 2005).

37

2 MATERIALS AND METHODS

Eggs of the great bustard have been taken from disturbed nests in Russia and incubated since 1982 (PONOMAREVA 1983). In previous years, chicks have been allowed to imprint on the human rearers, but success at reintroducing these chicks to the wild can be assumed to be poor (OSBORNE 2005). For the purposes of the reintroduction project this process has been altered to prevent this imprinting. Methods used are similar to those used in the rearing of the whooping crane (Grus americana). Thirty chicks of 33-50 days of age were shipped from Russia to the UK prior to spending 28 days in licensed quarantine. They were then moved to soft pens prior to release onto the Salisbury Plain. For the release site a large open pen was constructed. This was planted with game cover crops to provide a basic plant diet and to attract invertebrates for the bustards. Similar crops were planted around the pen and at other sites near the release site. This was to allow certain guaranteed feeding sites and to regulate the dispersal of the birds. Naturally there were concerns over entry of disease into wild and domesticated birds in the UK. In addition to compulsory testing for inuenza and paramyxoviruses during quarantine, testing for other pathogens was also carried out.

3 RESULTS 3.1 In Russia

a. b. c. Egg mortality. Most showed early embryonic death, probably coinciding with moving. There were two deaths due to malposition. Chick problems. Most common was failure to retract the yolk sac followed by infection. Angel wing was also seen frequently but was easily corrected using wing bandages. Delays in obtaining export visas in Russia meant that many of the birds were too old to travel. Therefore the pool of birds from which the reintroduction birds could be chosen was much reduced. This may explain the deaths of two birds in transit to Moscow, the death of one bird immediately post-transit and three others within three days of arrival. Post-mortem examination of those birds that died in Russia was not possible. Of those that died in the UK, three had a bacterial hepatitis (one possibly linked to previous yolk sac infection) and one had a ventricular foreign body and rupture.

3.2 In UK: quarantine (1-28 days post-arrival) 1. Faecal tests; performed at 2 and 10 days post-arrival
a. Parasitology. This did not reveal any parasites on either test. However, one dead bird did have agellates and trematode eggs in the gut. All birds were dewormed using fenbendazole and praziquantel.

38

b. c. d. e.

Bacteriology. No pathogens were detected. Acid Fasts. None detected. Chlamydophila PCR. Four birds were positive for this organism. Issues surrounding this include source of infection and zoonotic aspects. Biosecurity in the rearing unit will be improved. Virology. Swabs were taken from trachea and cloaca on arrival for storage and future testing. No inuenza or paramyxoviruses were isolated.

2. Blood Tests; performed at 2 and 10 days post-arrival

a. b. c. d. e. Serology. Paramyxovirus, inuenza and avipox virus serology was negative. Serum storage. For future virological investigation. Blood Parasites. None detected. Cell storage for future DNA analysis and/or sexing. Haematology. No obvious problems detected and ranges similar to other birds. A suggested normal reference range was obtained for birds aged 43-60 days.

3. Deaths. Four birds died in quarantine (see above)

1. 2. Ill on arrival at Heathrow airport and died that night. Hepatitis; probably bacterial. Three birds died the night after the rst handling session 2 days postarrival. a. Pre-existing gizzard foreign body, also, agellates and trematodes. b. Hepatitis. Probably bacterial. c. Hepatitis. Probably bacterial and probably secondary to yolk sac infection.

3.1 Soft pens (29-43 days post-arrival). Two birds were injured:
Fractured humerus. Male bird. Wing amputated. Dislocated elbow. Male bird. Retained. Problems were associated with the age and size of birds following delays prior to export from Russia. Therefore the period spent in soft pen was shortened so birds could be released earlier. The risk of injury meant that predator awareness trials could not be performed.

3.2 Post-release.
At the time of writing seven of the twenty-two released birds still survive in the wild. Two injured birds (humeral fractures) have had wings amputated and are maintained in captivity. Of the birds that died, causes of death were attributed to trauma (10 birds) and predation (5 birds). All birds that died

39

had fed and were in good body condition at time of death. Post-mortem examinations showed no sign of infectious disease. Removal of fences and marking of those that cannot be removed will now be carried out in the vicinity of the release pen.

4 DISCUSSION
To date, disease and mortality levels have been within acceptable levels for this type of project. The screening procedures appear to have indicated that birds being reintroduced are unlikely to constitute a serious health risk for indigenous UK birds. Over the next 5-10 years further groups of birds will be re-introduced from Russia. The disease screening programme will be continued for these with some modications.

5 ACKNOWLEDGEMENTS
The authors would like to thank Tom Bailey MRCVS, Dr Ruth Manvell and Chris Davis MRCVS for their help in designing the disease screening protocols; Chris Davis MRCVS and Kate Chitty MRCVS for assistance in treating and sampling birds; Saul Cowen, Karen Waters, Tanya Osborne and other members of the Great Bustard Group for the hard work in record keeping, observing, catching, handling and maintaining the birds in captivity; Dr D Welchman and colleagues at VLA Winchester for performing autopsies on dead birds in quarantine; Medlab Laboratories for clinical pathology work.

6 CITATION INDEX
1. 2. OSBORNE PE. Key issues in assessing the feasibility of reintroducing the Great Bustard (Otis tarda) to Britain. Oryx 2005; 39(1): 1 - 8 PONOMAREVA TS. Die Restitution naturlicher Populationen der Grosstrappe (Otis tarda) in der USSR. 4. Symposium uber die Grosstrappe (Otis tarda). 1983, Eberswalde, Germany.

AUTHORS ADDRESS
J. R. Chitty BVetMed, CertZooMed, MRCVS Strathmore Veterinary Clinic, London Road, Andover, Hants, SP10 2PH, UK Email: xmo32@dial.pipex.com

40

Johannesburg Zoo South Africa

DISEASES AND MANAGEMENT OF CAPTIVE CRESTED SCREAMERS (CHAUNA TORQUATA).

M. Barrows BSc BVMS CertZooMed MRCVS, M. Hartley BVetMed MapplSc (Wildlife Health) MRCVS, J. M. Pittman CVT.

KEYWORDS
Birds - Crested screamer - Chauna torquata - Diseases - Botulism - Avian gastric yeast - Proventricular impaction

ABSTRACT
This paper discusses the management and medical conditions of crested screamers, Chauna torquata in captivity. These large South American birds are members of the family Anhimidae in the order Anseriformes and have several unusual anatomical and physiological characteristics. Medical conditions discussed include botulism caused by Clostridium botulinum Type C toxin, avian gastric yeast Macrorhabdus ornithogaster and proventricular impaction.

1 INTRODUCTION
Crested screamers are one of three species of screamer in the order Anseriformes and family Anhimidae. The horned screamer Anhima cornuta is the sole member of the genus Anhima, while the near threatened black-necked screamer Chauna chavaria and the relatively abundant crested screamer belong to the genus Chauna. Crested screamers are widespread in South America occurring from Bolivia, Uruguay and Brazil south to Argentina and inhabit a variety of wetlands including swamps, marshes, rivers and lagoons. They are primarily herbivorous, ingesting grasses, seeds and the leaves, owers and roots of aquatic plants, although they will take some invertebrate prey, especially when chick rearing (AZA WATERFOWL TAG 2004). Screamers are large birds weighing from 3 to 5 kg when adult and have several interesting anatomical and physiological characteristics. They have no uncinate process, a lack of feather tracts or apterae and only a few developed lamellae on the

41

inner surface of the bill. They have a well developed system of air sacs beneath the skin which can feel on palpation like subcutaneous emphysema or oedema and can make jugular venepuncture difcult. They are also said to have the most pneumatic bones of any bird species (AZA WATERFOWL TAG 2004) and can soar on thermals at great heights. They can also swim but are mainly terrestrial. They have large toes with vestigial webbing at the basal portion and both males and females have sharp carpal spurs, used as a defence mechanism (PERRINS 1990). The proventriculus is large and sacculiform and the liver is predominantly on the right hand side so that on radiographs a symmetrical liver shadow is not seen. The moult is gradual unlike most other anseriformes. Screamers are named for their loud honking vocalisations and can live for up to 35 years in captivity. Clutch size varies from 2-7 (DEL HOYO et al. 1992) and both parents incubate the eggs for 42 to 45 days. The chicks are precocial.

2 DISEASES OF CAPTIVE SCREAMERS 2.1 Non-infectious diseases 2.1.1. Gastrointestinal impaction

Gastrointestinal (proventricular) impaction is the most common and signicant disease of captive screamers and is a major cause of chick mortality. Chicks are generally affected within the rst two months, however cases have been seen in juveniles almost a year old. Some collections regularly lose 50-75% of chicks to impaction and another author gives a general gure of 48% overall chick mortality within the rst 30 days (AZA WATERFOWL TAG 2004). Diet and husbandry, in particular substrate type are important aetiological factors and the presence of avian gastric yeast Macrorhabdus ornithogaster in several of these cases may also be signicant. Impaction has been seen with inappropriate dietary sources of bre such as alfalfa hay and with substrates such as sand and grit. Clinical signs include weight loss, weakness progressing to recumbency, dehydration and dyspnoea. Diagnosis is straightforward from standard lateral and ventrodorsal radiographs which typically show a massively distended and impacted proventriculus. The intestines may also be impacted or may show gaseous distension. Treatment is often unrewarding especially in young chicks. However a combination of intravenous and oral uids, antibiosis, cisapride and gavage with polyethylene glycol with electrolytes (Movicol, Norgine Ltd) and a maltodextrin and protein concentrate solution (Critical Care Formula, Vetark UK) has proved successful. Preventative measures should include paying attention to the formulation of chick diets, using plenty of water to hydrate pellets, and keeping screamers on a natural grass substrate rather than sand or loose soil. Feed dishes should be placed on solid mats so that when food spills over the side the chicks are not ingesting substrate. Plenty of exercise is also important for chicks.

42

2.1.2. Botulism
Like other waterfowl, screamers are very susceptible to botulism caused by Clostridium botulinum Type C toxin, although interestingly in an outbreak at the authors zoo where the toxin was waterborne through a series of adjacent aviaries with an interconnected water source, none of four one month old chicks were affected even though both parents succumbed to the disease. Clinical signs included typical accid paralysis of the legs, wings, neck and head. A clear ocular discharge and a thick mucoid oral discharge were also seen. The six year old female presented in good body condition but was weak and unable to stand with a thick mucoid oral discharge. She could move her legs slightly but at times was unable to lift her head. The adult male also displayed general paresis, a bilateral clear ocular discharge and a thick mucoid oral discharge. He was however, able to walk. Treatment was mainly supportive and involved initial gavage with 20ml/Kg warm electrolyte solution (Darrows solution, Kyron Laboratories Pty Ltd, SA) containing a probiotic (Avipro, Vetark, UK) and activated charcoal; lactated Ringers solution (20ml/kg SC to the male and 5ml/Kg IO per hour to the female), ivermectin at 0.2mg/ kg SC and enrooxacin at 15mg/kg IM q12h. They were also given a single dose of 0.5ml C. botulinum antitoxin (Bio Onderstepoort) intravenously. They were gavaged with ground up waterfowl pellets three times daily. The male screamer was treated in situ in his aviary so that he could continue to feed the chicks that the pair was rearing. On day two his oral discharge had decreased but he remained dull and inactive, however by day three he was much improved and the treatment was discontinued. He went on to successfully rear two of the four chicks. The female Screamer was holding her head up on day two but was still unable to stand. She was much the same by day three, unable to eat and still producing a thick oral discharge, although she was drinking. She was kept in a sling and physiotherapy carried out on her legs. She started eating a little on day four and was moving her legs more but did not show any further signs of recovery. On day seven she started regurgitating food after tube feeding and she died on day eight. On post mortem examination, there were no signicant gross lesions visible, other than a haemorrhagic appearance to the caecum. Histopathology revealed mild to severe congestion of several organs, including the caecum, suggestive of an acute shock reaction. Intestinal contents tested strongly positive with the mouse toxicity test for C. botulinum type C. Since this outbreak our screamers have been vaccinated annually with a single dose of 1ml C. botulinum vaccine (Bio Onderstepoort) given subcutaneously.

2.1.3. Trauma
Trauma has been reported as the most common problem of adult screamers (AZA WATERFOWL TAG 2004) and may result from intra or inter-specic aggression. Introduction of non-related adults should be done gradually in neutral territory

43

utilising a divider fence. Crested screamers are monogamous and territorial during the breeding season (SILVEIRA and FOWLER 2001) but are fairly gregarious during the rest of the year and have been kept successfully in family groups for up to a year and in single sex groups for longer periods.

2.1.4. Digit trauma

Digit trauma is particularly common in screamers and is most likely related to inappropriate substrate. One juvenile screamer at the authors zoo required amputation of three out of four toes on each foot after damaging the digits at the level of P3/P4. It was thought that this occurred after she had difculty getting out of the concrete pool in her enclosure. A young male screamer developed a 90 degree exion of his digits at P3/P4 at around 7 months of age and consequently developed ulcers on the dorsal surfaces and a bird hospitalised on rubberised ooring for treatment of gastrointestinal impaction developed plantar ulcers and worn nails on all digits. Again this can be prevented by keeping screamers on a natural grass substrate and utilising natural surfaces rather than concrete or abrasive materials for ponds.

2.1.5. Bumblefoot
Pododermatitis is common in screamers as in other waterfowl, particularly when forced to stand on concrete or abrasive surfaces and can follow on from the plantar trauma and ulcers mentioned above. The plantar surfaces of the feet are relatively thin and are easily damaged or bruised. Mixed bacterial infections are found, and treatment involves husbandry changes, antibiosis, dressings to take pressure off the affected area and surgery as in other species.

2.1.6. Capture myopathy

Capture myopathy has been reported in screamers after prolonged manual restraint.

2.1.7. Other non-infectious diseases

Other non-infectious diseases reported in screamers include renal gout and oxalate nephrosis.

2.2 Infectious diseases 2.2.1. Avian megabacteria gastric yeast (Macrorhabdus ornithogaster) or

Avian gastric yeast is a relatively common post mortem nding in screamer chicks and may occur concurrently with proventricular impaction. Clinical signs include weight loss and loose unformed faeces or diarrhoea. The mode of transmission is likely to be the faecal-oral route. Diagnosis may involve a Gram stain of faeces or of a proventricular wash, although false negative results are possible if affected birds

44

are not shedding the organism at the time of the test. In addition, proventricular dilatation may be seen on radiography or at post mortem examination, with or without proventricular impaction; however the proventriculus is large and may appear dilated even in healthy screamers. Histopathology typically reveals marked koilin dysgenesis in the ventriculus, sometimes with atrophy of the muscle layers and presence of the typical large rod-shaped yeasts in both the ventriculus and proventriculus. Treatment is likely to be palliative and as in other species (PHALEN and TOMASZEWSKI 2003; RAVELHOFER-ROTHENEDER et al. 2001), avian gastric yeast may not always be pathogenic, especially in adult screamers. In other species, factors such as age, nutritional status, immunologic status and the presence of concurrent disease may affect pathogenicity (LANGLOIS 2003). However an attempt should be made to treat it in screamer chicks especially where weight loss is present and because koilin dysgenesis may affect ventricular function and be a predisposing factor for proventricular impaction. A course of oral amphotericin at 10mg/kg bid for 4 weeks, along with an avian probiotic (Entero-Plus, Medpet Ltd, SA) resulted in resolution of diarrhoea and weight gain in a one year old juvenile screamer with a positive faecal Gram stain.

2.2.2. Suspected pox virus

Two siblings at the authors zoo have developed pox-like proliferative lesions at the commisures of the beak. These lesions were rst apparent at 3 months and 7 months respectively and have not regressed over the subsequent months. One of the birds has also developed raised crusty lesions on his feet and at one point had a focal 1mm whitish plaque adjacent to his glottis. He also had severely deformed feathers. In spite of the similarity of most of these lesions to pox virus, repeated biopsies from both birds have revealed only secondary bacterial (Staphylococcus aureus isolated) and food particle contamination. The foot lesions consisted of severe acanthosis and orthokeratotic crusts with multifocal areas of moderate heterophil inltration and degranulation in the dermis with associated overlying epidermal ulceration and serocrust formation. Electron microscopy also failed to reveal a viral aetiology. The commisure lesions in the female failed to respond to a week long course of systemic and topical antibiotics and debridement.

2.2.3. Other infectious diseases:

Other infectious diseases reported in screamers include Capillaria sp, intestinal coccidiosis, cryptosporidiosis, gastrointestinal candidiasis and fungal rhinitis.

3 DISCUSSION
Many of the diseases reported in screamers are husbandry related and can be prevented by good dietary management and by keeping them on a natural grass substrate. Collections which have had few problems with their screamers generally keep them on grass with access to a natural lake or pond where they can graze on aquatic vegetation. Captive diets are based around commercial waterfowl or gamebird

45

pellets with a variety of chopped greens. Chicks may require an increased percentage of protein (AZA WATERFOWL TAG 2004) and one collection routinely adds probiotics to the chick diet. Screamers are usually kept as pairs but can be successfully kept in groups, especially outside of the breeding season. They have been kept successfully in mixed exhibits with a variety of species including Leopard tortoises, camelids, tapirs, capybara and several bird species including other waterfowl, amingos and psittacines. Chicks are usually pinioned at around 3 days of age and a three foot barrier is suitable for containing pinioned birds.

4 CITATION INDEX
1. 2. 3. AZA WATERFOWL TAG Screamers In: Proc of Waterfowl Husbandry Workshop, Santa Barbara 2004. DEL HOYO J, ELLIOT A and SARGATAL J (eds): Handbook of the Birds of the World, Vol 1 Ostrich to Ducks. Barcelona, Spain: Lynx editions 1992; 696. LANGLOIS I. The anatomy, physiology and diseases of the avian proventriculus and ventriculus. In: HERNANDEZ-DIVERS and HERNANDEZ-DIVERS (eds): Veterinary Clinics of North America Exotic Animal Practice Internal Medicine 6 2003; 85 - 111. PERRINS C. The Illustrated Encyclopaedia of Birds. The denitive guide to birds of the world. International Council for Bird Preservation: Headline Book Publishing PLC 1990; 76. PHALEN D and TOMASZEWSKI E. Investigation into the identication, detection and treatment of the organism formally known as megabacterium. Proc European Assoc Avian Vet, Tenerife 2003: 79 83. RAVELHOFER-ROTHENEDER K, ENGELHARDT H, WOLF O et al. Megabacteria taxonomic classication and signicance as pathogen in various bird species. Proc Euro Assoc Avian Vet, Munich 2001: 189 - 190. SILVEIRA L and FOWLER M. Order Anseriformes (Ducks, Geese, Swans). In: FOWLER M and CUBAS ZS (eds): Biology, Medicine and Surgery of South American Wild Animals; Ames: Iowa State University Press, 2001; 103 - 114.

4. 5. 6. 7.

5 ACKNOWLEDGEMENTS
The authors would like to thank the staff of Professional Zoolife Consultants and Johannesburg Zoo as well as Drs Thelma Meiring and Liza Du Plessis of Vetpath and Dr Emily Lane for carrying out histopathology on several of these cases.

AUTHORS ADDRESS
M.Barrows, BSc BVMS Cert ZooMed MRCVS Professional Zoolife Consultants PO Box 956 Houghton 2041 Johannesburg South Africa Email: Vet@jhbzoo.org.za

46

Private Practitioner, United Kingdom

CLINICAL APPROACH TO THE DOWNER SWAN

G. Cousquer BSc(Hons), BVM&S, CertZooMed, MRCVS.

KEYWORDS
Swans Grounded Unable to stand Examination Diagnostics Treatment.

ABSTRACT
It is not uncommon for swans to be found on the ground. A healthy swan is unlikely to allow itself to be approached and will usually stand and make some sort of response. The term downer swan has been borrowed from cattle medicine to denote the swan that does not stand or respond when approached. Such swans should be investigated. This paper will review the history taking, clinical examination and assessment of such swans. History taking will include the recording of key information such as the location, ight paths, weather conditions, time of day and breeding history. The contact details of the nder should always be recorded. The bird should be identied, weighed and condition scored. Clinical examination will look for signs of acute versus chronic disease. A detailed examination will help establish a list of differential diagnoses. These will include shing tackle problems, lead poisoning, crash landing, power line injuries, chronic disease (tuberculosis and aspergillosis) and more unusual presentations such as parasitism, egg peritonitis and migration exhaustion. Further diagnostic tests will include blood sampling for haematology and biochemistry, radiography and endoscopy.

1 INTRODUCTION
Swans have an almost worldwide distribution, being indigenous to every continent except Africa and Antarctica (SCOTT et al, 1972). Of the eight species and subspecies, three are natives of the southern hemisphere (the South American black-necked swan, the coscoroba swan and the Australasian black swan), two are natives of North America (the trumpeter swan and the whistling swan) and three are natives of Eurasia (the mute swan, Bewicks swan and whooper swan). Most of the populations in the northern hemisphere are migratory but, except for vagrants, there is no movement between continents and none between the two hemispheres (SCOTT et al. 1972). Free-living populations of non-native species do exist, however, having arisen as the result of introduction programmes for sport, ornamental and other purposes. The

47

mute swan (Cygnus olor) is common in many parts of Europe and Asia, particularly in the British Isles, Holland, Germany, Poland and southern Sweden. It occurs less abundantly in Belgium, northern France, Switzerland, Austria and Turkey. In northern Europe, and particularly in the British Isles, the mute swans present status and distribution has been greatly inuenced by introductions and a long history of domestication. The domestication and farming of the mute swan in Britain dates back to medieval times as does the birds royal status; indeed, all unmarked swans on open or common land remain the property of the Crown to this day (SCOTT et al. 1972). Today, the mute swan is a common bird on British waterways and frequently presents to wildlife hospitals and rehabilitation centres (COOKE 2003). Many of these birds will present as weak or unable to stand. Limited funds and facilities, together with a wide range of underlying aetiological problems can make the investigation of such cases diagnostically challenging. This paper describes the clinical approach to these patients, drawing on the authors extensive experience to provide an illustrated review of this subject and a framework for the clinical approach to the downer swan.

2 DIFFERENTIAL DIAGNOSES
The range of possible differential diagnoses is expounded in Table 1. They fall broadly into traumatic and non-traumatic aetiologies. The latter include a range of conditions that have a predominantly neurological presentation but also include chronic disease and miscellaneous conditions such as egg peritonitis, grieving and migration exhaustion.

3 ASSESSMENT AND CLINICAL APPROACH

The assessment and clinical approach to the downer swan will, for the purposes of this article, be broken down into history taking (anamnesis), clinical examination and subsequent diagnostic tests.

3.1 Anamnesis
On admission it is essential that the opportunity be taken to record all key information; a standardised admission sheet will ensure that nothing is omitted. The keeping of detailed and accurate records is not only good clinical practice but can greatly assist future retrospective studies. Admission details should include the time and date of admission, species, age, identifying rings or markings, where and when found and the reason for admission. Use of an OS (Ordnance Survey) map (1:25000 or 1:50000) can prove helpful when identifying and recording the exact location at which the casualty was found. In addition, it is essential that the nders contact details be recorded to allow them to be contacted at a later date, should the need arise. In the case of a swan, attention should also be paid to the following: - Prevailing weather conditions. - Type of waterway - Habitat. - Local ight paths. - Breeding history. - Response when approached - Proximity to power lines. - Recent observations

48

A detailed history can shed light on the possible reason(s) for presentation. Thus, a swan ying in fog or into the setting sun can fail to see an obstacle and crash into it. Knowledge of local ight paths and an accurate record of all such incidents will help analyse trends in such incidents and allow preventive measures to be taken. This is particularly important where swans have crashed into power lines as electricity companies have a responsibility to respond to any such problems. The nature of the habitat and the type of waterway can be relevant: certain waterways, particularly inner city canals and lakes, are heavily and often irresponsibly shed. This can make shing tackle related problems particularly common. Certain waterways are also heavily contaminated with lead shot and clinical as well as sub-clinical lead poisoning will be common in swans from, or that have moved through, these areas. Breeding pairs can be badly affected by the loss of a mate. This should be noted if one of the pair has recently been killed, or if the capture and removal of one of the pair for treatment is being contemplated. When approached a healthy swan will usually stand and, especially if male, adopt an imposing threatening posture with its wings outstretched. If a swan has been on the ground for a period of time the surrounding vegetation may be attened. Failure to stand is likely to be signicant and should be noted and investigated further

3.2 Clinical examination

Attitude and appearance: The swan should be observed from a distance before a systematic head to toe examination is performed. The healthy swan will be bright, alert and responsive. Failure to lift up the head with the head remaining kinked over the back is suggestive of lead poisoning. As the bird is approached and stressed intention tremors of the head may become more obvious suggesting cerebellar damage or other CNS pathology. The birds ability and willingness to stand when approached should be noted. Body weight and condition score: The swan should be weighed and condition scored. Normal weights will vary between 7.3kg and 10.6kg (SNOW and PERRINS 1998). A condition score of 0 to 3 should be ascertained by palpating the pectoral musculature and assessing the prominence of the keel. Condition scoring can be difcult in deep barrel-chested birds such as swans and is best done adjacent to the keel. Concavity of the pectoral muscles is scored 0, attening of the muscles is scored 1, convexity is scored a 2, and rounding that makes palpation of the keel difcult is scored a 3. The body weight and condition score should be used together to assess the condition of the bird. A bird in poor condition is likely to have been ill for some time or failed to thrive through other circumstances such as bullying or limited food availability. Head and neck: A detailed examination of the head can be very informative: Examination of the oral cavity should be thorough to ensure that the presence of translucent shing line under the tongue is not missed. The commissure of the beak should be examined, for chinstrap injuries as shing line will often be found to have cut into the tissues, thus disappearing from view (CRACKNELL 2004). In cases of head trauma, bruising may be evident within the mouth and there may be bleeding from the choana. It is normal for a clump of weed to accumulate at the base of the

49

tongue, resulting in a visible and palpable distension of the inter-mandibular space. This should be differentiated from any inter-mandibular swelling arising as the result of shing tackle injuries to the base of the tongue and inter-mandibular space. The breath should be smelt. A necrotic smell may be present where shing tackle injuries have resulted in tissue necrosis or food impaction. Ketone bodies can also be smelt on the breath of swans that have been unable to feed and are therefore in negative energy balance. The nares should be examined for bleeding, either as the result of trauma or due to parasitism with leeches (Theromyzon spp.), which can invade the sinuses in large numbers. A full ophthalmological examination is indicated to establish the extent of any visual decits. Leeches may feed from the palpebral conjunctiva. Traumatic head injuries may involve the eyes and many swans will present with long-standing unilateral, and even bilateral, ocular pathology. Cataracts are not uncommon in swans and such swans will need this taken into account if they are to be released. The ears should be examined for bleeding following head trauma. The back of the head is often parasitised with lice (Mallophaga spp.) and the resulting feather damage should be differentiated from feather plucking that has arisen as the result of bullying. The neck should be carefully palpated. Fishing line and hooks may be palpated within the cervical oesophagus. Food impactions of the oesophagus may reect an obstruction or impaired crop emptying as the result of lead toxicosis. Soft tissue wounds are not uncommon, arising as the result of bites, power-line injuries, oesophageal tears and other traumas. Deformities and injuries of the cervical spine are not uncommon as a result of in-ight collisions. Rupture of the cervical air sac can result in accumulation of air within the tissues of the lower neck that will feel crepitant on palpation. Body and wings: As with most waterfowl, swans pass through an eclipse moult during the breeding season. This involves the loss of all the wing quills, rendering the swan ightless and relatively helpless until the new ones have grown (SCOTT et al. 1972). Parents normally moult at different times in order to better protect their brood. Whilst this condition renders the individual swan ightless and vulnerable to attack, it does not of itself render them unable to stand. Birds that have been grounded for a period of time are likely to have supercial soiling of the ventral abdomen. There may also be an accumulation of faecal material around the vent. Evidence of diarrhoea should be noted as this may reect lead poisoning, botulism, parasitism or bacterial enteritis. The keel should be carefully palpated and visually inspected for keel wounds. Sadly, keel wounds are a common nding in grounded, weak and emaciated birds. Such birds spend long periods sitting down and will fall heavily onto their keels through weakness. Debilitated waterbirds tend to allow their centre of gravity to move forward so that their keel is scraped along the ground (COOKE, 2003). Sustained contact with the ground will result in localised ischaemia and ulceration. The development of a pressure ulcer is a progressive process that will eventually result in bone necrosis (COUSQUER 2003a, COUSQUER 2003b). It is not unusual for dogs to attack waterfowl who are less mobile than passerines and therefore easier to chase. Female swans will remain on the nest despite receiving injuries to the neck and legs, while male swans will continue to defend a nest despite incurring bite wounds to the leading edge of the wing each time they strike out (ROUTH 2000). A swan poses a formidable adversary and will rear up and defend itself with its wings. Consequently dogs need

50

to attack from behind and it is perhaps for this reason that the tail and rump area is most often targeted (COUSQUER 2003b). Some of these injuries can be very severe and vulnerable to y strike. Infection tracks readily between the fascial layers of the tail muscles and euthanasia may be the only option in many cases (COUSQUER 2003b). For this reason it is essential that all bite wounds are thoroughly investigated and assessed. Collisions with power lines are common (SCOTT et al. 1972) and are likely to result in injuries to the neck and to other parts of the body. Injuries will not only arise as a result of the initial impact on the line, but also as a result of the subsequent crash-landing (ROUTH 2000). Electrical burns will occur where contact has been made between two wires. The manifestation of electrical injuries can be insidious (ROUTH 2000) and all birds found adjacent to power lines should be thoroughly and repeatedly examined for signs of burns to the feathers, skin or musculature over a period of up to ve days. Devitalised tissue will become cold and oedematous to the touch, before sloughing in a compartmentalised fashion, delineating the tract along which the electricity travelled (ROUTH 2000). The considerable associated tissue damage accompanying such electrical burns will dictate euthanasia in the majority of cases (COUSQUER 2003b). Legs and feet: Careful palpation of the legs is indicated to detect both soft tissue and orthopaedic injuries. It is not uncommon for swans to present with a range of lower limb injuries following a crash landing. Traumatic luxations of the femorotibial joint are probably sustained when the tibia is loaded and pushed proximal to the femoral condyles. Trauma to the foot when hitting a wire, vehicle or other object in ight can produce injuries to the dorsal surface of the foot and lower leg (COUSQUER 2003b, ROUTH 2000). Other common ndings include septic arthritis of the inter-tarsal joint and especially of the metatarsophalangeal joint. These can be very debilitating and render a swan extremely lame. Pododermatitis is common but is rarely likely to render a swan unable to stand and walk unless associated with a septic arthritis or tenosynovitis. Fishing tackle will often cut into the lower leg, causing considerable damage.

3.3 Further diagnostic tests

Blood sampling: All casualty swans should, in the authors opinion be blood sampled. The minimum data-base should include measurement of the birds PCV, total protein, total white blood cell count and blood lead. Finances will often limit the scope of any investigation but it should be possible to obtain reasonably accurate PCV, total protein and total white blood cell counts in house. Blood for lead testing will, however, have to be sent away to an approved specialised commercial laboratory, such as the VLA. In the downer swan, lead poisoning should always be suspected even if it be only at a subclinical level. PERRINS et al. (2003) found that slightly over 60% of all casualty swans sampled had blood lead levels in excess of 1.21mol/l. While these results were necessarily biased towards sick and injured birds, they reect the fact that a signicant proportion of these birds have been exposed to lead and may be suffering from the effects, either at a clinical or subclinical level. Chronic lead poisoning is likely to arise as the result of the absorption or the release of lead into the blood stream over a relatively long period. Some of this lead will be removed from the blood and stored in tissue such as bone and liver from where it can be released back

51

into the circulation following hepatocyte atrophy for example (WOBESER 1997). It is clear that the metabolism of lead in swans and the means by which it causes the observed clinical signs, are not fully understood. The possibility that subclinical lead poisoning may underlie the downer swans condition should not be overlooked. Further investigative blood work is indicated where abnormalities are found or the clinical picture demands further investigation. An elevated total white blood cell count should be investigated further to explore the possibility of chronic diseases such as avian tuberculosis and aspergillosis. A differential white blood cell count will prove very useful in such cases and will allow an assessment to be made of any response to therapy. An elevated white blood cell count will also be found in relatively uncommon conditions such as cholangiohepatitis (PATTERSON-KANE and COUSQUER 2005), egg peritonitis and chlamydiosis. Biochemical analysis can also be useful. Creatine kinase and aspartate aminotransferase levels will reect the extent of myonecrosis and can be monitored to provide some indication of the progression of disease and rate of recovery. Uric acid levels will be signicantly elevated in cases of renal failure. Radiography: Dorso-ventral radiographs are easily taken without the need for sedation or general anaesthesia. The neck should be weighted down and the head covered, thus calming the bird. The wings can then be extended away from the body and immobilised with sand bags. Finally, the lower legs should be extended and tied down to prevent the swan from standing. In this way it is possible to obtain a good quality symmetrical dorso-ventral x-ray: A view centred on the hips and dorsal midline will provide information on the digestive tract distal to the proventriculus, caudal airsacs (caudal thoracic and abdominal), liver, hips and knees. A view centred on the mid-scapular area and dorsal midline will provide information on the heart, thoracic oesophagus, pectoral girdle and cranial airsacs (cranial thoracic and clavicular). Lateral radiographs can be taken in the conscious swan but require the swan to adopt an unnatural position, which is far more stressful for the bird. The radiographs should be assessed carefully to glean the maximum amount of information: Are they symmetrical and of good diagnostic quality? A symmetrical radiograph will allow comparison of structures on the left and right sides of the bird. Comparison of the hips is, however, difcult as detail of the left hip is usually obscured by the underlying ventriculus. Is the picture an inspiratory or expiratory view? A max-inspiratory view will provide increased contrast and improved detail and is the gold standard. Is there any evidence of air-sac disease? Sacco-sacco membranes should not be identiable and there should be no lling of the air-sacs. Is there a general loss of denition? This may reect bleeding into or inammation within the coelomic cavity. Is the proventriculus distended? This may suggest loss of GI motility. Is there any evidence of shing tackle in the GI tract? The femoral cortices can be used to compare density of any material in the gizzard. Lead weights are considerably more radio-dense than bone and grit. New lead weights are recognisable by their shape (split shot). This is not, however, true of old weights that may have been weathered on the riverbed. The action of the gizzard can, in

52

time, result in new lead weights becoming polished and even breaking up. This should be born in mind as smaller lead particles have a greater surface area to volume ratio, are more easily absorbed and therefore potentially more toxic. Is there any evidence of liver pathology? An emaciated bird is likely to have a small liver whilst loss of the hourglass shape of the heart and liver suggests hepatic enlargement. The liver margins should be distinct; loss of denition may reect tears and bleeding into the coelomic cavity. Is there any evidence of joint disease? Stie injuries are particularly common. Is there any evidence of kidney disease? An increase in kidney density can be seen when urates build up in the kidneys as the result of kidney disease or in the dehydrated patient. Is there any evidence of spinal injuries?

Endoscopy: In many cases it will only be possible to conrm a diagnosis by visualising and biopsying lesions endoscopically. Standard techniques are indicated and will allow a suspected diagnosis to be conrmed. Rigid endoscopy is ideal for the investigation of air-sac disease, liver and kidney disorders, reproductive disorders (e.g. egg peritonitis), and the investigation of suspected avian TB. Flexible endoscopes can be used to examine the digestive tract; they may assist in the removal of shing tackle and are particularly useful in establishing the presence and extent of a tear in the wall of the oesophagus or proventriculus following removal of a shing hook. Exploratory coeliotomy: In certain cases, or where endoscopy is not available, it may be necessary to recourse to an exploratory procedure. This is performed under general anaesthesia. Anaesthesia is induced using a xylazine and ketamine combination (ROUTH and PAINTER 1999). Trial therapy: In certain situations, therapy may be initiated before a diagnosis is established. This will be the case when blood lead results are pending for example and a swan has presented with signs of acute lead poisoning that require urgent treatment. Trial therapy is, however, no substitute for an accurate diagnosis. Where lead poisoning is suspected, based on clinical signs and/or the presence of lead shot in the gizzard, treatment with sodium calcium edetate (NaCaEDTA) may be initiated. A blood sample should always be collected and submitted for lead testing prior to starting treatment. The patient will benet from intensive intravenous uid therapy and this will allow intravenous NaCaEDTA injections to be easily and aseptically administered. NaCaEDTA can be dosed at 35-50mg/kg i/v or s/c twice daily. A veday course of treatment is advocated with blood lead levels retested on day eight after a two-day rest from therapy (ROUTH 2000). NaCaEDTA does not contain any antimicrobial preservative and is prone to contamination. Consequently, a spike should be placed in the bottles septum to allow the drug to be removed aseptically without introducing any needles into the bottle. The drug is only licensed for intravenous administration. Intramuscular injections of NaCaEDTA should not be administered as they have the potential for causing considerable muscle necrosis (COOKE 2003) and may affect a birds ying ability and release prospects. Additionally hospital staff will nd it difcult to administer twice daily i/m injections aseptically in such a big bird. Where intravenous access is not possible, the next best alternative is an aseptic subcutaneous injection over the calf.

53

4 TREATMENT
The clinical approach outlined above should allow a diagnosis to be made and will facilitate the formulation of a therapeutic plan. Details of specic treatment protocols are available from CRACKNELL (2004), COOKE (2003), COUSQUER (2003b) and ROUTH (2000).

5 CITATION INDEX
COOKE SW. Waterfowl: swans, geese, ducks, grebes and divers. In: BSAVA Manual of Wildlife Casualties. Gloucester: BSAVA 2003; 219 - 231. 2. COUSQUER GO. Wound assessment in the avian wildlife casualty. World Wide Wounds. August 2003a. Available from: www.worldwidewounds. com/2003/august/COUSQUER/avian-wound-assessment.html 3. COUSQUER GO. Wound management in the avian wildlife casualty. World Wide Wounds. November 2003b. Available from: www.worldwidewounds. com/2003/november/COUSQUER/avian-wound-management.html 4. CRACKNELL J. Dealing with line and hook injuries in swans. In Practice 2004; 26: 238 - 245. 5. PATTERSON-KANE JC. and COUSQUER GO. Cholangiohepatitis and cholelithiasis in a mute swan (Cygnus olor). Accepted for publication in the Veterinary Record. 6. PERRINS CM, COUSQUER GO and WAINE J. A Survey of lead levels in mute swans (Cygnus olor). J Avian Pathol 2003; 32(2): 205 - 212. 7. ROUTH A. Veterinary care of the mute swan. In Practice 2000; 22: 426 443. 8. ROUTH A. and PAINTER KS. The use of intravenous ketamine and xylazine for induction of general anaesthesia in the mute swan (Cygnus olor): A review of 130 cases. In: Proc Autumn meeting of the British Zoological Society 1999: 17 - 20. 9. SCOTT P. The Swans. P. SCOTT and the Wildfowl Trust. London: Michael Joseph Ltd, 1972. 10. WOBESER GA. Lead and other metals. In: WOBESER GA. (ed): Diseases of Wild Waterfowl 2nd ed. New York: Plenum Press 1997; 151 - 159. 1.

AUTHORS ADDRESS
Glen COUSQUER BSc(Hons) BVM&S CertZooMed MRCVS. Private Practitioner

54

Table 1: Differential diagnoses for the Downer Swan.

Classication Trauma Sub Classication Acute Aetiology Crash landing Key Findings Orthopaedic injuries. Internal injuries and haemorrhaging. Soft tissue trauma. Orthopaedic injuries. Burns.Soft tissue trauma. Head and neck injuries. Internal injuries. Soft tissue trauma. Bite wounds to wing, neck and tail. Airgun injuries. Crossbow injuries. Shotgun injuries. Hook damage to the beak or GI tract. Cheese wire injuries. Impactions. Often accompanied by tissue necrosis. Stie luxations and arthritis. Hip luxations and arthritis. Septic arthritis especially of MTP joint. Pododermatitis. Limber (kinked) neck. Weakness and anorexia. GI stasis. Bright green diarrhoea. Ataxia. Flaccid paresis. Weakness. Respiratory distress. Usually fatal. UMN or LMN. Visual decits. Vestibular symptoms. Concussion. Ataxia. Head tremor. LMN signs.

Flown into power lines

Dog bites Shooting injuries Fishing hook injuries Fishing line injuries Chronic All of the above if not identied early. Degenerative joint disease Bumblefoot Neurological Intoxication Lead Poisoning

Botulism Traumatic Blue green algae Spinal Injuries Head Trauma Infectious Other Chronic disease Miscellaneous conditions Bacterial Fungal Encephalitis Kidney Disease Avian Tuberculosis Aspergillosis Egg peritonitis Hepatitis / Cholangiohepatitis Bullying Broken heart (grieving) Migration exhaustion Parasitism

Female birds.

Following loss of mate. Whooper and Bewicks. Gizzard worm (Amidostomum)

55

Aquila Foundation, ESVA, Asistencia Veterinaria sl, Centro de Estudios de Rapaces Ibricas CERI, JCCM, Instituto de Investigacin en Recursos Cinegticos IREC, Spain

TRICHOMONAS GALLINAE IN FREE-LIVING BIRDS OF PREY, PASSERINE BIRDS AND WOODPIGEONS (COLUMBA PALUMBUS)
U. He DVM, J. M. Blanco DVM, R. Valboa, D. Villana, C. Gortazar DVM

KEYWORDS
Trichomonas gallinae - Birds of prey - Passerine birds - Wood pigeons Sub-clinical infection - trichomoniasis

ABSTRACT
Avian trichomoniasis is a well known disease of the upper digestive tract of pigeons and other avian species caused by the protozoan parasite Trichomonas gallinae. The disease has recently emerged in some accipiter species as a factor in nestling mortality due to changes in the diet of these species. To obtain further information on the epidemiology of the disease, samples from hunter-harvested healthy woodpigeons, from mistnet captured passerines, from Bonellis and Imperial eagle nestlings and adults in the eld and from 14 other species upon admission to the CERI (Centro de Estudios de Rapaces Ibricas) were analysed for the presence of T. gallinae and associated lesions. Prevalence of T. gallinae in woodpigeons was 43%, while overall prevalence in birds of prey was 10.28%. T. gallinae could not be cultured from any of the passerines examined. Prevalence of lesions was low, but among the woodpigeons sub-clinical effects on fat deposition and bursal size were observed. The preliminary results suggest that parasitation by T. gallinae results in sub-clinical effects due possibly related to a physiological expense for the host. Host immune status appears to be an important factor in the development of clinical disease.

1 INTRODUCTION
The agellated protozoan Trichomonas gallinae is the causative agent of avian trichomoniasis, a disease affecting the upper digestive tract primarily in columbiforms (MEHLHORN et al. 1992). Birds of prey, especially species that catch avian prey are also known to be susceptible to the disease (COOPER and PETTY 1988, BOAL et al. 1998) and trichomoniasis has also been described in passerine, psittacine and

56

gallinaceous birds (COLE 1999). In the rock pigeon (Columba livia) parasitism is generally sub-clinical and development of lesions is supposed to depend on immune status of the host and pathogenicity of the infecting strain (STABLER 1954, COOPER and PETTY 1988). In clinical disease bronecrotic lesions typically develop in the upper digestive tract although occasionally involving the respiratory tract, sinuses or ear (STABLER 1954, SAMOUR 2000). Several authors have been concerned about the effect of the transmission of the parasite from reservoir to nave hosts, such as other endangered columbiforms after introduction of domestic pigeons from Europe into America (see STABLER 1954) or birds of prey (BOAL et al. 1998, HFLE et al. 2000, REAL et al. 2000). In birds of prey a direct relation was established between the consumption of urban pigeons and the appearance of trichomoniasis in the nestlings of Coopers hawks (BOAL et al. 1998), but relatively little is known about the epidemiology of trichomoniasis in susceptible avian species. In an ongoing study we evaluate the prevalence and effects of T. gallinae among free-living birds of prey, passerines and woodpigeons.

2. MATERIAL AND METHODS

Samples were obtained from birds of prey, passerine birds, and woodpigeons from central and southern Spain. We sampled 199 passerine birds of 16 different species at two sites in central Spain in both winter and summer. Samples were obtained from 61 hunter harvested woodpigeons from two different locations in their wintering grounds in central and southern Spain. To date 107 birds of prey of 16 different species have been sampled upon admission to the CERI in central Spain. In addition samples were obtained from ve nestling and ve adult Bonellis eagles in the eld as well as a total of 20 Imperial eagle (Aquila adalberti) nestlings. Each of the sampled birds was examined carefully for the presence of oropharyngeal or crop lesions and presence of T. gallinae was analysed by culture. Shortly, for culture samples were taken from the oropharynx, oesophagus and crop using sterile swabs and placed in sterile tubes containing 5 ml of CPLM medium supplemented with 10% foetal bovine serum and 10% antibiotic solution, incubated at 36,5 C and inspected daily for seven days for the presence of agellated protozoa. Identication as T. gallinae was achieved using morphology and PCR (HFLE et al. 2004). In addition, in the woodpigeons complete biometry and organ weights, as well as burdens of intestinal and external parasites were collected.

3 RESULTS
Prevalence of T. gallinae in the examined woodpigeons was 43% while the parasite was not cultured from any of the passerine birds examined. Overall prevalence of T. gallinae among birds of prey sampled was 10.28% with prevalence ranging from 100 to 0% (table 1). Most of the birds of prey in which the parasite was found were juveniles, while in the woodpigeons prevalence was higher among adults than among

57

juveniles. No difference appeared to exist in prevalence between sexes. All of the Bonellis eagle nestlings and none of the adults were positive for T. gallinae, and the parasite was cultured from half of the imperial eagle nestlings examined. 3.1. T. gallinae and disease In the sampled birds of prey only very few of the culture positive animals had clinical disease, as diagnosed from macroscopic lesions or typical clinical signs (table 1). Three of the culture positive Imperial eagles and one of the positive Bonellis eagle nestlings had clinical disease. Similarly in the woodpigeons prevalence of lesions (2.19%) was signicantly lower than prevalence of the parasite from culture. However when comparing body condition and organ weights of parasitized and non-affected woodpigeons, woodpigeons positive for T. gallinae had signicantly less body fat deposits (t-value= 2.13; p <0.05) and a signicantly larger cloacal bursa (t-value =2.6; p<0.02).

4 DISCUSSION
Our results suggest that, as in rock pigeons, sub-clinical infestations with T. gallinae do exist in birds of prey and woodpigeons and that development of disease depends on interaction between host immunity and strain pathogenicity as stated by other authors (STABLER 1954, COLE 1999). The ndings in the woodpigeons may provide evidence for the cost every parasite imposes on its host as dened for other host parasite systems (HUDSON and GRENMAN 1988). It could thus explain that T. gallinae positive pigeons aggregate more at easy food sources and are more easily predated and thus propagating the parasite. However the observations could also be related to the effect of a subjacent pathogen or factor that predisposes woodpigeons to infection by the parasite.

5 CITATION INDEX
1. 2. BOAL CW, MANNAN RW and HUDELSON, KS. Trichomoniasis in Coopers hawks from Arizona. J Wild Dis 1998; 34: 590 - 593. COLE RA. Trichomonosis. In: FRIEND, M. and FRANSON, J. C. (eds): Field Manual of Wildlife Diseases. U.S. Department of Interior. National Geological Survey 1999; 201 - 206. COOPER JE, PETTY SJ. Trichomoniasis in free-living goshawks (Accipiter gentilis gentilis) from Great Britain. J Wild Dis 1988; 24: 80 - 87. HFLE U, BLANCO JM, PALMA L and MELO P. Trichomoniasis in Bonellis eagle (Hieraaetus fasciatus) nestlings in South-west Portugal. In: LUMEIJ JT, REMPLE JD, REDIG PT, et al. (eds): Raptor Biomedicine III. Lake Worth: Zoological Education Network Inc 2000; 45 - 52. HFLE U, GORTZAR C, ORTZ JA and KNISPEL B. Outbreak of trichomoniasis in a woodpigeon wintering roost. European J Wild Dis 2004; 50: 73 - 77.

3. 4.

5.

58

HUDSON PJ and GRENMAN J. Competition mediated by parasites: biological and theoretical progress. Trends Ecol Evol 1988; 13: 387 - 390. 7. MEHLHORN H, DWELL D and RAETHER W. Atlas de Parasitologa Veterinaria. Barcelona: Ediciones Grass, 1992. 8. REAL J, MAOSA S and MUOZ E. Trichomoniasis in a Bonellis eagle population in Spain. J Wild Dis 2000; 36: 64 - 70. 9. SAMOUR JH. Supraorbital trichomoniasis infection in two saker falcons (Falco cherrug) Vet Rec 2000; 146: 139 - 140. 10. STABLER RM. Trichomonas gallinae: a review. Exp Par 1954; 3: 368 - 402.

6.

AUTHORS ADDRESS
Ursula He Instituto de Investigacin en Recursos Cinegticos IREC Ronda de Toledo s/n,13005 Ciudad Real, Spain Email: uhoe@irec.uclm.es Table 1. Culture results for T. gallinae and prevalence of clinical disease in birds of prey admitted to the CERI Species Scops owl Tawny owl Long-eared owl European Eagle owl Little owl Barn owl Marsh harrier Montagus harrier Peregrine falcon Common kestrel Lesser kestrel Goshawk Booted eagle Total n 5 10 8 13 12 10 7 9 3 18 10 1 1 107 Culture positive 0 0 0 0 2 1 3 2 2 0 0 1 0 11 Prevalence (%) 0 0 0 0 16,67 10 42,86 22,22 33,3 0 0 100 0 10,28 Clinical disease 0 0 0 0 0 1 0 0 2 0 0 1 0 4

59

Biomathematics and Epidemiology Unit, Veterinary University of Lyon; Station Biologique of La Tour du Valat, National Reference Center for Arboviruses, Institut Pasteur, France

A STUDY ON FREE-RANGING WILD BIRDS TO BETTER UNDERSTAND THEIR ROLE IN THE CIRCULATION OF WEST NILE VIRUS IN CAMARGUE, SOUTHERN FRANCE
E. Jourdain, DVM, PhD student, M. Gauthier-Clerc, DVM, PhD, D.J. Bicout, PhD , H. Zeller, PhD, S. Murri, Y. Kayser, P. Sabatier, PhD

KEYWORDS
West Nile Wild Birds Serology Camargue

ABSTRACT
West Nile (WN) disease is a mosquito-borne zoonosis transmitted in natural cycles between birds and ornithophilic mosquitoes. Often asymptomatic, the infection with this avivirus represents a threat to public and equine health. In Europe, outbreaks have been reported in humans or horses in numbers of countries including Romania (1996), Italy (1998), Russia (1999) and France (2000, 2003 and 2004). While several bird species have been proven to be very sensitive to the NY strain of WN virus in America, the picture appears to be different in Europe. Anyhow, birds are considered as the principal hosts of the virus and are suspected to play a major role in the transmission cycle. Recent outbreaks in the South of France enhance the need for further investigations on the role of wild birds in the ecology of WN virus in this area. In order to better understand and test different hypothetical roles of birds in WN virus circulation in Camargue, we set up a research program combining ornithological and serological approaches. In this study, we present preliminary results of serological investigations on different species of migratory passerine birds, cattle egrets (Bubulcus ibis), magpies (Pica pica), house sparrows (Passer domesticus) and tree sparrows (Passer montanus).

1 INTRODUCTION
Responsible for infections in humans and horses, West Nile (WN) virus is a avivirus with a transmission cycle involving birds and mosquitoes. The role of birds in WN epidemiology was rst studied in Egypt in the 1950s (WORK et al. 1955). Seroprevalence was higher in several species of wild birds and experimental

60

inoculations showed that sensitivity was variable depending on the species tested and was particularly high for house sparrows (Passer domesticus) and hooded crows (Corvus corone sardonicus). In these two species, viremia was high enough to infect mosquitoes. Since its introduction into North America in 1999, WN virus has been responsible for the death of thousands of wild and captive birds and is still spreading north, west and southward from its rst outbreak area (New York City). The spread of the disease is easily detected because of the high level of mortality observed in some species of birds, including American crows (Corvus brachyrhynchos) (EIDSON et al. 2001). Mortality in birds was also described in Israel in white storks (Ciconia ciconia) (MALKINSON et al. 2002). Nevertheless, the circulation of WN virus in bird populations is most often revealed by serological investigations only. Indeed, several outbreaks occurred recently in horses and humans in different European countries including Romania (1996), Italy (1998), Russia (1999) and France (2000, 2003) but no clinical sign was described in birds (MURGUE et al. 2002). Although most human and equine cases are asymptomatic, WN virus can be responsible for encephalitis and even death in sensitive hosts. As a consequence, it is important to study the circulation of this virus among birds in areas where recent outbreaks occurred, such as in Camargue in the year 2000 (MURGUE et al. 2001). Our objective in this study was to assess the seroprevalence of WN virus in selected wild bird species captured in the Camargue area.

2 MATERIAL AND METHODS

To select the species that we focused on, we made the hypothesis that WN virus was periodically introduced by migrating birds coming from endemic areas such as SubSaharan Africa. Next, we assumed that the virus was amplied by local birds mainly living in wet areas where mosquitoes are abundant. And nally, it could be spread further by dispersing birds only at the end of summer when outbreaks most often occur in Europe. Given these hypotheses, we chose to concentrate on: (1) migrating passerine birds just after their arrival on the beach after crossing the Mediterranean Sea; (2) cattle egret (Bubulcus ibis) chicks, because they lie exposed to mosquito bites in wet areas and the adults are often found in close contacts with horses; (3) tree (Passer montanus) and house (Passer domesticus) sparrows as they tend to disperse at the end of summer and are found numerously in the vicinity of horse farms and because they have been suspected to be a competent reservoir (KOMAR et al. 2001); (4) magpies (Pica pica) as they belong to the Corvidae family which is well-known for being highly susceptible to the virus on the Western Continent (YAREMYCH et al. 2004). The eld work consisted of capturing the birds, banding them in order to identify them individually and bleeding them. Small passerine birds were captured with mist nests and were identied with respect to species, sex and age. Magpies were caught with traps and cattle heron chicks were taken on the nest. They were all tagged with rings delivered by the National Museum of France. Blood was taken from the brachial vein

61

into heparin capillary tubes. We also looked for ticks but only on the head in order to reduce as much as possible the time during which birds were handled. The sample season lasted from April to mid-May for migratory passerine birds, from mid-June to mid-July for cattle egret chicks and from mid-August to the end of October for sparrows and magpies. The capture site of migrant birds in spring was located at the south of Salin de Giraud whereas at summer and autumn, two other main sites were used: one (site A) at the Station Biologique of La Tour du Valat, between Arles and Salin de Giraud, and the other one (site B) at the Marais du Vigueirat, on the other side of the Rhne river. Heron chicks were sampled from different colonies, the main one being located at the Charnier-Scamandre, north-east of Aigues-Mortes. Blood samples were screened for the presence of anti-avivirus IgG with an appropriate indirect ELISA method. We used horseradish peroxydase-conjugated goat anti-chicken IgG (KPL Europe, Guilford, UK), anti-duck IgG (KPL Europe, Guilford, UK) and anti-wild bird IgG (Bethyl Laboratories, Inc., Montgomery, TX) as described by EBEL et al. (EBEL et al. 2002). Condence intervals and other statistical results were calculated for a risk of error of 5% using the Binomial Law since we were out of the conditions of applicability of the Normal Law.

3 RESULTS
At the end of the eld work season, more than 500 hundred samples from migrating small passerine birds, 220 from cattle egret chicks, 196 from sparrows (144 from house sparrows and 52 from tree sparrows) and 34 from magpies were collected. The capture sites of sparrows and magpies are presented in Table I. Half of the sparrows were caught in site A (n=97) and half in site B (n=99). Magpies mainly came from site A but some of them were sampled from an outdoor cage where they were kept temporally in captivity by a trapper in the proximity of Les Saintes-Maries de la Mer. Table 1: Capture sites of Tree sparrows, house sparrows and magpies Species Tree sparrows House sparrows Capture site Site A Site B Site A Site B Site A Magpies Site B Stes Maries de la Mer Other sites Number captured 37 15 60 84 23 5 4 2 34 Total captured 52 144

62

The commercial anti-wild bird conjugate proved to be efcient to detect antibodies from tree sparrows, house sparrows and magpies. As shown in Table 2, the positive sparrow (of undetermined age) was captured in site A. If we assume that the captured sparrows belong to two different populations, the results in Table 2 lead to conclude to a serological prevalence with WN virus of 1.0% [0-5.6] for the sparrow population at site A and a prevalence smaller than 3.6% with a risk of error of 5% for the population at site B. The four positive magpies were hatch-year individuals with three of them coming from the area of Les Saintes Maries de la Mer. The fourth one came from site A where seroprevalence can be estimated to 4.3% [0-22]. Table 2: Sparrows and magpies serological results Species Capture site Site A Sparrows Site B All sites Site A Magpies Site B Stes Maries de la Mer Other sites All sites Number tested 97 99 196 23 5 4 2 34 Number positive 1 0 1 1 0 3 0 4 Seroprevalence (%) 1.0 [0.0-5.6] 0.0 [0.0-3.6] 0.5 [0.0-2.8] 4.3 [0.1-22.0] 0.0 [0.0-52.2] 75.0 [19.4-99.4] 0.0 [0.0-84.2] 11.8 [3.3-27.4]

We only found a few ticks: 23 on migratory passerine birds at spring and one on a tree sparrow in October. Most of them were nymphs of the genus Ixodes and three were Ixodes larvae. An unfed and unxed adult male of the genus Rhipicephalus was also found on a European robin (Erithacus rubecula) at spring: this species of tick was actually very abundant on the capture site.

4 DISCUSSION
Given the fact that an equine outbreak occurred during the same summer in the vicinity of Les Saintes Maries de la Mer, one could have expected a higher prevalence in local birds such as sparrows and magpies. In the year 2000, during WN epizootic in Camargue, similar results had been obtained with a seroprevalence in magpies and sparrows estimated respectively to 22% [6.4-47.6] and 0% [0-3.1] (HARS et al. 2004). As a comparison, 8% [0.2-38.5] of the Passeriformes sampled were found positive for WN virus during the outbreak that occurred in Bucharest in 1996 (SAVAGE et al. 1999) and 8% [4.15-13.4] two years after (GRANT et al. 2001). In Poland, where no recent outbreak had been described, 2.8% [0.9-6.4] of the house sparrows and 12.1% [3.4-28.2] of the tree sparrows tested between 1995 and 1996 were found positive to WN virus (JURICOVA et al. 1998).

63

Results of the serological investigations on wild birds are consistent with those from sentinel birds. Both studies indicate that WN virus was circulating among birds at the time of the epizootic in horses. Whereas ducks and domestic poultry were shown to have low viremia, sparrows are known for having high levels of virus in their blood in experimental infections (KOMAR et al. 2003), making them a potential reservoir and candidate for WN virus translocation. As they belong to the Corvidae family, magpies are also good candidates that could multiply and spread WN virus. Our results indicate that WN virus circulated in those two species at the end of summer 2004. Because of their dispersing behaviour, specially for young birds after leaving the nest, magpies and sparrows could be two species involved in the spread WN virus at a regional scale. Ticks have been suspected to play a role in the introduction of WN virus, if they are carried by migrating birds, and in the survival of the virus over winter. Even if we probably underestimated the real parasitic burden because small ticks such as larvae are difcult to see, our results seem to indicate that ticks are not really frequent in summer and autumn in sparrows and magpies. We found few hard ticks on migratory passerine birds and it will be interesting to check whether the birds from which ticks have been removed are positive in WN serology. Nevertheless, most of them were nymphs of the tick species Ixodes ricinus and experimental infections have suggested that this species of tick does not support replication of WN virus (LAWRIE et al. 2004). Actually, it is probable that we will not nd antibodies against WN virus in the species sampled at spring. Indeed, seroprevalence was low in birds captured during the outbreak in horses, when one would have expected it to be higher. Even if WN virus was circulating among birds populations earlier in the season, it was probably at a very low level. As a consequence, if we manage to nd antibodies in blood samples from cattle heron chicks, this will be in favour of the hypothesis that the virus was amplied in those birds before being responsible for clinical disease in horses. In the same way, if we nd antibodies in samples from migratory birds coming from Africa, the hypothesis of a new arrival of WN virus because of migratory birds will be reinforced as well. To conclude, we would like to underline the fact that eld work with free-ranging wild birds requires a huge investment of time and necessitates the participation of several qualied eld workers. The results obtained in turn are often disappointing in comparison with the involvement. This is nevertheless essential in order to understand the epidemiological cycle of diseases such as WN disease in non domestic species. That is why it is necessary to develop and adapt laboratory methods of analysis to make it possible to work on samples from wild species.

64

ACKNOWLEDGEMENTS
We want to thank the Parasitology Unit of the Veterinary University of Lyon for their help in the species identication of the ticks removed from birds. We also want to thank the Station Biologique of La Tour du Valat and the Marais du Vigueirat for allowing the capture of wild birds on their land.

5 CITATION INDEX
1. EBEL G D, DUPUIS A P and NICHOLAS D. Detection by enzyme-linked immunosorbent assay of antibodies to West Nile virus in birds. Emerg Infect Dis 2002; 8: 979 - 982. 2. EIDSON M, KOMAR N and SORHAGE F. Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis 2001; 7: 615 - 620. 3. GRANT L, CAMPBELL G L and CORNELIA S. Epidemic West Nile encephalitis in Romania: waiting for history to repeat itself. Ann N Y Acad Sci 2001; 951: 94 - 101. 4. HARS J, AUG P and CHAVERNAC D. Surveillance de linfection de lavifaune camarguaise par le virus West Nile. Faune sauvage 2004; 261: 54 - 58. 5. JURICOVA Z, PINOWSKI J and LITERAK I. Antibodies to alphavirus, avivirus, and bunyavirus arboviruses in house sparrows (Passer domesticus) and tree sparrows (P. montanus) in Poland. Avian Dis 1998; 42: 182 - 185. 6. KOMAR N, PANELLA N A and BURNS J E. Serologic Evidence for West Nile Virus Infection in Birds in the New York City Vicinity During an Outbreak in 1999. Emerg Infect Dis 2001; 7: 621 - 625. 7. KOMAR N, LANGEVIN S and HINTEN S. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 2003; 9: 311 - 322. 8. LAWRIE C H, et al. Ixodid and Argasid tick species and west nile virus. Emerg Infect Dis 2004; 10: 653 - 657. 9. MALKINSON M, BANET C and WEISMAN Y. Introduction of West Nile virus in the Middle East by migrating white storks. Emerg Infect Dis 2002; 8: 392 - 397. 10. MURGUE B, MURRI S and ZIENTARA S. West Nile outbreak in horses in southern France, 2000: the return after 35 years. Emerg Infect Dis 2001; 7: 692 - 696. The rest of references are available from the author.

AUTHORS ADRESS
E. Jourdain, DVM, PhD student Unit de Biomathmatiques & Epidmiologie, Ecole Vtrinaire de Lyon, 1 avenue Bourgelat, 69280 Marcy lEtoile, France Email: e.jourdain@vet-lyon.fr

65

Wildlife Center, National Veterinary School of Nantes, France

WILD BIRDS EXPOSURE TO ANTICOAGULANT RODENTICIDES IN LOIRE-ATLANTIQUE (FRANCE), A 2 YEARS STUDY ON WATERBIRDS AND RAPTORS (2002 - 2003)
O. Lambert, H. Pouliquen, B. Philippe., N. De la Cotte, M LHostis.

KEYWORDS
Raptors Waterbirds Exposure Anticoagulant rodenticides France

ABSTRACT
This presentation is a synthesis of two epidemiological surveys performed in Loire Atlantique (France, 44) on carcasses of birds (raptors and waterbirds) collected in the wildlife center of the national veterinary school of Nantes: birds were either dead when they were brought to the center or died within a week. The aim of these studies was to evaluate their possible exposure to anticoagulant rodenticides: In 2002, exposure of raptors (Falco tinnunculus, Buteo buteo, Tyto alba and Asio otus) to two anticoagulants (chlorophacinone and bromadiolone) was studied, In 2003, exposure of non migratory raptors (Falco tinnunculus, Buteo buteo, Tyto alba and Strix aluco) and waterbirds to ve anticoagulants (brodifacoum, bromadiolone, coumafen, coumatetralyl and difenacoum) was compared.

The anticoagulant rodenticides exposure is higher for raptors than for waterbirds; bromadiolone is the most frequently rodenticide detected or quantied in liver of birds.

1 INTRODUCTION
Anticoagulant rodenticides are traditionally used to control rodents worldwide. In France, they are used against rats, mice and particularly vole (Arvicola terrestris) and coypu (Myocastor coypus). They are presented as baits, carrots, apples or cereals. Many incidents of non-target wildlife mortality from anticoagulant rodenticides occurred, especially for predators and scavengers (BERNY et al 1997, STONE et al

66

1999). The purpose of the studies was to evaluate the exposure to such products of wildbirds (raptors and waterbirds).

2 MATERIALS AND METHODS

Birds of these surveys were collected by private individuals or wildlife protection associations because they were affected and were brought to the Wildlife Center of the National Veterinary School in Nantes for hospitalization. Complete necropsies were performed on birds immediately (due to the rapid degradation of anticoagulants) after their death either at their reception or during their hospitalization (within one week). The liver was removed from each carcass and stored at -18C until analyses. Analyses for anticoagulants were completed at the Toxicology Laboratory of the National Veterinary School in Nantes. Anticoagulant compounds were extracted from liver with mixture of organic solvents (acetone, diethyl ether and chloroform). Extracts were puried by a solid-phase extraction technique using C18 reversedphase cartridges. Puried extract were then injected onto a HPLC C18 end-capped reversed-phase column. Anticoagulant compounds were identied and quantied by using uorimetric detector set at an excitation of 318 nm and an emission wavelength of 390 nm. First study (2002). 51 carcasses were used: 19 kestrel falcons (Falco tinnunculus), 13 common buzzards (Buteo buteo), 9 barn-owls (Tyto alba) and 10 long-eared owls (Asio otus). Birds were anaylsed for two rodenticides: chlorophacinone and bromadiolone. Second study (2003). 58 carcasses were used : 15 diurnal raptors - 4 kestrel falcons (Falco tinnunculus), 11 common buzzards (Buteo buteo) 15 nocturnal raptors 10 barn-owls (Tyto alba), 5 tawny owls(Strix aluco) and 28 waterbirds 15 mallards (Anas platyrhynchos), 13 black coots (Fulica atra) and 1common moorhen (Gallinula chloropus). Birds were analysed for ve rodenticides: difenacoum, bromadiolone, coumattralyl, coumafene, brodifacoum.

3 RESULTS
3.1 First study (2002). Among the 51 raptors, 13 (26%) had rodenticides in liver: 5 with chlorophacinone, 7 with bromadiolone and one long-eared owl had both bromadiolone and chlorophacinone in detectable concentrations (respectively 1.4 and 2.5 g/g). Ten showed bromadiolone and chlorophacinone concentrations in liver above 0.4 g/g : 3 kestrel falcons (n=19), 2 common buzzards (n=13), 1 long-eared owl (n=10) and 4 barn owls (n=9). One kestrel falcon had chlorophacinone between limits of detection and quantication ( 0.11 et 0.33 g/g) and bromadiolone was detected in one common buzzard between limits of detection and quantication ( 0.04 et 0.12 g/g).

67

3.2 Second study (2003). Anticoagulant rodenticides were detected or quantied for 73% (n=22) of all raptors (n=30) and 14% (n=4) of all waterbirds (n=28). At least one anticoagulant rodenticide was detected in the liver of 15 birds or quantied in the liver of 11 birds. When anticoagulant rodenticide was quantied in the liver, its concentration was low (<1 g/g) except one common buzzard (coumafene liver concentration = 2 g/g) and one black coot (coumafene liver concentration = 23.52 g/g). For all the 26 contamined birds, 6 (only raptors) were contamined by 2 or 3 different anticoagulant rodenticides. Bromadiolone was detected or quantied in 15 birds, difenacoum in 8 birds, coumafene in 5 birds, brodifacoum in 4 birds and coumatetralyl in only one bird.

4 DISCUSSION
Our results show that the raptors exposure to anticoagulant rodenticides is relatively large and similar ndings were reported by STONE et al. (2003). However we cant determine the effects of such exposure on their health. Direct exposure of birds like raptors after ingestion of traited baits appears unlikely because of their feeding diet and the exposure seems to occur by the consumption of contaminated rodents (MERSON et al. 1984). According to our second study, the low exposure of waterbirds, which are more susceptible to eat ofcial baits, conrms a secondary exposure for raptors. Prior surveys also indicated that predators, such as raptors or carnivorous mammals (foxes, polecats) are commonly contaminated by anticoagulant rodenticides (SHORE et al. 1996, BERNY et al. 1997, STONE et al. 1999). The 3 most common anticoagulant rodenticides detected or quantied in the liver are respectively bromadiolone, difenacoum and chlorophacinone. In Loire Atlantique and especially in wetlands, bromadiolone is widely used as carrots or apples against coypu (Myocastor coypus), that is conrmed by our results. Other prior studies showed that these rodenticides (bromadiolone, difenacoum and chlorophacinone) were most frequently detected in non target wildlife in Europe (SHORE et al. 1996 ; BERNY et al. 1997). In contrast brodifacoum is implicated in 80% (STONE et al. 1999) and 84% (STONE et al. 2003) of the incidents of poisoning by anticoagulants in New York respectively from 1971 to 1997 and from 1998 to 2001; brodifacoum appears to have the greatest potential for non target wildlife mortality.

5 CITATION INDEX
1. BERNY P, BURONFOSSE T, BURONFOSSE F, et al. Field evidence of secondary poisoning of foxes (Vulpes vulpes) and buzzards (Buteo buteo) by bromadiolone, a 4-year survey. Chemosphere 1997; 35: 1817 - 1828. MERSON MH, BYERS RE and KAUKEINEN DE. Residues of the rodenticide brodifacoum in voles and raptors after orchard treatment. J Wildl Manage 1984; 48: 212 - 216. SHORE RF, BIRKS JDS, FREESTONE P and KITCHENER AC. Secondgeneration rodenticides and polecats (Mustela putorius) in Britain. Envir Pollut 1996; 91: 279 - 282.

2.

3.

68

4. 5.

STONE WB, OKONIEWSKI JC and STEDELIN JR. Poisoning of wildlife with anticoagulant rodenticides in New York. J Wildl Dis 1999; 187 - 193. STONE WB, OKONIEWSKI JC and STEDELIN JR. Anticoagulant rodenticides and raptors: recent ndings from New York, 1998-2001. Bull Environ Contam Toxicol 2003; 70: 34 - 40.

AUTHORS ADRESS
O. Lambert Centre de Soins de la Faune Sauvage, Ecole Nationale Vtrinaire de Nantes, Atlanpole La Chantrerie, 44307 Nantes cedex 03, France Email: cds.armauny@free.fr

69

Akron Zoological Park, Bird and Exotic Specialty Hospital, Ohio, United States of America

MYCOBACTERIAL INFECTION IN WATERFOWL COLLECTIONS : A CONSERVATION PERSPECTIVE.

G. Riggs DVM, ABVP (Avian)

KEYWORDS
Waterfowl Mycobacteria tuberculocidal SSP - White winged wood duck - UV light pH Band - Sharing coefcient

ABSTRACT
Although relatively rare in wild waterfowl (FRIEND 1999), mycobacterial infections can be devastating to captive waterfowl collections. Of particular concern is the captive management of endangered species, such as the white winged wood duck (WWWD) which represent the only anseriforme Species Survival Plan (SSP) of the American Zoo and Aquarium Association. There are currently approximately 60 WWWD individuals in captivity in the SSP managed program. A contributing factor to their low numbers in captivity is their historical predilection for mycobacterial disease, resulting in over 80% mortality in some collections. Ante mortem mycobacterial screening involving PCR, ELISA, CBC, and endoscopy has been attempted to help manage waterfowl mycobacterial infections but as of yet, has not proven sensitive or specic enough for adequate disease control (TELL et al. 2001). The ubiquitous nature of the mycobacterial organisms, poor performance of disinfectants in the eld, poor response to therapy, lack of early symptoms, and immune system deciencies (due to genetics or prior exposure), make disease prevention particularly difcult. Improved exhibit and holding design, optimization of husbandry, utilization of ultraviolet lighting, and altering soil pH (above 7) are being examined as possible tools to minimize organism build-up and thereby control the morbidity and mortality of exposed waterfowl. Of particular concern for the white winged wood duck is a presumed immune system deciency due to poor genetic diversity, resulting from limited population founders of unknown genetic relationship. Poor genetic diversity within species (ducks and others) has been implicated as contributing to immune system failures to resist mycobacterial infections (CROMIE et al. 1992). The WWWD population, previously

70

presumed to have 81% heterozygosity in a 1989 report (TOMLINSON et al. 1991) and to be 63.5% genetically diverse per the SSP studbook in 1992, has instead been shown by our study to have diversity no better than rst generation relatives (USA population), possibly contributing to their continuing sensitivity to mycobacterial infections in captivity. WWWDs in their natural habitat have limited exposure to mycobacteria and, therefore, are likely to have little historical immune system experience with the organisms. Captive holding techniques commonly employed have encouraged infection by maximizing both stress on the captive individuals and exposure to mycobacterial organisms. It is our feeling that the presumed increased sensitivity to mycobacteria of this population can be controlled with improved husbandry, enclosure, and quarantine protocols in the short term, and hopefully by improving genetic diversity in the future. 1 INTRODUCTION Mycobacterial organisms have been referred to as ducks of the microbe world (GRANGE 1987). This alludes to the fact that the organisms thrive in the shallow waters of the soil water interface, but can also reect the severe impact mycobacteria can have on waterfowl populations. Waterfowl mycobacterial infections are generally caused by organisms of the Mycobacterium intracellulare complex, most commonly by M. avium serotypes 1, 2, and 3 (TELL et al. 2001). Recently, an increasing number of mycobacterial isolates have been determined to be Mycobacterium genovense (TELL et al. 2001), in some surveys up to 70% of isolates. This evolving trend further complicates ante mortem diagnosis by requiring testing modalities to be sensitive to both M. avium and M. genovense in order to be effective screening tools. Mycobacterial infections are characterized as chronic, debilitating diseases usually seen in individual birds rather than in epizootics. They are characterized by numerous caseous lesions in the GI tract and major organs. There are no pathognomonic signs; symptoms relate to target organ and degree of damage. Mycobacterial infections are relatively rare in wild waterfowl, with a 0.3% incidence rate, compared to 3.6% incidence rate in total wild bird populations (FRIEND 1999). Incidence in captive collections can be much higher. All waterfowl are potentially susceptible to mycobacterial infection although ducks and swans are more represented than geese, due in large part to exposure variance. Dabbling ducks are also more likely to be infected than grazers since they feed in the favoured growth environment of the mycobacterial organisms (CROMIE et al. 1991). As with most diseases, stress can play a major role in determining which individuals within an exposed population will become chronically infected with mycobacteria. Reproductive stresses, overcrowding, poor ventilation, substandard nutrition, and management decisions (e.g. pinioning) can greatly exacerbate the disease process (CROMIE et al. 1992).

71

Early diagnosis of this relatively common disease can prove difcult. Currently no gold standard test exists. Acid fast faecal staining, long held as the test of choice, is at best positive in only 10%-30% of positive avian mycobacterial cases. Elevated white blood cell counts are often reported with marked monocyte shifts, although this is frequently unreliable early in the disease and can drop off late in the course of the disease, corresponding to immune system collapse. Mycobacterial cultures are relatively specic but do not often show high sensitivity and require long incubation times for results. Serology has also been historically unrewarding, although in one study (CROMIE et al. 1991), an ELISA did prove predictive in the mid to later stages of disease in selected duck populations. Radiology can be a benecial diagnostic tool (Bush et al 1978) but is effective in only the 10% of cases with bone lesions. Finally, endoscopy and biopsy are utilized by some institutions for mycobacterial screening. Even this is limited in its absolute ability to declare birds negative and presents time/ risk/cost implications. With the inherent diagnostic and treatment difculties, prevention and control are the mainstays of collection protection. The problem is made more difcult by the prevalence and resistant nature of the organism(s) involved. Mycobacteria (both pathogenic and non-pathogenic types) are ubiquitous organisms found routinely in most environments. They have been cultured in up to 40% of surface waters, and in nearly 100% of municipal water supplies in some studies (FALKINHAM 2003). They thrive in a variety of environmental conditions and can persist even in seasonal sub-zero temperatures for up to seven years (THOEN and RICHARDS 1977). They are normal soil inhabitants and can persist protected underground and multiply in earthworm and amoeba hosts. Compounding the problem, low level environmental exposure by young waterfowl has been found to decrease resistance to pathologic mycobacterial later in life (CROMIE et al. 2000). Once infected, waterfowl can also shed huge numbers of organisms into the environment for extended periods of time prior to displaying demonstrable clinical symptoms. This sub-clinical environmental contamination enhances and easily perpetuates the epizootic conditions in many waterfowl collections. Environmental disinfection is unfortunately not a viable solution to the problem. Mycobacteria, in particular M. avium, are notoriously resistant to most common disinfectants in the eld (FALKINHAM 2003). Mycobacteria have routinely been shown to grow in chlorinated water (500 times as resistant as E. coli), and are resistant to ozone and most common safe disinfectants. Even disinfectants labelled as tuberculocidal cannot be depended on for eld use. Tuberculocidal refers to organism destruction on non-porous surfaces with extended contact times at room temperatures (CARSON 1978). Natural surfaces are seldom non-porous and organic material, hard water, and lower temperatures will inactivate the disinfectant or require a much longer contact time than is feasible in the eld. Hot water is also not the answer. Mycobacteria thrive in hot water and will even grow in hospital hot water heaters. In addition, due to their adherent properties, they are difcult to physically wash off of contact surfaces.

72

Maintaining an alkaline soil, however, does show potential as a mycobacterial deterrent. A Johnes study (M. paratuberculosis) resulted in a ten-fold decrease in paratuberculosis cases after lime treatment of pasture soil increased the soil pH to over 7 (COLLINS 2001). This was not a controlled study and obviously more work is needed to determine successes and limitations, but this technique seems to hold promise to help minimize environmental build-up in exhibits. UV light may also be an effective anti-mycobacteriocidal agent for captive collections. Human hospital studies have demonstrated complete mycobacterial disinfection in water suspensions at 15 mW-s/cm2 (Manning unpublished). Many hospital ventilation sanitation systems utilize 100 mW-s/cm2 UVC devices for total mycobacterial airborne sanitation. UV light is only effective, however, with direct mycobacterial contact. Shaded areas or areas where organisms are shielded by soil will not be affected by UV radiation. At WWT, disease surveys found a signicantly higher mycobacterial infection rate in shaded pens as compared to pens directly exposed to sunlight (CROMIE et al 1992). To be successful at minimizing mycobacterial buildup, enclosure design must incorporate the ability to minimize persistent shading (or rotate shaded areas), and eliminate the shading of soil-water interface areas.

1.1. The white-winged wood duck story White-winged wood ducks are large, tree perching, deep forest ducks native to Assam, Thailand, Sumatra, and Java. Known in some areas as the Spirit duck for their haunting vocalizations, they live solitarily, in pairs, or small groups that feed nocturnally in shallow still waters. Due primarily to habitat pressures, the numbers in the wild are critical. They are listed CITES I and represent the only anseriforme SSP maintained by the American Zoo and Aquarium Association. Approximately 60 individuals currently exist in the USAs captive programme, down from the 120 birds previously maintained by the SSP. Mycobacterial disease has played a signicant role in the numbers decline in captivity. Currently, breeding recommendations are in place for all institutions holding pairs and it is hoped to build numbers back up to stable levels within the near future. WWWDs have had severe mycobacterial problems in captivity. Up to 84% of individual deaths in certain collections (CROMIE et al. 1992) have been linked to avian mycobacteriosis. We feel these high rates in captivity may not accurately reect the true sensitivity of the wild species, as poor genetics and exposure factors have played a signicant role in the problem. Due to the limited number of founders of the captive WWWD population (7), the genetic diversity within the SSP was felt to be only 63.5%. Our current study unfortunately has shown it to be much worse. Research in other species has shown that this lack of genetic diversity can have severe impact on the immune status of the individual, predisposing to mycobacterial infections. Our data has determined the DNA relatedness of the current breeding SSP population to be no better than rst order relatives.

73

Husbandry failures have also played a major role in the WWWDs mycobacterial problems. A solitary, tree perching duck in the wild, where mycobacterial contact is minimal, is now most frequently kept as a pinioned, ground bird in large mixed groups. The resultant increase in stress on the individual would further minimize resistance to the mycobacterial organisms it now routinely contacts. Natural husbandry traits also work against WWWDs in captivity. Their method of dabble feeding at the soil-water interface in shaded areas places them in contact with the highest mycobacterial concentrations in captivity. The introduction of the wild caught nave founder population into a large mixed collection endemic with mycobacterial has had tragic results. Not only did it lead to large numbers of individual losses, but more importantly, it led to losses in an already small irreplaceable genetic pool. Furthermore, due to endemic mycobacterial levels at the initial captive propagation site, infected individuals may well have been unknowingly disbursed to other collections to perpetuate the mycobacterial cycle. Currently, the WWWD SSP has undertaken several pilot projects in an attempt to better understand and improve the mycobacterial diagnostic testing (PCR, ELISA) and genetic status of white-winged wood ducks in captivity. Quarantine and necropsy protocols have been developed to investigate and screen for current infections and pilot projects are underway to determine genetic diversity and assess new ante mortem avian mycobacterial screening techniques.

2 MATERIALS AND METHODS

To investigate a genetic component contributing to the high infection rate of the species, samples were taken for DNA analysis from the pilot group (31 individuals) serving as the source for most individuals currently held in the US SSP. Therion International Labs performed the testing of the collected samples. DNA was isolated using non-organic extraction. DNA samples were cleaved with restriction enzyme Alu I. Digested fragments were separated by size with gel electrophoresis, then transferred to a nylon membrane and hybridized sequentially with radioactivelylabelled multi-locus DNA probes OPT-02 and OPT-05.

3 RESULTS
DNA prole analysis was performed on 31 white winged wood ducks to estimate relative relatedness of the birds in the captive conservation program. These ducks represent nearly all of captive breeding stock in the United States and are direct descendants from the original captive breeding stock in the UK. Arguably, all known captive WWWDs descend from the twelve individuals (TOMLINSON et al 1991) brought into captivity at the Waterfowl Trust in the late 1960s. Standard bird species assays showed little or no genetic variability within the population. Two additional assays were undertaken that yielded 72 genetic markers with an average of 45 markers per individual. The average band-sharing coefcient

74

(BSC) per pair was 0.80 and 0.75 for the respective assays and 0.77 for combined assays. The range of BSC for the tested individuals was 0.65-1.00. BSC values can be ranked as breeding guides to maximize genetic variability within a population. Within the tested population, estimated heterozygosity were 0.31-0.41 with only 2-2.3 alleles per locus. Results of this study displayed a very low genetic diversity within the population, much lower than was previously thought. The WWWD populations genetic variability appears no better than would be expected from rst-order relatives.

4 DISCUSSION
The results of the genetic analysis have major implications on the SSPs management of the species in captivity. Since no individual is farther apart than a rst order relative, specic pairings, other than for minor phenotypic variations, are not critical and would not be expected to improve genetic variability within the captive population. Our immediate goal is now to analyze any potential non-related individuals in collections outside of the United States to determine if signicant improvements in breeding can be accomplished with currently held individuals. It is imperative that genetic diversity of the captive species be improved within the foreseeable future if we are to truly function as a species survival plan. If we are to assume that the genetic inbreeding has also contributed to immune system deciencies that predispose the population to mycobacterial infections, it increases the importance of vigorously pursuing husbandry protocols and diseased bird culling to minimize environmental build-up of the mycobacterial organisms. To facilitate these goals, we will continue our pilot projects to submit samples for the development of a sensitive and specic ante mortem diagnostic screening tests. We will also implement faecal PCR screening into SSP quarantine protocols and continue aggressive necropsy screening to attempt to analyze collection contamination from new acquisitions. Husbandry recommendations to more closely mirror natural behaviour will be put into place via the SSP and we will continue to evaluate the efcacy of UV light and soil pH changes in the control of environmental mycobacterial contamination. Current white-winged wood duck SSP husbandry recommendations include: 1). 2). 3). 4). 5). 6). Maintaining non-pinioned birds where possible. Maintaining birds in breeding pairs or small non-breeding groups. Minimizing temperature extremes (50-80 degrees F). Minimizing soil-water interface on ponds. Constructing ponds to be cleanable with moving water. Minimizing shaded areas and allow rotation of shaded areas for periodic UV exposure where possible. 7). Routine health screening/aggressive work up on wasting and ill individuals. 8). Maximizing nutrition.

75

9). Encourage free ight exhibits with natural perching/nest boxes. 10). Exclude wild y-ins. 11). Minimizing shaded soil areas and maintain soil pH at 7 or greater. It is anticipated that achievable husbandry and genetic improvements can make demonstrable changes in the current and future captive SSP white-winged wood duck population. As improved ante mortem testing develops, and new genetic lines can be introduced into the SSP population, we look forward to simplifying husbandry guidelines and minimizing the impact of individual disease entities on the captive population of this important species.

5 CITATION INDEX
1. BUSH M, MONTALI R. Clinical experience with tuberculosis in exotic birds. In: MONTALI R. (ed): Mycobacterial infections of zoo animals. Smithsonian Institution Press 1978; 199 - 204. 2. CARSON L. Growth characteristics of atypical mycobacteria in water and their comparative resistance to disinfectants. Appl Environ Microbiol 1978; 36: 839 - 846. 3. COLLINS M. Survival. Johnes Information Center. 2001; University of Wisconsin, School of Vet Med.1 - 5. 4. CROMIE R. Avian immune response to mycobacterium avian: the wildfowl example. Dev and Comp Immun 2000; 24: 169 - 185. 5. CROMIE R. Susceptibility of captive wildfowl to avian TB: the importance of genetic and environmental factors. Tubercle 1991; 72: 105 - 109. 6. CROMIE R. The epidemiology of avian tuberculosis in White-winged wood ducks, Cairina Scutulata, at the Wildfowl and Wetlands Trust, Slimbridge Center (1976-1991). Wildfowl 1992; 43: 211 - 214. 7. FALKINHAM J. Mycobacterial aerosols and respiratory disease. CDC Emerging Infectious Diseases 2003; Vol 9: No. 7. 8. FRIEND M, ROFFE T. Tuberculosis. In: Friend M, Franson C (eds): Field Manual of Wildlife Diseases. US Dept. of Interior 1999; 93 - 98. 9. THOEN C, RICHARDS W. Mycobacteria isolated from exotic animals. J Am Vet Med Assoc 1977; 170: 987 - 990. 10. TOMLINSON C, MACE G. Improving the management of a highly inbred specie: the case of the White-winged wood duck Cairina scutulata in captivity. Wildfowl 1991; 42: 123 - 133.

AUTHORS ADDRESS
Gary Riggs DVM, ABVP (Avian) Akron Zoological Park, Bird and Exotic Specialty Hospital, 4873 Richland Ave, Norton, Ohio 44203, United States of America Email: garyriggs@aol.com

76

Abu Dhabi Falcon Hospital, Abu Dhabi, United Arab Emirates

HAEMATOLOGICAL VALUES OF GYR HYBRID FALCONS

M. G. Muller; A. R. George and A. T. Mannil

KEYWORDS
Birds - Gyr-saker hybrid falcon - Gyr-peregrine hybrid falcon - Haematology - Values

ABSTRACT
Haematological values for falcon species like gyr falcons, peregrine falcons and saker falcons are widely discussed in the literature. Due to the captive breeding of hybrid falcons, those falcons are seen in an increasing number in the Abu Dhabi Falcon Hospital, United Arab Emirates. The haematological parameters of two different hybrid species, gyr-saker hybrid falcons and gyr-peregrine hybrids falcons, had been studied to compare them with the purebred species in the literature and to establish reference parameters for those hybrid species.

1. INTRODUCTION
Although the saker falcon (Falco cherrug) is the traditional hunting bird in the MiddleEastern Countries, it is not the commonly used falcon for falconry in the United Arab Emirates anymore. In the United Arab Emirates, the number of captive-bred gyrsaker hybrid falcons and gyr-peregrine hybrids falcons used for falconry has rapidly increased over the past few years. One of the main reasons is the new law Cites of October 2002 allowing only captive-bred falcons to be used for falconry purposes inside UAE. This leads to a change in the patients frequenting falcon clinics and hospitals throughout UAE as well to a great need to enhance research on these hybrids for the sake of their health. Although the haematological parameters are wellknown in saker falcons (Falco cherrug) (SAMOUR et al. 1996; JENNINGS 1996), peregrine falcons (Falco peregrinus) (DTLINGER and BIRD 1995, JENNINGS 1996) and gyr falcons (Falco rusticolus) (WERNERY et al. 2004), only little information is available about those two hybrid species (WERNERY et al. 2004). The haematological blood picture can give signicant hints for the avian practitioner about e.g. anemia, dehydration, infections and aspergillosis (CAMPBELL 1988). Due to the importance of the haematological blood parameters for the early detection of diseases in falcons, this study covers the haematological parameters of complete blood counts (CBC) for gyr-saker and gyr-peregrine hybrid falcons.

77

2. MATERIAL AND METHODS

2.1 Material The haematological parameters of 689 clinically healthy hybrid falcons were evaluated. All falcons that were showing clinical signs or that were diagnosed with disease were excluded from in this study. The complete blood count (CBC) of 369 gyr-saker hybrid falcons and 320 gyr-peregrine hybrid falcons has been examined manually. After taking blood from the right or left basilic vein (Vena cutanea ulnaris supercialis) or the right or left caudal tibial vein (Vena metatarsalis plantaris supercialis), 0.5ml blood was stored in a 1.0 ml EDTA tube (Teklab). The blood count was performed directly after blood taking with a maximum delay time of 30 minutes. 2.2 Method Red blood cell count (RBC) (SAMOUR et al 1996) The working solution consists of 10ml 40% formaldehyde, 31.3g trisodium citrate and 1000ml distilled water. Four ml of this solution has been mixed in a plain sample tube with 20l of the falcon blood stored in the EDTA tube. This diluted sample has been lled via a capillary tube in the improved Neubauer haemocytometer. After 5 minutes waiting time, the cells of 5x16 squares have been counted in the center of the counting grid. The counted cells had been calculated as follows: N=Number of cells counted, then: N = RBC x 1012 / l 100 White blood cell count (WBC) (SAMOUR et al 1996) 1.9ml of a 1% ammonium oxalate solution had been mixed with 100l of the falcon blood sample and kept on a tube roller for 3 minutes. A small amount of the diluted samples has been lled via a capillary tube in the improved Neubauer haemocytometer. After 5 minutes waiting time, the cells of 4 outer large squares have been counted in the center of the counting grid. The counted cells had been calculated as follows: N=Number of cells counted, then: N = WBC x 109 / l 20 Packed cell volume (PCV)/ Haematocrit (Hct) (SAMOUR et al 1996) The PCV is measured by the used of microhaematocrit capillary tubes (Fortuna, Germany) and centrifuge (Hawsley, UK). In our study, the haematocrit is used as this might be the more accurate than the PCV (FUDGE, 2000) Mean cell volume: PCV x 10 = MCV() RBC Mean cell haemoglobin: Hb x 10 = MCH (pg) RBC Mean cell haemoglobin concentration: Hb x 100 = MCHC (g/l) PCV

78

Haemoglobin The haemoglobin is measured by capillary, venous or arterial blood used in EDTA. The optical eye of the HemoCue microcuvette contains reagents deposited on its inner wall and the blood sample is drawn in to the cavity by capillary action spontaneously mixed with the reagents. Used with the HemoCue photometer, the system provides a direct reading of the concentration of haemoglobin in a blood sample. Preparation of blood smear Take one small drop of blood sample in a clean microscopic slide. Position a spreader slide in front of the drop of blood at an angle of about 45 and move the spreader backwards and touch gently the drop of blood which will run across the edge of the slide. Push the spreader with a steady forward movement to create a blood smear. Staining method The HEMAstain test is for the rapid, differential staining of haematological smears that yields qualitative results similar to Wright-Giemsa stain. The slide is dipped in xative solution for 5 seconds, than in solution.1 for 5 seconds and than in solution 11 for 5 seconds. The excess solution is allowed to drain. The slide is rinsed with distilled or deionised water, then allowed to dry and examined under oil immersion lens. The examination of the blood smear for the shape of the blood cells and avian blood parasites is routinely performed for each blood smear, but is not part of this study. 3. Results The evaluation of the 369 (Falco rusticolus x Falco cherrug) hybrid falcons showed the following picture: Table 1: Haematological parameters for gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug)

Parameters RBC (x1012/l) HB (g/dl) Hct % MCV () MCH (pg) MCHC (g/dl) WBC (x 109 /l) Heterophils % Lymphocytes % Monocytes % Eosinophils % Basophils %

n=369 2.330.15 17.731.50 51.574.66 221.8521.64 76.246.83 34.421.57 7.502.22 49.903.88 44.112.91 4.421.58 1.290.92 0.400.15

range 2.18-2.48 16.23-19.23 46.91-56.23 200.29-243.49 69.41-83.07 32.85-35.99 5.28-9.72 46.02-53.78 41.20-47.02 2.84-6.00 0.37-2.21 0.25-0.55

79

The 320 evaluated (Falco rusticolus x Falco peregrinus) led to those results: Table 2: Table 1: Haematological parameters for gyr-peregrine hybrid falcons (Falco rusticolus x Falco peregrinus)

Parameters
RBC (x1012/l) HB (g/dl) Hct % MCV () MCH (pg) MCHC (g/dl) WBC (x 10 /l)
9

n=320
2.390.26 17.91.57 52.004.75 219.725.38 75.598.45 34.431.48 7.552.27 49.913.50 44.183.36 4.401.63 1.371.08 0.140.014

range
2.13-2.65 16.33-19.47 47.25-56.75 194.32-245.08 67.14-84.04 32.95-35.91 5.28-9.82 46.41-53.41 40.82-47.54 2.77-6.03 0.29-2.45 0-0

Heterophils % Lymphocytes % Monocytes % Eosinophils % Basophils %

4. DISCUSSION
Extensive work has been done on the haematological parameters of peregrine falcons. Although the range of some parameters is relatively close, signicant differences can be seen in MCV, WBC, heterophils, lymphocytes, monocytes and eosinophils. Table 3. Literature comparison of haematological parameters in peregrine falcons (Falco peregrinus)

Parameters
RBC (x1012/l) PCV % HB (g/dl) Hct l/l MCV () MCH (pg) MCHC (g/dl) WBC (x 109 /l) Heterophils % Lymphocytes % Monocytes% Eosinophils % Basophils %

JENNINGS 1996
n=70 2.95-3.94 37-53 11.80-18.80 ----188-146 40.00-48.4 31.90-35.20 3.30-11.00 14.00-85.50 11.00-33.00 1.00-8.60 0-0.3 0-0.6

DOETLINGER and BIRD 1995

n=48 3.490.21 ---14.821.32 0.400.38 117.517.70 --------12.563.06 45.2012.00 55.2013.60 2.500.30 2.300.90 ----

WERNERY et al 2004
n=138 3.180.50 ---14.672.06 0.440.06 138.436.60 46.132.10 ---9.324.69 60.9512.01 32.0011.81 6.323.65 0.52 0.26

80

A similar picture shows the literature comparison of the haematology values of saker falcons (Falco cherrug). Major differences can be found in MCV, MCH, WBC, heterophils, lymphocytes and monocytes. Table 4: Literature comparison of haematological parameters in saker falcons (falco cherrug) Parameters JENNINGS 1996 n=50 2.54-3.96 38-49 11.50-16.50 ---124-147 41.40-45.40 30.40-34.90 3.80-11.50 26.00-58.50 8.00-42.50 0-2 0-0.45 SAMOUR, DALOIA 1996 n=25 2.640.08 ---15.930.38 0.470.59 183.163.84 60.741.42 33.280.63 5.700.31 41.402.40 13.300.90 2.100.30 0 0.800.10 WERNERY et al 2004 n=146 3.180.29 ----15.001.46 0.450.04 141.505.89 47.171.73 ----10.213.53 61.7811.16 31.1111.46 4.723.02 1.12 0.4

RBC (x1012/l) PCV % HB (g/dl) Hct l/l MCV () MCH (pg) MCHC (g/dl) WBC (x 109 /l) Heterophils % Lymophocytes % Monocytes % Eosinophils % Basophils %

Only little information is available on the gyr falcons (Falco rusticolus). Table 5: Literature data of haematological parameters in gyr falcons (Falco rusticolus)

Parameters RBC (x1012/l) HB (g/dl) Hct l/l MCV () MCH (pg) WBC (x 109 /l) Heterophils % Lymphocytes % Monocytes % Eosinophils % Basophils %

WERNERY et al 2004 n=187 3.230.28 15.001.33 0.450.04 139.325.44 45.781.84 8.713.80 58.5312.90 37.5412.98 3.722.50 0.2 0 81

The value ranges of the gyr peregrine hybrid falcons (Falco rusticolus x Falco peregrinus) differ especially of RBC, MCV, WBC, heterophils and eosinophils (table 6). RBCs in the range of 3.35 are very rarely evaluated among our patient clientele. As per our observation at the Abu Dhabi Falco Hospital, falcons with a WBC of 12.55 and 70% heterophils, being the maximum range of WERNERY et al. (2004), show already clinical signs of reduced well-being like reduced appetite, not ying well and not gaining weight. Table 6: Comparison between haematologicalvalues in the literature and our laboratory results of gyr-Peregrine hybrid falcons (Falco rusticolus x Falco peregrinus) WERNERY et al Parameters RBC (x1012/l) HB (g/dl) Hct % MCV () MCH (pg) MCHC (g/dl) WBC (x 109 /l) Heterophils % Lymphocytes % Monocytes % Eosinophils % Basophils % n=267 3.350.12 15.330.62 46.002.0 137.314.15 45.703.43 ---9.313.24 60.0010.00 37.4311.18 2.571.23 0 0 Study results n=320 2.390.26 17.91.57 52.004.75 219.725.38 75.598.45 34.431.48 7.552.27 49.913.50 44.183.36 4.401.63 1.371.08 0.140.014

WERNERY et al. (2004) evaluated the gyr hybrid falcons by combining gyr-saker and gyr-peregrine hybrid falcons. The results of our study show that the haematological values of both hybrid species are relatively close to each other, yet far below the results measured in WERNERY et al. (2004). From our patients records hybrid falcons with such elevated blood parameters often display infections or aspergillosis. The high values might be related to the use of the Cell Dyn 3500 analyzer (Abbott Laboratory, North Chicago IL) used for the calculation of RBC, WBC and packed cell volume (WERNERY et al. 2004). When using the Cell Dyn 3500 analyzer (Abbott Laboratory, North Chicago IL) we experienced that this equipment can process only a low number of daily falcon blood samples with accurate results. For the examination of more than approx. 10 samples per day, the Cell Dyn 3500 analyzer is not able to run the falcon blood with its very thick consistency thus leading to unreliable results due to blockage of the washing function of the analyzer. This technical problem resulted in parameters showing very high values that were contradicting clinical conditions of the falcons. Due to these unreliable results, the Abu Dhabi Falcon Hospital returned to the manual haematology examination.

82

Table 7: Comparison between haematological values in the literature and our study in gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug) and gyrperegrine hybrid falcons (Falco rusticolus x Falco peregrinus) Gyr hybrid (GS and GP) Parameters RBC (x10 /l)
12

WERNERY et al 2004 GS and GP n=990 3.140.31 -----14.671.33 44.004.00 140.138.10 46.212.71 ----9.435.15 60.4214.68 34.3714.23 4.733.67 0.31 0.04

Study results GP n=320 2.390.26 17.91.57 52.004.75 219.725.38 75.598.45 34.431.48 7.552.27 49.913.50 44.183.36 4.401.63 1.371.08 0.140.014 GS n=369 2.330.15 --17.731.50 51.574.66 221.8521.64 76.246.83 34.421.57 7.502.22 49.903.88 44.112.91 4.421.58 1.290.92 0.400.15

PCV % HB (g/dl) Hct % MCV () MCH (pg) MCHC (g/dl) WBC (x 10 /l)
9

Heterophils % Lymphocytes % Monocytes % Eosinophils % Basophils %

The evaluation of the haematological parameters shows a big variety of value ranges especially of RBC, WBC, MCV, heterophils, lymphocytes and eosinophils. Major differences between our study and WERNERY et al. (2004) can be observed in hybrid falcons. These ndings may caused by the hand-count method on the other hand and the use of the Cell Dyn 3500 analyzer (Abbott Laboratory, North Chicago IL) (WERNERY et al. 2004) which does not produce accurate results as soon as it is used for processing a large number of falcon blood samples per day on one hand. The relatively high ranges in the haematological parameters produced by the Cell Dyn 3500 analyzer were contradicting the clinically healthy picture of the falcons tested. Haematology results as one important piece of the disease picture should never be interpreted alone but always in connection with the clinical disease symptoms of the examined falcons.

5. CITATION INDEX
1. DTLINGER HS and BIRD DM. Haematological parameters in captive peregrine falcons (Falco Peregrinus). Falco Newsletter No 4, 1995. The Middle East Falcon Research Group, National Avian Research center, United Arab Emirates. SAMOUR JH, DALOIA M-A and HOWLETT JC: Normal haematology of captive saker falcons (Falco cherrug). Com Haem Inter 1996; 6: 50 - 52.

2.

83

3. 4. 5. 6. 7.

JENNINGS I.B. Haematology. In: BEYNON P.H., FORBES N.A. and HARCOURT-BROWN N.H. (ed): Manual of raptors, pigeons and waterfowl. Gloustershire: BSAVA 1996; 68 - 78. WERNERY R., WERNERY U., KINNE J and SAMOUR J: Colour atlas of falcon medicine. Hannover: Schluetersche Verlagsgesellschaft; 2004: 18. SAMOUR J.H., BAILEY T.A., HOWLETT J.C., et al. Handbook of bustard haematology. National Avian Research Center 1996. CAMPBELL T.W. Avian hematology. In: CAMPBELL T.W. Avian hematology and cytology. Ames, Iowa: Iowa State Press 1988; 3-17. FUDGE A.M. Avian complete blood count. In: FUDGE A.M. Laboratory medicine: avian and exotic pets. Philadelphia: W.B. Saunders, 2000: 9-18.

AUTHORS ADDRESS
M. G. Muller Dr. med. vet. MRCVS Abu Dhabi Falcon Hospital/Environmental Research and Wildlife Development Agency (ERWDA) P.O.Box 45553, Abu Dhabi, United Arab Emirates Email: mmuller@erwda.gov.ae

84

Falcon Specialist Hospital and Research Center, Fahad bin Sultan Falcon Center, Riyadh, Kingdom of Saudi Arabia

CAUSES OF MORBIDITY AND MORTALITY IN CAPTIVE FALCONS IN SAUDI ARABIA

J. Samour, MVZ, PhD, Dip ECAMS, J. L. Naldo DVM

KEYWORDS
Morbidity Mortality - Saudi Arabia Newcastle - Infectious diseases - Lead toxicosis Parasitic - Viral

ABSTRACT
The medical records from 12523 falcons from different species presented for examination to the Falcon Specialist Hospital and Research Institute of the Fahad bin Sultan Falcon Center, Riyadh, Kingdom of Saudi Arabia from 1st September 1998 to 31st December 2004 were reviewed to determine the causes of morbidity and mortality amongst falcons used in the sport of falconry in Saudi Arabia. This report intends to contribute to the scanty information on morbidity and mortality in captive falcons in the Kingdom of Saudi Arabia and the Middle East as a whole.

1 INTRODUCTION
Falcons and falconry have formed an integral part of Arab desert life for thousands of years. In the past, Bedouins would trap, train, and hunt with passage falcons during the winter months in order to supplement their basic diet. In early spring, they would release the falcons, as caring for birds throughout the summer could strain their already limited resources. Nowadays, Arab falconers keep their falcons in air-conditioned rooms or in free-ying aviaries during the moulting months so that they can be used again for the next season. The need to maintain falcons in captivity prompted the establishment of falcon hospitals in the different countries of the Arabian Peninsula to cater for their medical needs. Although falcon hospitals have existed in the Middle East for over 25 years, very little information is available about falcon morbidity and mortality (AL TIMMIMI 1987, REMPLE 1988, RIDDLE and HOOLIHAN 1993, REMPLE and GROSS 1993, SAMOUR 1996). Knowledge of the causes of morbidity and mortality of falcons in the Middle East is essential

85

to understand diseases, improve therapeutic regimes and to establish suitable preventative medicine programmes. The main objective of this report is to provide an account of the causes of morbidity and mortality in falcons in the Kingdom of Saudi Arabia (KSA). It is our desire that the information presented in this report could contribute to provide the basis for future veterinary research.

2 MATERIALS AND METHODS

The medical records from 12523 falcons from different species presented for examination to the Falcon Specialist Hospital and Research Institute of the Fahad bin Sultan Falcon Center (FSFC), Riyadh, Kingdom of Saudi Arabia from 1st September 1998 to 31st December 2004 were reviewed. The falcon species included 9722 (77.6%) saker falcons (Falco cherrug), 1547 (12.3%) peregrine falcons (Falco peregrinus), 576 (4.6%) hybrid falcons, 394 (3.1%) gyr falcons (Falco rusticolus), 267 (2.1%) lanner falcons (Falco biarmicus) and 17 (0.1%) Barbary falcons (Falco pelegrinoides). Most falcons presented to the Hospital belonged to Saudi falconers, but an unknown number of falcons originated from Kuwait, Qatar, Bahrain and the United Arab Emirates (UAE). The post-mortem records from 471 falcons that died in the hospital during this period were also reviewed. The falcon examined post-mortem included 329 (69.8%) saker, 57 (12.1%) peregrine, 37 (7.8%) gyr, 31 (6.6%) hybrids and 17 (3.6%) lanner falcons. All falcons presented were subjected to a complete physical examination. Selective diagnostic procedures included faecal testing for endoparasites, haematology analyses, plasma chemistry and blood lead level analyses, bacterial and fungal culture and sensitivity testing and cytology, endoscopy and radiography examinations. Falcons examined by endoscopy and radiography were anesthetised with isourane administered via a face mask. In general, whole body survey radiographs were obtained in both ventrodorsal and laterolateral positions. All the above analyses were carried out in our own laboratory. A comprehensive post-mortem examination was carried out in all deceased falcons within 24 hours of death. Swabs from affected organs were collected for microbiology examination and examined in our own laboratory. Selective tissues were collected and preserved in 10% buffered formalin for routine histopathology examination. In selected cases tissue samples, including brain, liver, spleen and kidneys, were also collected and stored at -20C for virus isolation. Histopathology and virus isolation analyses were carried out at the Central Veterinary Research Laboratory, Dubai, UAE.

86

3 RESULTS
The total number of falcons presented for examination is shown in Table 1 and a breakdown of the total number of falcons per species is shown in Table 2. The classication of the causes of morbidity was based on the primary medical condition identied on clinical examination (Table 3). The number of falcons deceased at the Hospital is shown in Table 4. The causes of mortality were based on the primary pathologic lesion found at post-mortem examination (Table 5).

4 DISCUSSION
It is beyond the scope of this paper to discuss in detail the different causes of morbidity and mortality in falcons during the study period. Nevertheless, the most common causes of morbidity were infectious diseases, notably Newcastle disease, followed by traumatic injuries, toxicosis and metabolic or nutritional diseases (Table 3). The most common cause of mortality was Newcastle disease followed by septicaemia, aspergillosis, amyloidosis/hepatitis, visceral gout, severe trauma and sour crop (Table 5). The incidence of some diseases varied during the study period due to improved health awareness and improved diagnostic techniques. For instance, the number of lead toxicosis cases diagnosed at the Hospital decreased signicantly over the six year period due to public awareness campaigns and a better understanding of the toxic effects of lead. In a preliminary study for example, 96 cases of lead toxicosis were recorded over a period of 2.5 years (NALDO and SAMOUR 2004). In contrast only four cases of lead toxicosis were recorded in 2004. In addition, only one case of Newcastle disease (ND) was diagnosed at post-mortem in 2001. This number increased to 14 in 2002, to 44 in 2003 and 42 in 2004. This was the result of increased awareness of the two clinical presentations of Newcastle diseases in falcons, the neurotropic and viscerotropic forms (SAMOUR and NALDO 2004) and routine virus isolation analysis from every suspected case of ND. It is hoped that the information presented in this paper could serve as a base for other similar studies in the region. Knowledge of the most common diseases affecting falcons in the region could provide a better understanding of the diseases and effective ways to prevent them. In addition, every effort should be made to conrm the diagnosis by using either in-house laboratory facilities or commercial laboratories. The need to keep and maintain a medical record database cannot be over-emphasised.

87

Table 1. Total number of falcons presented for examination at the Fahad bin Sultan Falcon Center from 1st September 1998 to 31st December 2004 Period 1998 1999 2000 2001 2002 2003 2004 TOTAL Total no. of falcons 314 912 1834 2340 2101 2346 2676 12523 No. of new falcons 314 863 1684 2046 1675 1655 1656 9893 Total no. of visits 420 1352 2880 4321 4113 4483 5957 23526

Table 2. Number of falcons per species presented for examination at the Fahad bin Sultan Falcon Center from 1st September 1998 to 31st December 2004 Species Saker Peregrine Gyr Hybrid Lanner Barbary
a

1998 282 (89%) a 27 (8.6%) 2 (0.6%) 1 (0.3%) 2 (0.6%) 0

1999 740 (81%) 132 (14.5%) 9 (1%) 14 (1.5%) 17 (1.9%) 0

2000 1505 (82%) 237 (13%) 29 (1.6%) 32 (1.7%) 30 (1.6%) 1 (0.05%)

2001 1886 (81%) 291 (12.4%) 58 (2.5%) 60 (2.6%) 45 (1.9%) 0 2340

2002 1599 (76%) 242 (11.5%) 79 (3.8%) 119 (5.7%) 60 (2.9%) 2 (0.1%) 2101

2003 1743 (74.3%) 270 (11.5%) 104 (4.4%) 163 (6.9%) 58 (2.5%) 8 (0.3%) 2346

2004 1967 (73.5%) 348 (13%) 113 (4.2%) 187 (7%) 55 (2.0%) 6 (0.2%) 2676

314 912 1834 TOTAL Percentage of the total number of birds.

88

Table 3. Causes of morbidity diagnosed in falcons at the Fahad bin Sultan Falcon Center from 1st September 1998 to 31st December 2004 Categories Infectious diseases Parasitica Bacterialb Viralc Fungald Undetermined respiratory diseasee .Undetermined hepatophaty Traumatic injuries Bumblefoot Collision-type injuriesf Gunshot injuries Eye and eyelid abnormality Bursitis Miscellaneous traumag Toxicosis Lead Ammonium chloride Pharmacological toxicosis Metabolic or nutritional diseases Low condition Gout Sour crop GIT obstruction or impaction Uropygial gland abnormality Moulting abnormality Metabolic bone disease Hernia Pancreatic abnormality Degenerative disease Amyloidosis Neoplastic disease Lipoma
a b

No of cases 5,151 285 380 398 901 265 355 183 15 293 7 234 207 49 15 68 25 31 59 17 102 10 5 7 15 3

Endoparasites, trichomoniasis, haematozoa E. Coli. P. aeruginosa, C. perfringes, Proteus sp., Salmonella sp., Klebsiella sp., Shigella sp. c Newcastle disease, avian pox, herpesvirus hepatitis, avian inuenza d Aspergillosis, candidiasis e Rhinitis, rhinoliths, air sacculitis, pneumonia f Avulsion, dislocation, fractures g Ring constriction, burns, distal digital necrosis, detached claws, lacerations, abrasion

89

Table 4. Number of falcons deceased per species at the Fahad bin Sultan Falcon Center from 1st January 1999 to 31st December 2004
Species Saker Peregrine Gyr Hybrida Lanner Total
a

1999 36 3 1 2 42

% 85.7 7.1 2.3 4.8 -

2000 52 11 2 4 69

% 75.4 15.9 2.9 5.8 -

2001 34 2 3 3 2 44

% 72.3 4.5 6.8 6.8 4.5 -

2002 69 19 6 4 4 102

% 67.6 18.6 5.9 3.9 3.9 -

2003 85 12 14 10 2 123

% 69.1 9.7 11.4 8.1 1.6 -

2004 53 10 11 14 3 91

% 58.2 11.0 12.0 15.4 3.3 -

Primarily gyr-sakers and gyr-peregrines.

Table 5. Causes of mortality diagnosed in falcons at the Fahad bin Sultan Falcon Center from 1st January 1999 to 31st December 2004
1999 Causes Newcastle disease Aspergillosis Severe trauma Amyloidosis /hepatitis Visceral gout Septicaemiaa Bumblefoot (severe) Lead toxicosis Sour crop Other infectious diseasesb Miscellaneous diseasesc Total
a b

2000
No 3 6 6 1 8 24 3 4 3 6 5 69 % 4.4 8.7 8.7 1.5 11.6 34.8 4.4 5.8 4.4 8.7 7.2 -

2001
No 1 8 2 2 3 11 2 1 6 8 44 % 2.3 18.2 4.6 4.6 6.8 25.0 4.6 2.3 13.6 18.2 -

2002
No 14 10 7 8 9 13 5 5 5 13 13 102 % 13.7 9.8 6.9 7.9 8.8 12.7 4.9 4.9 4.9 12.7 12.7 -

2003
No 44 12 4 10 10 10 3 4 3 16 7 123 % 35.8 9.8 3.3 8.1 8.1 8.1 2.4 3.3 2.4 13.0 5.7 -

2004
No 42 9 4 9 6 7 3 1 2 3 5 91 % 47.2 9.9 4.4 9.9 6.6 7.7 3.3 1.1 2.2 3.3 4.4 -

No 1 5 4 2 14 1 2 13 42

% 2.4 11.9 9.5 4.8 33.3 2.4 4.8 31 -

Causes of septicaemia included Escherichia coli, Clostridium perfringens and Salmonella species. Other infectious diseases included herpesvirus hepatitis, avian inuenza, falcon pox, trichomoniasis, pseudomoniasis, candidiasis, tuberculosis. c Miscellaneous diseases included lower GIT impaction, pharmacological toxicosis, ammonium chloride toxicosis, scorpion sting, inhalation pneumonia, pancreatic atrophy, low body condition.

90

5 ACKNOWLEDGEMENTS
The authors would like to thank HRH Prince Fahad bin Sultan bin Abdulaziz Al Saud for continuing support to the clinical and research programme of the Hospital and to Dr Niel Nuguit, Wilson Joseph, Mohammed Shiyan, Mohammed Foaud, Salem Al Yami and Merle Apo for help with the practice statistics.

6 CITATION INDEX
1. 2. AL TIMIMI FA. Falcons and Falconry in Qatar. Doha: Ali bin Ali Printing Press; 1987. REMPLE JD. An overview of Arab falconry, its medical lore and the introduction of avian medicine in the Arabian Gulf. In: CADE TJ, ENDERSON JH, THELANDER CG and WHITE CM, (eds): Peregrine Falcon Populations: Their Management and Recovery. Boise: The Peregrine Fund Inc; 1988; 825 - 830. RIDDLE KE and HOOLIHAN J. A research hospital for falcons: design, operation and admissions summary. In: REDIG PT, COOPER JE, REMPLE JD and HUNTER DB (eds): Raptor Biomedicine. Minneapolis: University of Minnesota Press; 1993; 188 - 193. REMPLE JD and GROSS C. Falconry and Birds of Prey in the Gulf. Dubai, United Arab Emirates: Motivate Publishing; 1993. SAMOUR JH. Veterinary medicine, falcons and falconry in the Middle East. Proc Ass Avian Vet, Orlando 1996: 233 - 239. NALDO JL and SAMOUR JH. Causes of morbidity and mortality in falcons in Saudi Arabia. J Avian Med Surg 2004; 18(4): 229 - 241. SAMOUR JH and NALDO JL. Newcastle disease in falcons in Saudi Arabia. Proc Assoc Avian Vet, New Orleans 2004; 131 - 133.

3.

4. 5. 6. 7.

AUTHOR ADDRESS
Jaime Samour, MVZ, PhD, Dip ECAMS Fahad bin Sultan Falcon Center P.O. 55, Riyadh 11322 Kingdom of Saudi Arabia Email: falcon@shabakah.com

91

Dubai Falcon Hospital1, Dubai, Project for Falcon Conservation2 (Profalcon), Al Ain, United Arab Emirates

HISTOPLASMA SP. - INCIDENTAL FINDING, UNDERDIAGNOSED, OR OCCASIONAL OPPORTUNISTIC PATHOGEN OF FALCONS IN THE MIDDLE EAST ?
T. Bailey1, C. Silvanose1, E. Pesci2, A. Di Somma1

KEYWORDS
Falcon - Fungal infection - Histoplasmosis - Aspergillosis - Histoplasma sp Voriconazole - F10

ABSTRACT
Histoplasmosis is an infectious, but not contagious mycotic disease that has been occasionally reported in non-domestic birds, pigeons and poultry. We reviewed the clinical ndings, diagnosis, treatment and outcome of nine falcons with lower respiratory tract disease from which H. capsulatum was isolated alone or as part of a mixed infection with Aspergillus sp. In order to determine the prevalence of Histoplasma sp. in isolates from airsac biopsies, mycology records from two falcon hospitals were analysed. Histoplasma sp. was isolated 6 times (prevalence of 5.1%) from 116 fungus positive airsac biopsies that were collected over a one year period at the Abu Dhabi Falcon Hospital. Histoplasma sp. was isolated once (prevalence of 1.2%) from 86 fungus positive airsac biopsies that were collected over a one year period at the Dubai Falcon Hospital. Our observations demonstrate that Histoplasma sp. is a rare isolate from falcons with lower respiratory tract disease, although its signicance remains unclear.

1 INTRODUCTION
Histoplasmosis is an infectious, but not contagious mycotic disease of animals that has been reported in a wide variety of wild and captive mammals and occasionally in nondomestic birds, pigeons and poultry (BAUCK 1994; VAUGHN 1996; BUREK 2001). The causative agent of histoplasmosis is a dimorphic fungi, Histoplasma capsulatum, which occurs naturally in soil in warm, humid areas of the world (BUREK 2001). The

92

organism lives as a soil saprophyte, particularly where animal faeces accumulate (WALSH et al. 1995). Infection of the respiratory tract occurs when dust containing spores are inhaled. Clinically apparent histoplasmosis can be a localised, benign, pulmonary disease, or a disseminated, progressive and potentially fatal disease. In mammals the disease is characterised by primary and secondary foci of infection becoming established in the lungs, followed by lymphohaematogenous dissemination via the monocyte-macrophage system. Our retrospective study reviews the clinical ndings, diagnosis, treatment and outcome of nine falcons with lower respiratory tract disease from which H. capsulatum was isolated alone or as part of a mixed infection with Aspergillus sp.

2 MATERIALS AND METHODS 2.1 Clinical history and investigations

Histoplasma sp. was isolated from the lower respiratory tract of eight falcons seen by the authors at the Abu Dhabi Falcon Hospital [ADFH] (2000-2001) and the Dubai Falcon Hospital [DFH] (2002-2003). A further bird with airsacculitis and cytological ndings consistent with Histoplasma sp. was included in the study. The birds were females; 7 birds were juveniles and 2 were adults. Six affected falcons were presented with a history of; loss of weight (2), inappetance (1), vomiting (1), poor exercise performance (3) and dyspnoea (1). Two birds were admitted for pre-purchase examinations and no clinical history was available. One bird was admitted for a premoult health check and no clinical signs had been noted by the owner. The birds were given a clinical examination that included: 1) physical examination, 2) faecal and crop parasitology, 3) haematology, 4) plasma biochemistry, 5) survey whole body radiographs and 6) endoscopic examination of the lower respiratory tract. During endoscopy biopsy samples were collected from affected areas of the respiratory system of each individual. The samples were obtained directly with biopsy for culture and cytology. Impression smears on slides were air dried, xed and stained with Neat stain (Astal Diagnostics, USA).

2.2 Bacteriology and mycology

Biopsy samples were cultured in sheep blood agar, MacConkeys agar (MCA), for bacteriology and Sabourauds agar (SA) for mycology. Blood agar plates were incubated at 37C in a 5% CO2 incubator for 48 hrs and MCA plates were incubated aerobically at 37C for 48 hrs. The SA plates were incubated at 37C for 48 hrs and further 8 to 14 days at room temperature (25 C). The fungi were identied by culture appearance and morphological characteristic under microscope with the help of lactophenol aniline stain (QUINN et al. 2002). In order to determine the prevalence of Histoplasma sp. in isolates from airsac biopsies, mycology records over a one year period from January 2000 to January 2001 at the ADFH and from June 2003 to June 2004 from the DFH were analysed.

93

2.3 Treatments, clinical outcome and follow-up

The birds were seen over a four year period at two hospitals by 3 veterinarians. The different treatment regimens are summarised in Table 1. Parasitic infections (coccidia, trematodes and Serratospiculum sp.) were treated. Treatment ended and birds were discharged from the hospital according to the following criteria: 1) resolution of the presenting signs, 2) weight gain, 3) marked improvement of the lower respiratory tract lesions during endoscopy, 4) negative culture or cytology of endoscopic biopsies, and 5) haematology within normal ranges. After discharge, follow-up was possible at 3 and 12 months in one bird and at 5 months in another. Follow-up procedures included the examination previously described.

3 RESULTS 3.1 Clinical investigations

Physical examination ndings included poor pectoral muscle condition (5), low pectoral muscle tone (4) and dehydration (2) in 5 birds. No abnormalities were noted in 4 birds. Other underlying diseases diagnosed included: coccidiosis (2), bumblefoot (1), serratospiculiasis (1) and trematodes (1). Radiographic abnormalities were present in the two of the three DFH birds. The radiological abnormalities included prominent bronchial pattern, asymmetry of the air sacs because of air sac consolidation or hyperination, visible caudal airsac line, focal densities within the lungs or the air sacs and splenomegaly. In one of these 3 cases that was cytology positive for Histoplasma sp. only, and from which Aspergillus sp. was not isolated, radiological ndings included air sac consolidation and splenomegaly. In all 9 cases endoscopy revealed a generalized air sacculitis including; thickened, opaque and vascularised air sacs (6), diffuse cheesy plaques (6), small (<5mm) multifocal white to yellow granulomas (3), extensive, large disseminated granulomas (>5mm) (1), hepatic inammation (1), hepatomegaly (1) and pulmonary congestion (1). In the 3 cases that were cytology and/or culture positive for Histoplasma sp. only, and from which Aspergillus sp. was not isolated, endoscopy ndings included opaque and vascularised air sacs (2), diffuse cheesy plaques (1) and small (<5mm) multifocal granulomas (1).

3.2 Laboratory investigations

Haematology results from 8 of 9 birds showed from mild to severe leukocytosis (6.7-22.7 x109/l) with mild to marked heterophilia. Haematology parameters were within normal ranges in one bird. Cytological ndings included; inammatory cells including heterophils, macrophages, multinucleated giant cells (6), the presence of fungal spores (coniodospores, bulb like structures) and /or septate hyaline hyphae consistent with Aspergillus sp. infection (4), an inammatory response in the air sacs lining cells including vacuolation, giant cell formation and metaplasia (4) and the

94

presence of multinucleated giant cells containing globular structures, up to 4m in size, consisting of a central spherical basophilic body surrounded by an unstained halo, morphologically consistent with Histoplasma organisms (3). Histoplasma sp. was isolated from airsac biopsy specimens from 8 of 9 cases. After 10 days of incubation, fungal colonies appeared white to cream in colour with cottony ariel hyphae. Microscopically the hyphae are delicate, averaging 1-2m in diameter with small lollipop-like microconidia. Histoplasma sp. was the only organism isolated in 2 birds. In one bird organisms consistent with Histoplasma were seen in cytology preparations, but bacterial and fungal cultures were negative. In 6 birds mixed cultures of Histoplasma sp. and Aspergillus sp. were isolated. The strains of Aspergillus included A. fumigatus (1), A. nidulans (2), A. avus (1) and A. niger (1). In one bird the strain of Aspergillus was not determined. No bacteria were isolated from biopsy specimens. Histoplasma sp. was isolated 6 times (prevalence of 5.1%) from 116 fungus positive airsac biopsies that were collected from falcons over a one year period from 1/1/00 to 1/1/01 at ADFH. Other fungi isolated at ADFH comprised; A. fumigatus (45), A. avus (45), A. niger (13), Peciliomyces sp. (3), Scediospium sp. (2), A. nidulans (1) and A. versicolor (1). Histoplasma sp. was isolated once (prevalence of 1.2%) from 86 fungus positive airsac biopsies that were collected from falcons over a one year period from 1/6/03 to 1/6/04 at DFH. Other fungi isolated at DFH comprised; A. fumigatus (40), A. avus (14), A. terreus (12), A. niger (9), Aspergillus sp. (7), A. nidulans (1), Candida albicans (1) and Mucor sp. (1).

3.3 Treatments and follow-up

A summary of the treatment regimens, clinical response and follow-up are presented in Table 1. Two cases from which Histoplasma sp. and Aspergillus sp. were isolated died 7 and 36 days after hospitalisation. Unfortunately the owners would not allow post-mortem. A further case was improving clinically, but was removed from the hospital against medical advice after 10 days treatment by an owner who wished to train his bird and go hunting. Another case treated as an outpatient was not seen again. Five hospitalised cases were discharged after 21-70 days (median 36 days) treatment. In these ve birds: 1) the presenting clinical signs had resolved, 2) the birds had gained weight (7-13% from time of presentation), 3) endoscopic examinations revealed marked improvement of the surface of air sacs and lung, 4) no bacteria and fungi were cultured from biopsies, 5) cytology biopsies were negative and 6) haematology results were within normal ranges. Two of the four birds discharged after what was considered to be a full course of treatment were both presented for follow-up examination at 3 and 5 months after rst diagnosis. Both birds had a complete examination, including endoscopy and were considered fully clinically recovered. One of these birds was examined 12 months later, no abnormalities were detected and the falconer reported that the bird was ying well.

95

4 DISCUSSION
In the majority of cases (66%) described in this report Histoplasma sp. and Aspergillus sp. were isolated together and it is not possible to relate pathology to either organism. Only in two cases was Histoplasma sp. isolated as the sole organism from airsac biopsies and both birds showed signs of airsacculitis. However, in these cases the failure to culture Aspergillus sp. does not conclusively rule out concurrent infection with aspergillosis. It is not uncommon to fail to culture Aspergillus sp. from biopsies collected from cases with endoscopically apparent lower respiratory tract disease, and where cytology of biopsies is consistent with a fungal infection. It is known that some strains of Aspergillus (e.g. A. fumigatus) produce toxins (e.g. gliotoxins) that are highly immunosuppressive (WERNERY et al. 2004). It is possible that a primary aspergillosis infection could predispose falcons to infection with other pathogens, such as Histoplasma sp. Another interesting nding is the wide range of fungi that are isolated from falcons with lower respiratory tract disease (Table 2). Some isolates (e.g. A. terreus) are recognized pathogens in humans and animals. Other isolates (e.g. Scediospium sp.) are considered emerging pathogens in human medicine or as atypical causes of infection in humans and domestic animals (e.g. A. nidulan, Mucor sp.). Studies to assess if these less commonly isolated fungi are clinically relevant or are simply contaminants are warranted. In humans it is estimated that more than 95% of cases of histoplasmosis are self-limiting and the small number of infection episodes that advance to overt disseminated disease are associated with predisposing risk factors, particularly defects in cell-mediated immunity (WALSH et al. 1995). In wild animals, additional immunosuppressive factors including the stress of capture, transportation, connement and altered habitat are thought to trigger overt histoplasmosis (BUREK 2001). Fungal infections in falcons are similarly thought to occur secondary to an immunosuppressive event. In the Middle East, aspergillosis typically occurs in captive bred birds shortly after they arrive from Europe and North America and are exposed to high temperatures, high humidity, new environments and falconry training methods. The various stresses associated with these changes contribute to predisposing birds to systemic fungal infections. The Histoplasma organism thrives in soil mixed with faeces and especially hen houses and bird/bat roost. In humans, outbreaks of histoplasmosis are seen in people who have demolished buildings infested with bird guano. The frequent nding of Histoplasma organisms in association with bird droppings is considered to be due to environmental enrichment by the bird faeces rather than propagation and transmission by birds (BUREK 2001). Decreasing exposure to substrates enriched with bat or bird droppings is important in minimising infection with Histoplasma sp. Spraying infected soil and faecal deposits with 3% formalin destroys some spores. Histoplasmosis has zoonotic potential and in humans may cause pneumonitis that progress to a disseminated disease of the reticuloendothelial system (BAUCK 1994). Clients should be warned of the risks of the disease, because the environment where the bird was exposed may be a risk to humans. The cases reported in this study could have been linked to poor hygienic conditions in the environment where the birds were housed.

96

Cryptococcus has been more widely reported as a cause of disease in psittacines and pigeons and BAUCK (1994) reports that histoplasmosis is similar to cryptococcosis in many ways, but has been less commonly reported in birds. Some authors consider that histoplasmosis is not a clinical problem in birds because the body temperature of birds is too high to support the growth of this mycotic organism (CARPENTER and GENTZ 1997). While our observations are far from comprehensive, with no post-mortem/histopathology examinations conducted on the dead birds, they do demonstrate that Histoplasma sp is a rare isolate from falcons with lower respiratory tract disease. It is possible that this condition is underdiagnosed because in routine SA cultures it is easily overgrown by other fungi such as Aspergillus sp. and because it is slow growing, requiring 2-4 weeks of incubation (QUINN et al. 2002). Aspergillus sp. cultures usually grow in less than 5 days at 37C and it is routine to discard SA plates after 4-5 days incubation. The use of deep-lled culture plates or enriched media (brain heart infusion) may improve the chances of culturing this organism. Table 2 presents minimum inhibitory concentrations (MIC) taken from the literature of two commonly used antifungal agents against H. capsulatum and for comparative purposes against A. fumigatus (PRESCOTT 2000). The MIC of antifungal agents against H. capsulatum are 4-5 times lower than the equivalent MICs for A. fumigatus, suggesting an increased susceptibility of Histoplasma to these drugs. In our study a positive clinical response was seen in cases given itraconazole, itraconazole and F10, itraconazole and amphotericin B and voriconazole and amphotericin B. Itraconazole has been successfully used to treat cats with histoplasmosis (HODGES et al. 1994). Recurrences occurred in the cats after completion of therapy requiring a further 2-3 months of treatment. Antifungal regimens in humans are often extended for 6-12 months and in veterinary species treatment duration recommendations vary from 2 to 6 months and one month past the resolution of the disease. Unfortunately, client compliance in outpatients with extended antifungal therapy is difcult in the Middle East and extended therapies, while desirable, may not always be realistically achievable. At the current time we must leave unanswered the title of our talk, the question of whether Histoplasma sp. is an incidental nding, underdiagnosed, or just an occasional opportunistic pathogen of falcons in the Middle East? Further investigations including histopathological and immmunohistopathology diagnosis from conrmed cases are needed to conclusively demonstrate the clinical signicance of this interesting fungus that has been isolated from falcons.

5 ACKNOWLEDGEMENTS
We thank H.H. Sh. Hamdan bin Rashid al Maktoum and Mr Humaid Obaid al Muhairi, Dubai Falcon Hospital Director, for their support of the work of Dubai Falcon Hospital and all of the falcon hospital team for their assistance with cases. We thank H.E. M. Al Bowardi for his support of the veterinary science programme of Abu Dhabi Falcon Hospital.

97

6 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. 9. BAUCK L. Mycoses. In: RITCHIE BW, HARRISON GJ, HARRISON LR. (eds): Avian Medicine: Principles and Application. Lake Worth, Florida: Wingers Publishing Inc 1994; 997 - 1006. BUREK K. Mycotic disease. In: WILLIAMS ES, BARKER IK. (eds): Infectious Diseases of Wild Mammals. London: Iowa State University Press 2001; 518 - 520. CARPENTER JW and GENTZ EJ. Zoonotic diseases of avian origin. In: ALTMAN RB et al. (eds): Avian Medicine and Surgery. Philadelphia: W.B. Saunders Co, 1997; 350 - 363. HODGES RD, LEGENDRE AM, ADAMS LG, et al. Itraconazole for the treatment of histoplasmosis in cats. J Vet Int Med 1994; 8: 409 - 413. PRESCOTT JF. Antifungal chemotherapy. In: PRESCOTT JF et al. (eds): Antimicrobial Therapy in Veterinary Medicine. Iowa: Iowa State University Press, 2000; 367 - 395. QUINN PJ, CARTER ME, MARKEY BK, CARTER GR. (eds): The dimorphic fungi. In: Clinical Veterinary Microbiology. London: Mosby International Ltd 2002; 402 - 408. VAUGHN S. What is your diagnosis? J Avian Med Surg 1996; 10: 37 - 39. WERNERY R, WERNERY U, KINNE J, SAMOUR J. Colour Atlas of Falcon Medicine. Hannover: Schlutersche, 2004; 134 WALSH TJ, MITCHELL TG, LARONE DH. Histoplasma, blastomyces, coccidioided, and other dimorphic fungi causing systemic mycoses. In: MURRAY PR (ed): Manual of Clinical Microbiology. Washington: ASM Press 1995; 749 - 764.

AUTHORS ADDRESS
Tom Bailey, BSc, BVSc, MRCVS, Cert Zoo Med, MSc (Wild Animal Health), PhD, Dip ECAMS Dubai Falcon Hospital PO Box 23919 Dubai, United Arab Emirates Email: tom.bailey@dfh.ae

98

Central Veterinary Research Laboratory1, Dubai, Dubai Falcon Hospital2, Dubai, United Arab Emirates

MYCOBACTERIOSIS IN HUNTING FALCONS IN THE MIDDLE EAST

U. Wernery1, DVM, PhD, F.A. Hassan1, MSc, T. Bailey2, BVSc, MSc, PhD, Dip ECAMS, CertZooMed, B. Johnson1, MSc, J. Kinne1, DVM

KEYWORDS
Falcon Mycobacteriosis Mycobacterium Culture PCR

ABSTRACT
One thousand two hundred and three falcons were tested for mycobacteriosis. The samples originated from 702 falcons necropsied at Central Veterinary Research Laboratory (CVRL), from 200 falcons necropsied outside CVRL, and 301 biopsies. In total 23 (1.9%) falcons were diagnosed with avian mycobacteriosis due to typical granulomas and positive Ziehl-Neelsen stains of histological slides. The organs mainly infected were liver followed by spleen and peritoneum. Due to the unavailability of PCR and culture facilities at the beginning of our investigations, only a small number of samples were tested using PCR and culture. PCR was able to detect DNA from nontuberculous mycobacteria from formalin xed, parafn embedded tissue samples in 2 out of 10 cases. PCR from strains cultured on Loewenstein/Jensen or Herrolds egg yolk slant medium identied strains as M. avium paratuberculosis (2x) and M. avium (2x). Eight falcons with mycobacteriosis were examined at Dubai Falcon Hospital. Clinical signs as well as results of radiographs, endoscopy and haematology are presented.

1 INTRODUCTION
Mycobacteriosis is a common bacterial disease among wild and particularly captive birds of prey (HEIDENREICH 1995) with a worldwide distribution. The disease has been called avian tuberculosis, but classical tuberculosis lesions are only one of the many manifestations of mycobacterial infection in birds. Therefore, mycobacteriosis is a more appropriate term for this disease (TELL et al. 2001). Several mycobacterial species can cause the disease in birds, principally Mycobacterium (M.) avium, M. intracellulare and M. genavense. As M. avium and M. intracellulare share some common antigens, these species are often grouped and termed as M. avium-

99

intracellulare complex (MAI) (GRANGE et al. 1990). Sometimes M. avium subsp. paratuberculosis and M. lepraemurium which rarely cause avian mycobacteriosis are also included in the MAI complex because of their close relationship to M. avium. Other mycobacterial species, belonging to the M. tuberculosis complex (MTC, LACHNIK et al. 2002), may also cause lesions in birds including M. tuberculosis and M. bovis (VAN DER HYDEN 1997). The disease becomes apparent in a variety of clinical forms, and the bacteria remain infectious in the environment for many months. This paper describes the diagnosis of mycobacteriosis in hunting falcons from the Middle East.

2 MATERIAL AND METHODS

A clinical and laboratory study was performed on falcon mycobacteriosis at Dubai Falcon Hospital (DFH) and Central Veterinary Research Laboratory (CVRL). Over a period of 9 years (1995 2004) 702 falcons were necropsied and tuberculosis suspicious organ samples stained according to Ziehl-Neelsen (ZN). Furthermore, organ samples from 200 falcons necropsied outside of CVRL in different countries of the Middle East and 301 biopsies collected from sick falcons were also included in this study, totalling 1,203 falcons. Clinical investigations were performed on 8 falcons, suspicious for mycobacteriosis including physical examination, endoscopy and haematology before being euthanased due to poor prognosis. Besides histological investigation, culture was performed on 4 cases using LowensteinJensen (LJ) medium slants (BBL, BD221387) and Herrolds egg yolk (HEY) slants with mycobactin J (BBL, BD222233) after decontamination and concentration of the samples according to TELL et al (2003). The suspension was centrifuged for 20 minutes at 1500 rpm, the supernatant discarded and the sediment used as inoculum. The LJ and HEY slants were incubated at 37oC for 6 8 weeks and inspected weekly for any growth. For DNA extraction 1 suspicious colony was picked from the LJ or HEY agar and suspended in 1 ml of sterile distilled water. This suspension was boiled for 30 min. Extracted DNA from suspected colonies and from fresh tissue was processed using High Pure PCR Template Preparation Kit (ROCH 1796828) according to the manufacturers recommendation. Furthermore, DNA was extracted from formalin xed, parafn embedded tissue samples (airsac, liver, spleen, intestines) of 10 falcons according to CHRISTOPHE et al. (2000). For PCR amplication three pairs of primers were used according to VITALE et al. (1998). PCR was performed on 10 ZN-positive falcons using formalin samples, as well as on fresh tissue samples from 4 out of these 10 falcons. Furthermore, 4 strains cultured on LJ agar were analysed by PCR.

100

3 RESULTS
Twenty-three cases (1.9%) of falcon mycobacteriosis from a total of 1,203 birds were diagnosed by histology from 1995 to 2004 at CVRL. Acid fast rods were found in 8 of 702 necropsies, in 10 of 200 organ samples received from outside, as well as in 5 of 301 biopsies through ZN staining of histology slides. A steady increase of mycobacteriosis in hunting falcons was noticed with a single case in 1995, ve cases in 2000, eight cases in 2003, and six cases in 2004. The species mostly involved were saker falcons (9), gyr x saker hybrids (4) followed by gyr falcons (3), gyr hybrids (3), and peregrines (3). Gross pathology of carcasses and organs of all 23 tuberculosis cases revealed yellow-whitish caseous nodules 1 to 4 mm in diameter in the liver (17), spleen (14), peritoneum (7), skin (5), lung (4) and kidney (2). In airsac and heart, tuberculous lesions were only seen once. The granulomas in peritoneum, airsac, heart and skin were diffuse. Acid-fast rods were observed by histopathological examination in all macroscopic suspicious organs. Histopathology revealed central necrotic granulomas with numerous intralesional acid-fast rods surrounded by giant cells. No mineralisation of the lesions was seen. Suspicious skin biopsies showed chronic granulomatous-necrotizing dermatitis containing some acid-fast rods. In one of these cases the lesion was found in the conjunctival-lacrimal gland. Concurrent aspergillosis was diagnosed in 12 of 18 ZN-positive cases from necropsied falcons. In 9 of these 18 cases systemic amyloidosis was also found during histopathological investigation. Eight tuberculous suspicious falcons were examined at DFH. The following clinical signs were observed: Loss of weight (3), inappetance (2), vomiting (1), change in faecal consistency (2), biliverdinuria (2), blood in the faecal (1), depression (1), poor exercise performance (1) and dyspnoea (3). Physical examination ndings included poor pectoral muscle condition (4), the presence of palpable subcutaneous nodules in the neck/crop region (1), carpal bursitis (2), an enlarged hardened liver (1), ascites (1) and dehydration (2). Other underlying diseases diagnosed included: Trichomonosis (1), Caryospora sp. (3), Porrocaecum sp. (1), Chlamydophilosis (1), lead toxicosis (1), bumblefoot (1), serratospiculiasis (1), Syngamus sp. (1) and candidiasis (1). Haematology results from 5 birds showed moderate to severe leucocytosis (from 15.5 to 57.5 x 109/l) with marked heterophilia and moderate to severe monocytosis. All birds were anaemic with low packed cell volumes and haemoglobin values. Radiographs from four cases were taken. Radiographic abnormalities were present in all four investigated birds and included: multiple granulomas in the crop and neck (1), hepatomegaly (3), radiodensities within the alimentary tract (1), focal densities within the lungs or the air sacs (2), a lead pellet (1) and splenomegaly (1). Five cases were endoscoped. Endoscopy revealed hepatomegaly (2), pulmonary inammation (1), generalized airsacculitis including; thickened, opaque and vascularised air sacs (1), small (< 5 mm) multifocal white granulomas in the surface of abdominal organs and liver (3), pus oozing from the ostium (1), brinous adhesions (1) and splenomegaly (1).

101

Mycobacteria-suspicious colonies from 4 falcons (Table 1) grew 4 6 weeks after inoculation on LJ or HEY slants, which stained acid fast. Primary isolation of M. avium paratuberculosis was possible on HEY slant from one case. However, subsequent subcultures grew on LJ slants as well. The isolates from the other three cases (Table 1) grew on both agars. PCR did not detect mycobacterial DNA from fresh tissue samples in 4 ZN-positive falcons and identied only 2 of 10 formalin xed ZN-positive specimens belonging to the non-tuberculous complex (NTC). PCR identied 2 isolates from culture as M. avium paratuberculosis and 2 as M. avium (Table 1). Table 1. Results of culture and PCR from 10 ZN-positive falcons. Primary culture Species Peregrine Saker Peregrine Gyr hybrid Saker Gyr Saker Gyr x Saker Saker Peregrine Sample n.r. n.r. n.r. n.r. Spleen Intestine Liver n.r. Liver n.r. Airsac LJmedium Negative Positive Positive Positive HEYmedium Positive Positive Positive Positive Formalin fixed tissue NTC* Negative Negative Negative Negative Negative NTC* Negative Negative Negative PCR Fresh tissue n.r. n.r. n.r. n.r. Negative Negative n.r. Negative n.r. Negative Culture n.r. n.r. n.r. n.r. M. avium paratuberculosis M. avium n.r. M. avium n.r. M. avium paratuberculosis

* NTC = non-tuberculous complex nr = not received

4 DISCUSSION
From 1,203 falcons tested at CVRL since 1995, 23 (1.9%) cases of mycobacteriosis were diagnosed. The diagnosis was based on typical tuberculous granulomas, and the detection of acid-fast rods in histology sections using ZN staining. Mycobacterial strains grown on LJ and HEY agars were identied by PCR as M. avium (2x) and M. avium paratuberculosis (2x). Also, with PCR, mycobacteria were identied from a peregrine and a saker falcon from formalin xed and parafn embedded tissues as belonging to the non-tuberculous complex (NTC) which also includes M. avium, M.

102

intracellulare and M. avium paratuberculosis (LACHNIK et al. 2002, CHRISTOPHE et al. 2002). PCR has clearly identied the cultivated mycobacterial strains. It is not completely clear why PCR failed to identify mycobacterial DNA from 4 fresh and 8 formalin xed tissues. One explanation could be the sample size. The bigger parts of the small granulomas were used for histology and culture, leaving little material for PCR. At the beginning of our diagnostic procedure the PCR used was also limited to identifying only M. avium and M. avium paratuberculosis and not the tuberculous complex (MTC). Until a decade ago, most cases of mycobacteriosis in birds were caused by mycobacteria of the MAI complex. However, nowadays a great number of different mycobacteria species have been identied from other avian species using modern laboratory technologies. So far only M. avium has been isolated from raptors (TELL et al. 2001). M. avium paratuberculosis, the causative agent of Johnes disease has not been reported to occur in raptors, but in other avians (TELL et al. 2001). However, we isolated M. avium paratuberculosis from a saker falcon. Johnes disease is very common in small ruminants in the UAE. It is therefore believed that feeding contaminated mutton to falcons may cause infection. Mycobacteriosis is common in free-living birds of prey as well as in raptors from zoological collections and those used for falconry (LUMEIJ et al. 1980). According to various authors mycobacteriosis accounts for 1 to 30% of the cases examined at postmortem. The highest incidence is from zoological collections. Compared to FABIAN and VETESI (1980) who reported 10% Falconiformes infected with mycobacteria in the Budapest Zoo between 1971 and 1978, the incidence of 1.9% in the Middle East hunting falcons is low. However, an increasing number of cases were observed during the last years, culminating in 8 cases in 2003. The clinical signs (particularly weight loss, carpal bursitis, palpable subcutaneous nodules) described in this case report are typical for this disease in falcons (HEIDENREICH 1995). The haematology ndings (leucocytosis, monocytosis, anemia) in the clinical cases described in this paper are typical of mycobacteriosis (FUDGE and JOSEPH 2000), although aspergillosis and chlamydophilosis would need to be considered as possible differential diagnoses. HEIDENREICH (1995) states that the usual route of infection is oral and therefore the most common lesions in falcons are found in the gastrointestinal tract. However, other authors (TELL et al 2001) observed characteristic features in the liver spleen and intestines although any tissue may be affected. Our investigations clearly showed that the liver (17 x) was the prime organ, followed by spleen (14 x), peritoneum (7 x), skin (5 x) and lung (4 x). LUMEIJ et al. (1982) described tuberculous meningitis and encephalitis as well as knee joint involvement in raptors. Raptors seem to be more susceptible to mycobacterial bone infections than other avians. In buzzards (Buteo buteo) mycobacteria were isolated from bumble foot cases (TELL et al. 2001). It is worth noting that in our study mainly saker falcons (9 x) and saker hybrids (4 x) were infected with mycobacteria representing more than 50% of the cases. Three cases were seen in gyr falcons as well as in gyr hybrids and peregrines. In two cases, the species was not reported.

103

Due to the variation of the clinical appearance, avian mycobacteriosis is often not recognized on initial presentation and therefore remains challenging to diagnose in live birds. In the clinical cases described in this report other conditions, importantly chlamydophilosis and aspergillosis were diagnosed concurrently. The presence of other diseases can complicate the diagnosis of mycobacteriosis. Diagnosis is generally performed by ZN staining. To date, very little research has been conducted to develop appropriate tests for diagnosing mycobacteriosis in falcons. Although some diagnostic methods are considered more reliable than others, a direct comparison of diagnostic assays on a great number of samples is still lacking. In view of the zoonotic capacities of this infection and the close relationship between Arab falconers and their birds, therapy is not recommended by the authors. Other falcons that have been in close contact with affected birds should also be investigated and may need to be quarantined.

5 CITATION INDEX
CHRISTOPHE C, VANNUFFEL P, BLONDEEL N, et al. Duplex PCR for differential identication of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin-xed parafn-embedded tissues from cattle. J Clin Microbiol 2000; 8: 3048 2054. 2. FABIAN L and VETESI F. Analyse der Vogelverluste (1971 1978) im Zoo Budapest. Verh. Erkrankungen der Zootiere 1980; 22: 215 221. 3. HEIDENREICH M. Birds of prey. Medicine and management. Blackwell Science. 1995; 117 120. 4. GRANGE JM, YATES MD and BROUGHTON E. A review: the avian tubercle bacillus and its relatives J Appl Bacteriol 1990; 68: 411 431. 5. LACHNIK J, ACKERMANN B, BOHRSSEN A, et al. Rapid cycle PCR and uorimetry for detection of mycobacteria. J Clinical Microbiol 2002; 40: 3364 3373. 6. LUMEIJ JT, DORRESTEIN GM and STAM JWE. Recent advances in the study of raptor diseases. Observations in tuberculosis in raptors. Proc. int. symposium on diseases of birds of prey, 1st 3rd July, London, 1980. 7. TELL LA, WOODS L and CROMIE RL. Mycobacteriosis in birds. Rev sci tech off int Epiz 2001; 20: 180 203. 8. TELL LA, FOLEY J, NEEDHAM ML and WALKER RL. Diagnosis of Avian Mycobacteriosis: Comparison of culture, acid-fast stains, and PCR for the identication of Mycobacterium avium in experimentally inoculated Japanese quail (Coturnix coturnix japonica). Avian Dis 2003; 47: 444-452. 9. VAN DER HEYDEN N. Clinical manifestations of mycobacteriosis in pet birds. Semin. avian exot Pet Med 1997; 6: 18 24. 10. VITALE F, CAPRA J, MAXIA L, et al. Detection of Mycobacterium tuberculosis complex in cattle by PCR using milk, lymph node aspirates, and nasal swabs. J Clin Microbiol 1998; 36: 1050 1055. 1.

104

6 ACKNOWLEDGEMENTS
The authors are grateful to Dr. P. McKinney, Dr. E. Pesci, Dr. A. Jawad, Dr. J.D. Remple and Dr. J. Samour for sending the samples. Furthermore we thank Mr. Chellappan Vishwanathan for his excellent histological slides.

AUTHORS ADDRESS
U. Wernery Central Veterinary Research Laboratory, PO Box 597, Dubai, United Arab Emirates. Email: cvrl@cvrl.ae

105

Eco-Ouest, France

EPIDEMIOLOGY OF AVIUM BOTULISM IN FRANCE : EXAMPLE OF GRAND LIEU LAKE

Le Dran-Quenechdu S1 DMV, PhD, Marion L2 PhD, Popoff M3 DVM PhD

KEYWORDS
Botulism - Waterbirds - Epidemiology - Management ABSTRACT Botulism is dened as a nervous affection, characterized by asks paralysis and induced by the ingestion of the toxin of Clostridium botulinum. This bacterium has the faculty to produce extremely resistant spores in the environment: what are today the risks to see appearing such an outbreak? The authors present the epidemiological characteristics of the disease and the situation in the Loire Atlantique (France). They present the results of a study aiming at a better understanding and managing of the development of epizooty on a natural reserve.

1 INTRODUCTION
The botulism is a nervous disease; characterised by a limb paralysis; this disease is due to a neurotoxin, produced by Clostridium botulinum. In the case of Grand Lieu Lake, a major wetland in France, the epizooty were due to type C toxin, so called avian botulism. This disease is a typically multifactor disease: it appears when environmental conditions allow the toxin production. These conditions are mainly anaerobic conditions. The Grand Lieu Lake is a wetland of international importance, located near the Loire estuary in France. An epizooty in summer 1995 provoked the death of several thousands of birds among which, birds of patrimonial importance, such as spoonbill or great egret. In this paper, after a brief review on the disease and the local context, we present the results of a study aiming to understand the epidemiology of botulism in Grand Lieu Lake and to make management and prevention propositions.

2 THE DISEASE
The toxin of C. botulinum provokes the disease: the presence of both bacteria and favoured conditions for toxin production are needed to trigger of the disease.

106

2.1. The bacteria and its toxin C. botulinum is a soil bacterium, anaerobic bacterium that forms dormant spores, and toxin producing bacterium. Its classically divided into 7 toxinotypes, A B C D E F G, in relation with antigenic specicities of the toxin (WOBESER 1981, CATO et al. 1986). The resistance of the spore explains the persistence of the disease in a given environment. The neurotoxins inhibit the neurotransmitters transmission, on the neuromuscular junction: the muscular contractions are blocked. 2.2. The disease The disease is characterised by limb paralysis. Wild birds show ight difculties, particularly for taking ight and landing. The paralysis seems to begin on the legs, then the wings, the neck and the eyelids. Spasmodically movements of eyelids may be seen. When the muscles of neck and head are reached, the head of the birds stays either on the ground or on the bird back: the disease has been for a long time called limberneck. Botulinic birds are enable to walk, to swim and can drown up. They let them easily catch (POUANT 1997). Heavy and green diarrhoea is often seen. The death appears after respiratory paralysis; for wild birds, the death is often due to predation or the drowning for aquatic birds. Generally, there are not specic lesions. For birds there may be enteritis lesions, sometime haemorrhagic. 2.3. Epidemiological aspects C. botulinum is a bacterium normally present in the sediment. Apparition of botulism epizooty should be interpreted as an indicator of environmental disequilibria: the massive death of waterbirds is often in relation with pollution of the environment (MOUTOU 1993, LAMARQUE 1995). Concerning avian botulism, that is to say type C botulism, more than 174 species of bird species, form 22 families have been implicated in an epizooty. There are several theories on the needed conditions to have an outbreak. In the sludge bed theory, the outbreak is in relation with the environment conditions, favourable to the spore germination, bacterial growing and toxin production: there a lot of organic matter, anoxia, high temperatures, pH between 7.5 and 9, redox negative. In the microenvironment theory, there is a relatively independence of the environment conditions. The toxin production is due to local parameters. The presence of mammalians or birds corpses is very often the departure point of the outbreak because: Clostridium botulinum is normally present in the intestinal microore Corpses are suitable environment for bacterial growing and toxin production Scavenger invertebrates, such as ies larvae, concentrated in a very high way the toxin; theses larvae ma be eaten by birds, and then may amplify the phenomena.

107

3 CASE STUDY : LAKE OF GRAND LIEU IN LORE ATLANTIQUE (FRANCE)

3. 1 Local context Mass mortality on waterbirds may occur in several areas in France, according to meteorological conditions and local context. Since 1994, 24 French departments have been reached by botulism outbreaks. In Grand Lieu Lake, 1995 is the year of the rst heavy outbreak described: 36 species have been contaminated, without to count the passerine birds. 73 % of the diseased / death birds were ducks, but all the families were affected, even those uncommonly affected, such as raptors. MARION (1995) estimated that 90% of the Marsh Harrier died. Following this outbreak, the water levels were increased on the whole lake and management measures were recommended (for example clean up of the streams). Summer 2003 was very hot summer. Another outbreak has occurred in Grand Lieu Lake and in several areas in France. In Grand Lieu Lake, more than 800 corpses were collected, but mainly mallards (see BORET and REEBER 2003). The systematic collect of the corpse, each day, with a speedboat, allow probably limiting the extension of the mortality. 3. 2. Materials and methods In June 2001, a rst study has been conducted, aiming to map the bacteria presence. 102 samples of mud have been made. On each point, the GPS position was measured; we measured also water level, oxygen in surface water, in depth water and in mud, pH, redox, organic matter in the mud, water and mud temperature. Samples of mud were sent to Pasteur laboratory in Paris, in order to search for Clostridium botulinum and its type C and D toxin. In summer 2003, during the botulism epizooty, 34 samples have been made, amongst which, several were made on the same location than in 2001. The same parameters were measured. Analyses were performed by mouse test after and before enrichment and by double PCR as described by FACH et al. (1995, 1996). 3. 3. Results The results of 2001 study shown that 17.6% of the samples were positive for type C or/ and type D. The distribution of positive stations was not due to chance. There were 3 sites of risk, according to the presence of bacteria: Snaigerie, Ruby and La Morne. However, the physicochemical conditions in June 2001 did not allow toxin production: any positive station, except one, showed at the same time, conditions of oxygen content, pH, redox and temperature suitable for toxin production. There were not signicant differences between positive and negative stations for any physicochemical parameters. In summer 2003, 48% of the sampled stations were positive but the sample was not made according random way. The results conrmed the presence of 3 risk sites: Moreover, a forth sites La Capitaine appeared to be a risk site. There were not signicant differences between positive and negative station according to water level, surface and depth parameters, except for depth oxygen parameters:

108

the oxygen content of depth water is signicantly lower for negative station (mean = 0.873, standard deviation negative station = 0.382; mean positive station = 1.864, negative station standard deviation positive station = 2.127, t = -2.118, p = 0.045). However, for all the positive station except one, pH is suitable for toxin production (lower than 7.5) and oxygen content is very low (lower than 1.5). Moreover, the mud temperature was higher than 23 C for all positive station except one. In 2003, the physicochemical conditions were suitable to toxin production and then for the outbreak. These conditions lead the high mortality observed. The presence of dead birds on La Capitaine, despite the absence of bacteria in 2001, may suggest that the birds did not primarily contaminated in this site: the contamination may be done on the roosting place, such as in pounds (Snaigerie, Morne), were birds stays in very large ocks, mainly during moulting season, in June. When the contaminated birds died on the feeding place, such as La Capitaine, they contaminated this site. We can then assume that: the conditions i.e the bacteria in the sludge bed theory are present in the lake, but not in the whole lake. It is possible to identify risk sites but further analyses are needed to improve these sites. When physicochemical conditions of environment are suitable for toxin producing AND birds are present in high density (such as during moulting period), an outbreak may occur like in summer 2003. For some site, like La Capitaine, the microenvironment theory explains the amplication of the outbreak.

4 MANAGEMENT AND PREVENTION OF BOTULISM EPIZOOTY

4.1. Treatment of the birds In benign cases, the bird can recover in 2 in 3 days if it is removed from his environment: the recovery is downward, by opposition to the installation of the paralysis which is ascending. We shall administer a treatment of support: rehydratation, vitamins, possibly antibiotic. There is no immunity post-infection because the toxic dose is lower than the immunizing dose: the immunization against the disease can thus obtain only with the use of specic anatoxins (inactivated toxin) (POUANT 1997). Other vaccines were tested with more or less of success to birds. MARTINEZ and WOBESER (1999) showed for example that the administration in ducks mallards of a vaccine on base of inactivated Clostridium botulinum (usually used in minks farms) can protect birds and suggested to administer it to birds in the same time as the treatment. 4.2. Management during the epizooty In case of outbreak, it is fundamental to proceed to a systematic collect of corpses to avoid the enrichment of the environment. This collect has to appeal to appropriate means, notably speedboats that allow reaching the zones of weak depths. This was made during summer 2003 on the Grand Lieu Lake but it is essential to nalize a protocol of reproducible collect: it will also allow following the mortality from one year

109

to the next. Corpses are cremated. Birds alive are directed to a wildlife centre. The wildlife centre must be equipped with swimming pools as it is the case of the wildlife centre of Nantes. If it is possible (it is not the case of the Grand Lieu Lake) it is necessary to go to raise brutally the levels of water in order to lower the temperature of the sediment and possibly make inaccessible the contaminated sediments. 4.3. Prevention of the epizooties Concerning the organic matter content, when it is possible, we can limit the vegetable production, to clean out ponds, and possibly when banks are weakly tilted to modify their hillside. In term of water level, the most radical solution is the complete dryness of the site, outside the periods of strong temperature, that is to say in winter. If the dryness is not complete, in particular if sediments remain rich in water, we have on the contrary the risk of favouring the toxin production by allowing the increase of sediment temperature. On the same way if strong rains arise in spring. We can recommend thus on the contrary maintaining water on a sufcient level and especially stable from the beginning of the warm season. BARRAS and KADLEC (2000) recommended to divide, when it is possible, sites into small subunits, hydraulically independent, so as to be able to go up or lower quickly the water level on these micro-sites according to the outbreak risk.The preservation of a predatory and scavengers population can help to limit the number of corpse and thus the risk of outbreak. It is also necessary to identify and to ght the other causes of mortality.

5 CITATION INDEX
1. 2. BARRAS SC and KADLEC JA. Abiotic predictors of avian botulism outbreaks in Utah. Wildlife Society Bulletin, 2000; 28: 724 - 729. BORET P and REEBER S. Lac de Grand Lieu: rsultats des operations de ramassage des oiseaux atteints de botulisme sur la reserve naturelle en 2003. Le Courrier de la Nature 2003; 209: 17-24. CATO EP, GEORGE WL and FINEGOLD SM. Genus Clostridium Prazmowski 1880. In: SNEATH PHA, MAIR NS, SHARPE ME and HOLT JG. (eds): Bergeys Manual of Systematic Bacteriology, vol 2, Baltimore: Williams & Wilkins 1986; 1157 - 1161. FACH P, GIBERT M, GRIFFAIS R, et al. PCR and gene probe identication of botulinum neurotoxin A, B, E, F, and G-producing Clostridium spp. and evaluation in food samples. Appl Environ Microbiol 1995; 61: 389 - 392. FACH P, GIBERT M, GRIFFAIS R and POPOFF MR. Investigation of animal botulism outbreak by PCR and standard methods. FEMS Immunol Med Microbiol 1996; 13: 279 - 285. LAMARQUE F. Mortalits massives; Botulisme. Au service de SAGIR, Note dinformation de lONC, 1995; 78: 2 - 3. LE DREAN-QUENECHDU S. Etude du risque sanitaire reprsent par le botulisme sur le lac de Grand Lieu. 2001. Rapport non publi pour la SNPN. MARION L. Le botulisme Grand Lieu : une catastrophe cologique majeure. Le Courrier de la Nature, 1995; 154: 18 - 23.

3.

4.

5.

6. 7. 8.

110

9.

MARTINEZ R, WOBESER G. Immunization of ducks for type C botulism. J Wildl Dis 1999; 35: 710 - 715. 10. MOUTOU F. Les animaux sauvages sentinelles de lenvironnement. Le Point Vtrinaire, 1993; 24: 667 - 672.

The rest of the references are available from the author

AUTHORS ADRESS
Le Dran-Quenechdu S, DVM, PhD Eco-Ouest 3 rue de la Janaie, 35520 Melesse, France Email: sldq@club-internet.fr

111

Deptartment of Pathobiology1, The Ontario Veterinary College, University of Guelph; Health Canada2, Canadian Science Center for Human and Animal Health, National Microbiology Laboratory, The Owl Foundation3, Canada.

A WEST NILE VIRUS OUTBREAK IN NORTH AMERICAN OWLS

A.Y. Gancz1, DVM, MSc, I.K. Barker1, DVM, PhD, R. Lindsay2, PhD, A. Dibernardo2, K. McKeever3, and B. Hunter1, DVM, MSc.

KEYWORDS
West Nile virus - Epidemiology - Owl - Icosta Americana - Hippoboscida - Ontario - Canada.

ABSTRACT
Between July and September 2002, an outbreak of West Nile Virus (WNV) caused high mortality of captive owls at The Owl Foundation, Vineland, Ontario, Canada. Peak mortality occurred in mid August and corresponded with mortality of wild corvids in the surrounding region. Most of the survivors (75.8%) were seropositive for WNV. Species with northern breeding range were at signicantly higher risk of WNVrelated mortality, and died earlier during the outbreak. Species with northern breeding range and of medium/large body-size were at signicantly higher risk of exposure to WNV. The outbreak occurred in the midst of a louse y (Icosta americana, Family Hippoboscidae) infestation. Flies collected off sick or dead owls tested positive for WNV RNA.

1 INTRODUCTION
Since its initial detection in the New York City area in 1999, West Nile Virus (WNV) has emerged as a health risk to humans and has been associated with morbidity and mortality of a wide variety of North American birds, mammals, and reptiles (McLEAN et al. 2002, MILLER et al. 2003). While in humans and horses only a minority of WNVinfected individuals develop severe clinical disease, some bird species appear to be highly susceptible to this virus (EIDSON et al. 2001, KOMAR et al. 2003). To date, the factors that make some species highly susceptible to WNV remain unknown.

112

Little is known about the effect of the hosts taxonomic, geographic and demographic background on its susceptibility to WNV. Taxonomy alone offers limited help in predicting susceptibility to WNV, as some closely related species (e.g., American crows (Corvus brachyrhynchos) and sh crows (Corvus ossifragus) show different susceptibility (KOMAR et al. 2003). From July to September 2002, high mortality occurred in captive owls kept at The Owl Foundation (TOF), Vineland, Ontario, Canada. At the time, many of the birds were infested with adult haematophagous louse ies (order Diptera, family Hippoboscidae). Initially the deaths were attributed to this infestation and only on August 9 did the authors examine three dead owls. Necropsy ndings included marked hepatomegaly, splenomegaly and cerebral haemorrhage. On August 16, tissue samples from eight owls, including the initial three, were found to be WNV positive based on reverse transcription polymerase chain reaction (RT-PCR). Vaccination of the remaining birds with a killed WNV vaccine was attempted at the face of the outbreak, however, between July 26 and September 28, 108/245 (44%) owls died. To the authors knowledge, this is the largest WNV outbreak in a captive wildlife collection in North America, and the rst one in Canada. These outbreaks are of special interest as they offer a unique opportunity to study the impact and epidemiology of WNV infection in multiple species under quasi-natural conditions. The objective of this study was to test the effect of outdoor housing, age, body size, taxonomy (at the sub-family level) and geographical range on exposure to WNV and on WNV-related mortality. The role of louse ies in transmitting WNV was also examined.

2 MATERIALS AND METHODS

Study site, records and observations The Owl Foundation specializes in breeding and rehabilitating North American owls. Its facility in the Niagara region (Vineland, Ontario; 4310 N, 7920 W) has approximately 3340 M2 of specially designed outdoor cages and a few indoor cages. The organisation maintains detailed records of all birds in the facility. This data was used in the analysis of this outbreak. Data on dead Corvidae sightings in the Niagara region was obtained from the Canadian Cooperative Wildlife Health Center national WNV surveillance database. This data was gathered by the Niagara Region Health Unit between May 14 and October 12, 2002. Samples of tissue, blood and ies Complete diagnostic necropsies were performed at the Ontario Veterinary College on 94 owls and one falcon that died at TOF between April 15 and December 25, 2002. For each bird, samples (approximately 1mm3 in size) of brain, lung, liver, spleen and kidney were pooled and tested for WNV RNA by real time RT-PCR. Using a 20G spinal needle, small core samples of brain, kidney and liver were collected from three additional carcasses that were not available for necropsy.

113

During the outbreak louse ies were collected at TOF off sick, dead and asymptomatic birds. In addition, pupae were collected of the bottom of the cages. Six adult ies were submitted for species identication (courtesy of Nixon Wilson, University of Northern Iowa). Adult ies and pupae were pooled by cage and tested for WNV RNA by real time RT-PCR. A selected sample of ies, collected off WNV-positive birds, were dissected and specic body parts (e.g. salivary glands, hind gut, abdomen and thorax) tested for WNV by RT-PCR and immunohistochemical (IHC) staining. A serological survey of all outbreak survivors was conducted. Blood samples placed in heparinised tubes. Plasma was then separated by centrifugation and frozen at -70C until analysed. Real-time (TaqMan) RT-PCR Real-time RT-PCR assay was used to detect WNV RNA as previously described (LANCIOTTI et al. 2000). Positive controls consisted of three ten-fold dilutions of Egypt 101 (E101) strain of WNV. Three sets of negative (water) controls were used; two during the extraction procedure and one during amplication. Extracts were screened using the generic 3NC primer set. Positive samples underwent a second RNA extraction and were then tested by both the 3NC and ENV primer sets (LANCIOTTI et al. 2000). Samples that had CT 37 with both primer sets were considered positive. Homogenates prepared from whole louse ies were tested using the same procedures described for owl tissues. Serology We used an enzyme-linked immunosorbent assay (ELISA) shown to detect avian antiWNV immunoglobulin G (IgG) in 23 avian species of 12 orders, including a barred owl (Strix varia) (EBEL et al. 2002). The assay was performed as previously described with slight modications. A subset of 20 plasma samples representing 8 species of owls and 4 species of raptors was tested both by ELISA and by plaque reduction neutralisation tests (PRNT). The assay was performed as described previously (KOMAR et al. 2001). The controls used were back titrations of WNV (NY strain) at 2.5x107 PFU/ml diluted to 100, 10 and 1 PFU and a negative control (media only). Study Population The study population (SP) included 245 birds of 17 species that were present at TOF on the rst day of the WNV outbreak (based on RT-PCR results). Most were permanently disabled birds of wild origin; some had spent many years at TOF while others were recent additions or hatchlings of the year. Ten birds were housed indoors while 235 were kept outdoors. Statistical analysis Logistic regression was used to test the effect of outdoor housing, taxonomy (at the subfamily level) geographical range, age and species body size on exposure to WNV and on WNV-related mortality (among exposed birds). Species were considered Northern if their reported natural breeding range was mainly north to latitude 48N or Other if it was not so (MARKS et al. 1999). Species were further classied as

114

small, medium or large if their average body weight was <250g, 250-500g, or >500g respectively (MARKS et al. 1999). The two age groups compared were <1year and >1year. A general linear model was used to test the effect of the same factors on the date of death (i.e. the number of days to death from the index case). Regression procedures were performed using the SAS software (Version 8.2, SAS Institute Inc., Cary, NC, USA). The Kappa statistic (Quickcalcs, GraphPad Software, Inc. CA) was used to test for agreement between ELISA and PRNT results.

3 RESULTS
Based on RT-PCR results, the rst and last cases of WNV-related mortality at TOF were on July 26 and September 28, respectively. The rst ve cases occurred over a period of 11 days in ve different cage complexes scattered throughout the facility. The patterns of daily mortality at TOF and dead corvid sightings in the Niagara region during this period show close resemblance. Within the outbreak period, 79/85 (92.9 %) birds that died tested positive for WNV. Mortality rates (MR) differed greatly between species. Three distinct groups were seen: high MR (90-100%), low MR (MR< 16.7%), and no mortality (MR = 0%). All species in the high MR group have northern native range, while species that had MR of 0% have relatively southern range. Species in the low MR group have intermediate or very broad range of distribution (Table 1). A total of 91 outbreak survivors, kept outdoors during the entire outbreak and not vaccinated against WNV, were tested for anti-WNV IgG. Of these, 69 (75.8%) were seropositive based on ELISA. Agreement between ELISA and PRNT results was good (Kappa = 0.857, 95% CI 0.58-1.13). The overall exposure rate (ER) was 84.3 %. Being kept outdoors during the outbreak was found to be a highly signicant risk factor (P <0.0001) for WNV-related mortality. Species northern breeding range and large/medium body size were signicant risk factors for exposure to WNV (P<0.05) with odds ratio (OR)=52.56 (3.13-881.84) and OR=16.82 (3.79-74.67) respectively. Age and taxonomy (at the subfamily level) did not signicant inuence exposure. Among exposed birds, northern range was a highly signicant risk factor for WNVrelated mortality (P<0.0001) with OR=1507 (85.51- 26,557). Birds older than 1 year were also more likely to die of WNV (P<0.05) with OR=4.87 (2.46-9.62). Size and subfamily were not found to be signicant risk factors for WNV-related mortality. Geographical range was signicantly associated with the date of death (P<0.01). Northern species died earlier during the outbreak (mean 23.8 9.8 days, n=93) compared to other species (mean 35.0 12.9 days, n=8). Large or medium birds also died earlier during the outbreak (mean 21.9 8.5 days, n=77) compared to small species (mean 33.9 11.2 days, n=24) (P<0.05). Subfamily and age were not signicantly associated with the date of death.

115

The six louse ies examined were identied as Icosta americana (Order Diptera, Family Hippoboscidae). WNV RNA was detected by real-time RT-PCR in 16/18 (88.9%) ies collected off dead or sick owls during the outbreak. Five adult ies that were collected off healthy appearing birds were all WNV negative. Further data, including IHC results will be presented as it becomes available.

4 DISCUSSION
West Nile virus RNA was present in the vast majority of owls that died at TOF during the outbreak and antibodies against WNV were detected in most outbreak survivors (overall ER 84.3%). The similarity between the mortality pattern at TOF and that of dead corvids in the Niagara region and the fact that the initial cases occurred in several cages scattered throughout TOF, suggests that the outbreak was part of a regional WNV activity. As none of the birds kept indoors were affected, the major route of WNV transmission likely was vector borne. Northern geographical range and large/medium body-size were signicant risk factors for exposure to WNV. Birds that attract more vectors have a higher risk of exposure to WNV, and the hosts body size may be an important determinant of vector attraction. Northern species due to their thicker feathering may attract more feather-dwelling arthropods. WNV RNA was detectable in Icosta americana louse ies collected off dead or sick birds. If this parasite can transmit WNV, this would explain the overall high ER seen at TOF in light of the wide spread louse y infestation. Looking at the species specic MR, it is clear that the distribution of WNV-related mortality was uneven. Given the high overall ER, this nding suggests marked differences in species susceptibility to the virus. Taxonomic afliation at the subfamily level did not signicantly affect MR. Susceptibility to WNV-related mortality crossed taxonomic lines and was strongly related to geographical range. This link is intriguing, especially since a similar relation with regards to susceptibility to other pathogens (e.g. Aspergillus sp.) has been documented. T-cell mediated immunity is essential for ghting fungal pathogens (LEHMANN 1985) and is also believed to play an important role in the immune response against aviviruses (ANRAKU et al. 2002). It is possible that northern species have a less effective cell-mediated immune response to pathogens, which are scarce or nonexistent in their natural environment. Immunity to WNV, as for other pathogens, can be either innate or acquired. If immunity in this case was acquired, it would be expected that all owls that spent years at TOF would have similar susceptibility. Furthermore, if this was the case, young birds, regardless of species, would have been less likely to have acquired immunity. Our data shows the opposite. Innate immunity could potentially have evolved through selection if some of the species have coexisted over long periods of time with agents similar to WNV. Northern owls may have little or no exposure to aviviruses (e.g. St. Louis Encephalitis Virus)

116

and may therefore be particularly susceptible to WNV. Northern distribution and large/medium body-size were signicantly associated with earlier death during the outbreak. This could be a result of infection at earlier date, larger infective dose, shorter incubation, shorter disease course, or a combination of the above. Further investigation is needed in order to answer this question.

5 CONCLUSION
There is a strong link between geographic range and susceptibility to WNV in North American owls. This relationship crosses taxonomic lines at the subfamily level and may be related to differential immune competence. Factors such as size and age may (to a lesser magnitude) affect exposure and susceptibility respectively. Louse ies, common avian haematophagous parasites, may play a role in transmitting WNV, however this requires further examination. As WNV continues to spread, free ranging populations of susceptible owl species may be signicantly affected.

6 ACKNOWLEDGEMENTS
The authors thank Annick Gionet, Kara Kristjanson, Jody Crossingham, Chris Enright, and Amanda Low for their assistance in data collection; Heather White for technical assistance; Douglas Hodgins, and Hillary Voet for assistance in statistical analysis; Nathalie Lemieux for help in documentation; Dale A. Smith for critical reading of the manuscript; The Rathlyn Foundation for the stipend that made this study possible.

7 CITATION INDEX
1. 2. ANRAKU I, HARVEY TJ, LINEDALE R, et al. Kunjin virus replicon vaccine vectors induce protective CD8+ T-cell immunity. J Virol 2002; 76: 3791 - 3799. EBEL GD, DUPUIS AP II, NICHOLAS D, et al. Detection by enzyme-linked immunosorbent assay of antibodies to West Nile virus in birds. Emerg Infect Dis 2002; Available from: URL: http://www.cdc.gov/ncidod/EID/vol8no9/020152.htm EIDSON M, KOMAR N, SORHAGE F, et al. West Nile Virus Avian Mortality Surveillance Group. Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis 2001; 7: 615 - 620. KOMAR N, PANELLA NA, BURNS JE, et al. Serologic evidence for West Nile virus infection in birds in the New York City vicinity during an outbreak in 1999. Emerg Infect Dis 2001; 7: 621 - 625. KOMAR N, LANGEVIN S, HINTEN S, et al. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 2003; Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no3/02-0628.htm LANCIOTTI RS, KERST AJ, NASCI RS, , et al. Rapid detection of west nile virus from human clinical specimens, eld-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol 2000; 38: 4066 - 4071.

3. 4. 5. 6.

117

7.

LEHMANN PF. Immunology of fungal infections in animals. Vet Immunol Immunopathol 1985; 10: 33 - 69. 8. MARKS JS, CANNINGS RJ, MIKKOLA H, et al. Family Strigidae (Typical owls). In: Del Hoyo J, Elliott A, and Sargatal J, editors. Handbook of the birds of the world, Vol. 5 Barn-owls to Hummingbirds. Barcelona: Lynx Edicions 1999; 34 - 243. 9. MCLEAN RG, UBICO SR, BOURNE D, KOMAR N. West Nile virus in livestock and wildlife. Curr Top Microbiol Immunol 2002; 267: 271 - 308. 10. MILLER DM, MAUEL MJ, BALDWIN C, et al. West Nile virus in farmed alligators. Emerg Infect Dis 2003; Available from: URL: http://www.cdc.gov/ ncidod/EID/vol9no7/03-0085.htm

AUTHORS ADRESS
Ady Y. Gancz, DVM, MSc. Dept. of Pathobiology, The Ontario Veterinary College, University of Guelph, Guelph, ON, CANADA, N1G 2W1 Email: agancz@uoguelph.ca

118

Table 1. Mortality at The Owl Foundation property during a West Nile virus outbreak - (July 26 - September 28, 2002)

Species Snowy owl (Bubo scandiacus) Northern hawk owl (Surnia ulula) Great gray owl (Strix nebulosa) Boreal owl (Aegolius funereus) Northern saw-whet owl (Aegolius acadicus) Northern pygmy owl (Glaucidium gnoma) Short-eared owl (Asio ammeus) Flammulated owl (Otus ammeolus) Long-eared owl (Asio otus) Great horned owl (Bubo virginianus) Barn owl (Tyto alba) Barred owl (Strix varia) Burrowing owl (Athene cunicularia) Eastern screech owl (Megascops asio) Elf owl (Micrathene whitneyi) Spotted owl (Strix occidentalis) Tawny owl (Strix aluco) American kestrel (Falco sparverius) Peregrine falcon (Falco peregrinus) Total:
MR = Mortality rate (calculated when n>6). 1 Based on real-time RT-PCR.

At Risk 20 26 27 11 13 6 16 9 13 22 10 8 10 36 1 1 2 2 2 235

WNV-related1 MR % 100.0 100.0 91.3 90.9 92.3 16.7 12.5 11.1 7.7 4.5 0.0 0.0 0.0 0.0 43.0

119

Ofce National de la Chasse et de la Faune Sauvage Unit Sanitaire de la Faune, France

SURVEILLANCE OF WEST NILE VIRUS IN THE AVIFAUNA OF SOUTHERN FRANCE

J. Hars, P. Aug, J. Pradel, M. Mortamais, D. Chavernac, G. Balanca, S. Zientara, I. Schuffenecker, H. Zeller

KEYWORDS
West Nile virus - Avian disease - Epidemiosurveillance - France

ABSTRACT
West Nile fever is a mosquito-borne viral disease for which wild birds are amplifying hosts, and humans and horses sensitive hosts (accidental victims). West Nile virus (WNV) was recognized for the rst time in southern France (Camargue) in 1962-1965. It reappeared in 2000 and 2004 in the same region involving only horses. In 2003, seven human and four equine cases were observed in the Var district, 200 km East from the Camargue region. In 2000, the French game and wildlife agency (ONCFS) conducted a serological study of 5 bird species in the Camargue, whereby low seroprevalences were detected in mallards (8%) and magpies (22%). Since 2001, there is an epidemiosurveillance program for avifauna. It is based on the detection of abnormal mortality in wild birds and on a serological monitoring of sentinel birds, mallards and chickens located in 30 sites distributed along the coastal region. The monitored area was extended in 2004. Among the avifauna, no abnormal mortality due to the WNV virus was reported. In 2001 and 2002, only 2 seroconversions were detected in sentinel birds suggesting a very low circulation of WNV. In 2004, with the help of several seroconversions the presence of the virus was revealed in sentinel birds, before the occurrence of equine cases. These results show the interest of monitoring WNV circulation in birds, as an early warning system.

1 INTRODUCTION
The West Nile virus is an arbovirus (arthropod borne virus), belonging to the Flaviviridae family like the yellow fever virus. It was isolated for the rst time in 1937 in Uganda, West of the Nile (after which it has been named) in the serum of a young woman with a febrile syndrome. The avifauna is the virus main reservoir, since birds are the hosts that will amplify the viral circulation. Many wild, sedentary or migratory species, (Corvids,

120

Laridae, Anatidae, Waders, Raptors, Passerines) but also domestic ones (Turkey, Goose, Chicken) may host and transport the virus (ZELLER and MURGUE 2001). In general, birds are asymptomatic carriers. However, neurological manifestations with high mortality rates have been observed among pigeons in Egypt (TAYLOR et al. 1956, WORK et al. 1953) and Corvids in the United States (STEELE et al, 2000). Like any other arbovirus infection, West Nile fever is transmitted by a stinging arthropod vector. Generally, this is a mosquito of the Culex genus (C. pipiens or C. modestus in Europe), that contaminates itself when it bites an infected bird. The virus multiplies in the arthropod and then reaches its salivary glands. When, later on, the mosquito will feed on the blood of its host, it could transmit the virus to another sensitive host. The length of this cycle depends on the climate (temperature, humidity) which, in turn, is determined by the activity of the mosquito vectors. When an intense bird-mosquito cycle is going on, some vectors may bite horses as well as humans and transmit the virus to them. Among horses, the clinical signs of the disease are extremely variable, and may go from a simple u syndrome to encephalomyelitis with a high mortality rate. In Man like in birds, West Nile fever is usually subclinical. In some cases, however, humans also may develop a pyretic syndrome which, mainly among the older ones, may lead to complications like encephalitis or aseptic meningitis that might be lethal. Accidental hosts like horses and Man, are generally considered to be epidemiological culs de sacs: i.e. through them the virus can neither be multiplied, nor transmitted. Today, no curative treatment nor vaccine is available against the West Nile virus. The occurrence of the disease has been described many times in Africa (MC INTOSH et al, 1976), the Middle East (WEINBERGER et al. 2000) and Asia, but also in Europe (Portugal, Rumania, Italy, France) (TSAI et al. 1998, HUBALEK and HALOUZKA 1999, CANTILE et al. 2000, PLATONOV et al. 2001, MURGUE et al. 2001). The virus appeared for the rst time in 1999 in the United States in the region of New York and circulates now throughout the whole North American continent (STEELE et al. 2000). In 2002, 4,156 clinical human cases out of which 284 cases of deaths were led while 15,000 horses were infected, out of which 4,500 died. In 2003, 9,862 human cases were described and 264 of the sick died (CDC 2003). In North America, the West Nile virus was isolated in 234 bird species (HUBALEK 2000, BALANCA and HARS 2005), but the cases of mortality were mostly observed in corvids which, most often, were American crows (Corvus brachyrhynchos) and blue jays (Cyanocitta cristata) (ZELLER and SCHUFFENECKER 2004). In France, West Nile virus was described for the rst time in 1962-63 in the Grande Camargue area, where it had contaminated horses (500 clinical cases with a morbidity of 25% and a lethality of 10%) and humans (19 clinical cases, 10 of which were severe)., The virus was isolated in 1964-65 in men and in mosquitoes of the Culex modestus species (JOUBERT et al. 1970, 1971). After a phase of dormancy that lasted for more than 35 years, it suddenly reappeared in August 2000 in the area of the Petite Camargue, east of the city of Montpellier, where, between the month of August and the month of November the horse population was contaminated (76 conrmed cases, 21 deaths) (ZIENTARA et al. 2001). An exhaustive serological survey revealed that 8.3% of the 5.133 horses that had been tested in the regions of the Camargue, Hrault, Gard and Bouches-du-Rhne carried antibodies (source

121

AFSSA Alfort). It seems that only equids were clinically infected by the virus, since not a single human case of meningitis-encephalitus was recorded and no abnormal or unusual mortality was observed in the wild and domestic birds (CHEVALIER et al. 2002). In late autumn 2000, a serological survey in ve avian species was made in the outbreak area with the help of ELISA tests conrmed by seroneutralisation. Birds tested positive for the West Nile virus were found in three species with low prevalences: in 8% of the mallards (Anas platyrhynchos, n = 100), 0.9% of yellowlegged gulls (Larus cachinans, n = 114) and 22% of black-billed magpies (Pica pica, n = 18). However, all tested House sparrows (Passer domesticus, n = 117) and blackheaded gulls (Larus ridibundus, n = 81) were negative (HARS et al. 2001, 2004)

2 MATERIAL AND METHODS

Since 2001, a national surveillance programme for the West Nile virus has been implemented by the DGAl (General Food Administration, Ministry of Agriculture) and the DGS (General Direction of Health Affairs, Ministry of Health). This programme is based on the surveillance of the cases of human encephalitis in hospitals and equine encephalomyelitis occurring throughout France, with an enhancement in the Camargue region and in the Mediterranean dpartements. As soon as the occurrences of human or equine cases are conrmed, an entomological survey based on captured mosquitoes, their identication and the virus isolation, is carried out in the infected area. Concerning avifauna, the objective of the surveillance is to be able to rapidly detect any circulation of the West Nile virus in the Camargue so as to give an early warning before any clinical signs in equids or humans are revealed, and to take the appropriate measures to inform the public and prevent the disease. The implementation of this avian surveillance program is performed by the wildlife sanitary Unit of the French Hunting and Wildlife Agency (Unit sanitaire de la faune. Ofce national de la chasse et de la faune sauvage, ONCFS), in collaboration with the National Reference Center for Arboviruses and Haemorrhagic Viral Diseases of the Pasteur Institut (CNR-IP), the Center for International Cooperation in Agronomic Research (Centre de coopration internationale en recherche agronomique pour le dveloppement, CIRAD-EMVT), the dpartemental hunter federations, the dpartemental veterinary laboratories (LDAV), the dpartemental veterinary services, and practising veterinarians. In 2001, 2002 and 2003, surveillance was based on: A passive system based on a closer surveillance of the cases of avifauna mortality in the dpartements of the Camargue region (Hrault, Gard et Bouches du Rhne), and on virological analyses of all dead birds found by the members of the SAGIR network (the national network for the surveillance of the causes of mortality in wildlife). This work was carried out thanks to an awareness campaign for the general public based on an important advertising campaign, a large spread of posted public notices and a Freefone telephone number (Fig 1). The research of the virus in the brain of dead birds is carried out by RT-PCR and viral isolation on a cell culture.

122

an active system that is based on a serological survey of sentinel birds: i.e. an average number of 200 calling ducks (mallards breed by hunters and released on the ponds and in the marshes to attract the wild ducks) and domestic chicken, distributed over 20 sites . There were 10 to 12 birds per site which were identied with two coloured rings on one of their legs. An individual code number was attributed to each bird in order to monitor the changes in its serological status throughout the whole monitoring campaign. From each bird, blood samples were monthly taken (from June to November). The sera, collected and processed in the LDAV, were transmitted without delay to the CNR where they were treated by ELISA IgG with conrmation of positive results by sero neutralisation. The rst blood sample taken allowed to control whether a bird was serologically negative. A sero-conversion, i.e. the appearance of antibodies in an initial seronegative bird, was then considered as an incontestable sign of its recent contact with the virus.

Fig 1. Poster used for advertising campaign

In 2004, the protocol was modied because of the emergence of several cases of human and equine encephalomyelitis in September 2003 in the Var dpartement, where no aviary West Nile surveillance had been set up.

123

a passive system has been applied in ten mediteranean dpartements . A leaet to inform and incite the public to collect the dead birds has been distributed to the institutions or associations concerned by avifauna. an active system has been extended to three other dpartements: Pyrenes Orientales, Aude and Var. A total number of 320 birds, distributed over 31 sites (Fig 2) was controlled every month.

Fig 2. Geographical distribution of sentinels birds sites in the South of France in 2004 The collection, treatment and circulation of all data collected through the surveillance of the avifauna is done with a data base that was set up by, and is located at, CIRADEMVT. It may be consulted online on an Internet site with protected access: http:// west-nile.cirad.fr. by all partners of the program.

3 RESULTS
3. 1. Bird mortality See table 1 for the list of wildfowl mortality cases that were registered in the area of surveillance through a toll-free telephone number and by the SAGIR network.

124

Table 1: Reference table for the cases of avian mortality recorded in the area under surveillance between 2001 and 2004
Date July 2001 August 2001 August 2001 August 2001 July - August 2002 August 2002 August 2002 August 2002 August 2002 August 2002 August 2002 May 2003 August 2003 AugustSeptember 2003 August 2003 August 2003 August 2003 September 2003 October 2003 July 2004 August 2004 August 2004 September 2004 Species Waterfowl Wood pigeons Mallards Mallards Coots Waterfowl House sparrows Mallards Mallards Mallards, Garganeys, Teals Mallards Gulls, Waders Gulls Mallard Waterfowl House sparrow Mallards Pigeon Turtle doves Ducks DucksTurtle dove Partridge Pheasant Chicken Sheldduck Pigeons Place - Commune (dpartement*) Several communes (13) Lansargues (30) Vauvert (30) Marignane (13) Several communes (13 and 30) Vauvert (30) Ste (34) Marsillargues (34) Fleury dAude (11) St Laurent dAigouze (30) St Nazaire de Pzan Arles (13) Villeneuve les Maguelones (34) Several communes (30 and 34) Montpellier (34) Carcassonne (11) Nmes (30) Martigues (13) Carcassonne (11) Le Caylar (30) Nmes (30) Draguignan (83) Montpellier (34) Carcassonne (11) Presumed (P) or conrmed(C) cause of mortality Botulism (C) ? Intoxication pesticides (C) Botulism (C) Botulism (C) ? ? Ulcerative enteritis (C) ? ? Paralysis (P) Botulism + diarrhea (C) Salmonellosis (C) Botulism (C) ? Intoxication (P) ? ? ? ? ? ? ? West Nile virological ndings Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Ngative

* dpartements : 11 = Aude ; 13 = Bouches du Rhne, 30 = Gard ; 34 = Hrault ; 83 = Var

125

First of all, we can notice that the number of dead birds that was declared and for which an autopsy in the laboratory was made, is very small in spite of all the information and awareness campaigns organized in the dpartements concerned by the birds surveillance (cf 2). In total, 72 virological analyses were made in the brain of dead birds. All the PCR tests were negative and the West Nile virus has never been isolated. These results suggest that since 2000, no abnormal mortality due to the West Nile virus has been observed in the avifauna of the Mediterranean dpartements. 3. 2 Serological monitoring of sentinel birds The results of the serological monitoring campaign of calling ducks and poultry which were surveyed for four years are presented in the table 2. Table 2. Results of the serosurvey of sentinel birds.
Year Nb of controlled birds 288 286 185 % of birds found Nb of sites to be seroposive seroconversions (dpartements) at the rst annual control 28 (dp. 13, 30, 34) 25 (dp. 13, 30, 34) 16 (dp. 13, 30, 34) 9% 2.4% 3.2% 1 duck 1 hen 0 Stes Maries de la Mer (dp. 13) 1 in July 1 in August 6 in September 1 in October St Just (dp. 34) August St Laurent dAigouze (dp. 30) October Arles La Tour du Valat (dp 13) October Place and date of seroconversions Arles (dp. 13) Octobre 2001 Gallargues (dp. 30) August 2002

2001 2002 2003

2004

320

31 (dp. 11, 13, 30, 34, 66, 83)

3.1% (all situated in dp. 83)

9 hens

1 duck 1 duck 2 ducks

In the Camargue, between 2001 and 2003, not a single human or equine clinical case has been observed, although a very small circulation of the virus would have been revealed in 2001 and 2002, based on of two cases of seroconversion in birds. Moreover, it is interesting to note that 9% of the birds that have been tested early in the summer of 2001, and 2 to 3% of the birds tested in 2002 and 2003, still showed some serological traces of the 2000 outbreak; this proves a persistance of antibodies that, in birds, may last for several years (the same duck, positive in 2001 was controlled positive in 2003) as well as a lesser degree of seroprevalence after an epizootic peak. In 2003, WN monitoring of sentinel birds of the Camargue was

126

negative. Meanwhile a cluster of WN encephalitis cases was detected in humans (7 cases) and horses (4 cases) in the Var district, located more than 100 km east from the Camargue, in a drier area. Like in the Camargue, no avian mortality was observed in this region. These events showed that, in addition to these known areas, WNV could also appear in other locations in France. In 2004, in the Var region, not a single case of viral circulation was detected anymore, neither in man nor in horses or the avifauna. Nevertheless, the presence of residual antibodies from the 2003 outbreak was still observed in 3.1% of the tested birds that had been checked early in the summer of 2004 in this dpartement. On the contrary, the virus reappeared in the Camargue where the rst proof of its presence was noted by the seroconversion of a sentinel bird as early as in the month of July 2004 and then by the seroconversion of 12 other birds in the three dpartements of the Camargue. The circulation of the virus was conrmed by the emergence, in the same area, of 32 clinical cases among horses (10 of which died or have to be euthanized) from September to November.

4 DISCUSSION
It seems that the West Nile virus strain that circulates in France since 2000, is not very, or not at all pathogenic for birds because, in the Mediterranean dpartements which are considered to be areas at risk , not a single case of mortality due to this virus has been recorded by our surveillance network. The French strain is probably not the same than the one circulating in North America (i.e. this strain is phylogenetically very close to the Israelian one and its pathogenicity for birds has been established) where the presence of dead corvids allow to be on the virus track , something that at the present time is impossible in France. To overcome this difculty, we have to use sentinel birds that were monitored with serological assays, a method that apparently was efcient and sensitive enough to detect the virus at an early stage and alert the medical and veterinary services. In 2004, the appearance of bird seroconversions before the equine clinical cases, proves that. We can discuss about the lack of sensitivity of the network of sentinel birds that was set up in France and that, with only 320 birds distributed over 31 sites in 6 dpartements (a limited number because of technical and nancial constraints), is, from a numerical and spatial point of vue, theoretically insufcient. Thus, we can suppose that, as soon as it encounters the required environmental conditions, the virus will circulate rather among the mosquitoes and avifauna to be detected in birds by a not very sensitive surveillance network and before the appearance of any clinical signs in horses and humans. From an epidemiological point of view, the results of four years of surveillance make us think that the south of France is an epizootic area where limited outbreaks can develop and may be responsible for the contamination of accidental hosts (horses, man), contrary to enzootic areas (Africa, Middle East, North America) where the virus largely circulates whereby, each year, new clinical cases appear as well as antibodies that may be found in a great number in its hosts (birds, horses, man.). Even if wetlands, like the Camargue, seem to be more favourable areas for the development of WNV in the reservoirs, vectors and amplifying species, the appearance of the West Nile virus and the intensity of its circulation, that may generate a more

127

or less important risk for human and animal health, are totally unforseeable in our present state of knowledge since they depend on the conjunction of many ecological factors. The phenomenon of global warming could be one of the elements that would explain the emergence of the disease in Europe. Several research programmes have been launched in France and in Europe with the objective to improve our knowledge of the epidemiology of this virus.

5. REFERENCES
BALANCA G., HARS J.. Bird reservoirs and indicators of West Nile Fever. Game & Wildlife Science. 2005; in press. 2. CANTILE C., Di GUARDO G., ELENI C., ARISPICI M. Clinical and neuropathological features of West Nile virus equine encephalomyelitis in Italy. Equine Vet. J.2000; 32:31-35 3. CDC. Epidemic/epizootic West-Nile virus in the United States : Guidelines for Surveillance, Prevention and Control. 3rd revision. Centers for Disease Control, 2003; 77 pp. Available online at: http://www.cdc.gov/ncidod/dvbid/westnile/ publications.htm. 4. CHEVALIER V., DURAND B., GERBIER G., BABINOT M., MICHEL JF., TOURE I., ZIENTARA S. Analyse spatiale de linfection virus West Nile chez les chevaux de Camargue en 2002 : rsultats et perspectives. Epidemiologie et Sant Animale. 2002 ; 42 : 123-131. 5. DURAND B., CHEVALIER V., POUILLOT R., LABIE J., MARENDAT I., MURGUE B., ZELLER H, ZIENTARA S. West Nile outbreak in horses in southern France : results of a serosurvey. Emerg. Information. Dis. 2002 ; 8:777-782. 6. HARS J., AUGE P., De VISSCHER M-N., FRUITET L., KECK N., MURGUE B., POURRUT X., ZELLER H., ZIENTARA S. Etude prliminaire sur linfection de lavifaune du dpartement de lHrault par le virus West Nile en 2000. Rapport ONCFS/DGAl. 2001 : 17 p. 7. HARS J., AUGE P., CHAVERNAC D., BALANCA G., KECK N., PRADEL J. & ZELLER H. Surveillance de linfection de lavifaune camarguaise par le virus West Nile. Revue ONCFS Faune sauvage. 2004 ; 261 : 54-58. 8. HARS J., ZELLER H., ZIENTARA S. West Nile virus in France in 2003. J. Wildlife Disease. 2004; Vol 40: 2 supplement. 9. HUBALEK Z. European experience with the West Nile virus ecology and epidemiology: could it be relevant for the New World. Viral Immunol. 2000; 13:415-426. 10. HUBALEK Z. & HALOUZKA J. West Nile fever: a reemerging mosquito-borne viral disease in Europe. Emerg. Inf. Dis. 1999; 5 : 643-650. 11. JOUBERT L., OUDAR J., HANNOUN C., BEYTOUT D., CORNIOU B., GUILLON JC., PANTHIER R. Epidmiologie du virus West Nile : tude dun foyer en Camargue. IV. La mningo-encphalomylite du cheval. Ann. Inst. Pasteur . 1970 ; 118:239-247. 12. JOUBERT L., OUDAR J., HANNOUN C., CHIPPAUX M. Reproduction exprimentale de la mningo-encphalomylite du cheval par larbovirus West Nile. III Relations entre la virologie, la srologie et lvolution anatomoclinique. Consquences pidmiologiques et prophylactiques. Bull. Acad. Vet. 1.

128

1971; tome XLIV, 159-167. 13. MC INTOSH B.M., JUPP P.G., DOS SANTOS I., MEENEHAN G.M. Epidemics of West Nile and Sindbis viruses in South Africa with Culex (Cx) univittatus Theobald as vector. South Afr. J Sci. 1976; 72 : 295-300. 14. MURGUE B., MURRI S., ZIENTARA S., LABIE J., DURAND B., DURAND J.-P. & ZELLER H. West Nile in France in 2000: the return 38 years later. Emerg. Inf. Dis. 2001 ; 7: 692-696. 15. MURGUE B., MURRI S., TRIKI H., DEUBEL V. and ZELLER H. West Nile in the Mediteranean basin : 1950-2000. Annals of the NY Academy of Sciences. 2001; 951: 117-126. 16. PLATONOV A. E., SHIPULIN G. A., SHIPULINA O.Y., TYUTYUNNIK E. N., FROLOCHNIKINA T. LANCIOTTI R.S., YAZYSHINA S., PLATONOVA O.V., OBUKHOV I.L., ZHUKOV A.N., VENGEROV Y.Y., POKROVSKII V. Outbreak of West Nile Virus in Russia, Emerg. Inf. Dis. 2001; 7(1) :128-30. 17. STEELE K. E., LINN M. J., SCHOEPP R. J., KOMAR N., GEISBERT T. W & MANDUCA R. M. Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York city. Vet. Pathol. 2000; 37 : 208-224. 18. TAYLOR R. M., WORK T. H., HURLBUT H. S. & RIZK F. A study of the ecology of West Nile virus in Egypt. Am. J. Trop. Med. Hyg. 1956; 5 : 579-620. 19. TSAI T. F., POPOVICI F., CERNESCU C., CAMPBELL G. L., NEDELCU N. I. West Nile encephalitis in southeastern Romania. The Lancet 1998 ; 352:767-71. 20. WEINBERGER M., PITLIK S. D., GANDACU D., LANG R., NASSAR F., BENDAVID D., RUBINSTEIN E., IZTHAKI A., MISHAL J., KITZES R., SIEGMANIGRA Y., GILADI M., PICK N., MENDELSON E, BIN H, SHOHAT T, CHOWERS MY, West Nile fever outbreak, Israel, 2000: epidemiologic aspects. Emerg. Inf. Dis. 2001; 7:679-685. 21. WORK T.H., HURLBUT H.S., TAYLOR R.M. Isolation of West Nile virus from hooded crow and rock pigeon in the Nile Delta. Proc Soc Experiment Biol Med .1953; 84: 719-72. 22. ZELLER H.G., SCHUFFENECKER I. West Nile Virus: An Overview of Its Spread in Europe and the Mediterranean Basin in Contrast to Its Spread in the Americas. Eur.J. Clin. Microbiol. Information. Dis. 2004 (in press). 23. ZELLER H. & MURGUE B. Rle des oiseaux migrateurs dans lpidmiologie du virus West Nile. Med. Mal Infect. 2001 ; 31 : 168-174. 24. ZIENTARA S., MURGUE B., ZELLER H., DUFOUR B., MURRI S., LABIE J., DURAND B. & HARS J. Maladie virus West Nile en France. Epidmiologie et Sant Animale 2001 ; 11 : 295-298.

AUTHORS ADRESSES
Jean Hars Unit sanitaire de la faune, Ofce national de la chasse et de la faune sauvage, 5 Alle de Bethlem, 38610 Gieres, France Email: j.hars@oncfs.gouv.fr

129

Great Western Referrals1, Avian and Exotic Department, Swindon, United Kingdom; Veterinary Medical Research Institute2, Hungarian Academy of Science, Budapest; Central Veterinary Institute3 Budapest, Hungary; Institute for Virology4, Faculty of Veterinary Medicine University of Leipzig, Germany

ADENOVIRUS INFECTION IN RAPTORS

P. Zsivanovits1 DMedVet MRCVS, D. J. Monks1 BVSc (Hons) MACVSc (Avian Health) CertZooMed MRCVS, N. A. Forbes1 BVetMed CBiol MIBiol Dip ECAMS FRCVS, M. Benk DMedVet PhD2, K. Ursu MRCVS2,3, R. Raue4 DMedVet

KEYWORDS
Adenovirus - Ventricular ulceration - Intranuclear inclusion bodies - Raptors

ABSTRACT
Although previously described as a cause of mortality, adenovirus remains an under-reported disease in raptors. This report describes adenoviral outbreaks in two separate collections at similar times, involving a Harris hawk (Parabuteo unicinctus), a Bengal eagle owl (Bubo bengalensis), and a Verreauxs eagle owl (Bubo lacteus). The outbreaks were diagnosed by necropsy, histologic examination, and PCR. Attempts to isolate virus or to detect viral particles by electron microscopy were unsuccessful. PCR with consensus primers resulted in amplicons of specic sizes. DNA sequencing identied the detected virus as a member of the genus siadenovirus. To the authors knowledge this is the rst report of disease related to adenovirus infection in these species. This report highlights the clinical and histologic presentation of adenoviral infections, and the difculty of diagnosis.

1 INTRODUCTION
Adenoviruses are non-enveloped double-stranded DNA viruses, 70-90 nm in diameter, that replicate in the cell nucleus, forming basophilic intranuclear inclusion bodies. Based on genetic and phylogenetic analysis, four genera of adenoviruses are recognised (BENK et al. 2004). Mastadenoviruses affect mammals. Aviadenovirus (former group I) are isolated from numerous bird species including poultry. One novel genus, Siadenovirus, consists of the Turkey haemorrhagic enteritis virus (Turkey adenovirus type 3), Marble spleen disease virus and Chicken splenomegaly virus, originating from different diseases in different species but indistinguishable on

130

serology, together with an adenovirus isolated from a frog. Egg drop syndrome76 virus (Duck adenovirus type 1) together with multiple adenoviruses isolated from reptiles, birds, ruminants and a marsupial form the other new genus, Atadenovirus (BENK et al. 2004). Adenoviruses are reported to be opportunistic, depending on triggering factors (such as immunosuppression by bacterial, fungal, viral or parasitic pathogens) to cause clinical disease. (RITCHIE 1995). There have been sporadic reports of adenovirus infections in raptors with varying clinical signs: a Northern goshawk (Accipiter gentiles) was found with central nervous signs (STEHLE 1965, cited by GERLACH 1994); American kestrels (Falco sparverius) (SILEO et al. 1983) and a tawny frogmouth (Podargus strigoides) (REECE and PASS 1985) were diagnosed with haemorrhagic enteritis and splenomegaly; a merlin (Falco columbarius) showed adenoviral hepatitis (SCHELLING et al. 1989); and fatal adenovirus infections were reported in Mauritius kestrels (Falco punctatus) (FORBES et al. 1997). This paper discusses adenoviral outbreaks in two raptor collections. In one collection Harris hawks (Parabuteo unicinctus) were primarily affected, while in the second collection a Bengal eagle owl (Bubo bengalensis) and a Verreauxs eagle owl (Bubo lacteus) died. Identication of the causative agent was attempted based on the results of clinical examination, necropsy, histologic examination, virus isolation, electron microscopy and PCR. This report highlights the difculties in diagnosing adenoviral infection, particularly in the acute stages. To the best of the authors knowledge this is the rst report of adenovirus infection in a Harris hawk, a Bengal eagle owl or a Verreauxs eagle owl.

2 CASE REPORT
The death of the 20-weeks old Harris hawk was preceded by approximately ten minutes of tting. The 3-year-old European eagle owl showed 24 hours of increasing depression and the 1-year-old Bengal eagle owl showed 48 hours of depression and anorexia prior to death. Necropsy ndings of the Harris hawk and the eagle owls included hepatomegaly; splenomegaly; proventricular and ventricular dilation, ulceration and erythema; and renomegaly. The Harris Hawk had a Syngamus spp. infestation, while the Verreauxs eagle owl had a suspected protozoan infection. Histological ndings of all three birds consisted of hepatic necrosis, hepatitis, splenic necrosis, proventricular and ventricular ulceration and necrosis. Besides the above-mentioned organs, basophilic intranuclear inclusion bodies were also seen in the pancreas and the kidneys of the Verreauxs eagle owl. Virus isolation on chicken embryo liver cells and electron microscopy of pooled tissue samples (liver, spleen, ventriculus, kidney, heart) of the Harris hawk and the Eagle owls were negative for adenovirus. PCR was performed on pooled tissue samples in two laboratories, one in Leipzig, Germany, and one in Budapest, Hungary. Both laboratories carried out Aviadenovirus-specic PCR to detect the 12 fowl adenovirus

131

serotypes (former group I) using hexon gene-targeting primers (RAUE and HESS 1998), even in a nested system. None of these detected adenoviral DNA in the samples. The Budapest laboratory also performed nested PCR with a primer targeting a partial sequence of the polymerase gene (WELLEHAN et al. 2004). This PCR detected adenoviral DNA in tissue samples of all three carcasses submitted. DNA sequencing of the PCR products revealed that all three birds were infected with the same virus and alignment of amino acid sequences classied it as a member of the novel genus, siadenovirus. On the phylogenetic tree (based on the results of distance matrix analysis), the virus was grouped with Siadenoviruses, in a common branch with the Turkey adenovirus 3, and with the frog Siadenovirus representing a separate branch. Consensus PCR for circovirus and polyomavirus and a nested PCR for herpesvirus were also performed on the pooled tissue samples in the Leipzig laboratory and no viral DNA was detected.

3 DISCUSSION
The large basophilic intranuclear inclusion bodies of varying size coupled with the distribution in epithelial and lymphoreticular tissue, karyomegaly, hepatic and splenic necrosis and ventriculitis combine to generate a histological pattern that is highly suggestive of adenoviral infection in the presented cases. Intranuclear inclusion bodies were identied in the liver, spleen, ventriculus, pancreas, small intestines and kidneys. This pattern has also been observed in poultry infected with adenovirus (NAKAMURA et al. 2002; RITCHIE 1995). The Harris hawk belonged to a clutch of four edglings that were kept free-lofted in a large aviary with their parents. Prior to the death of this Harris hawk the other three edglings died acutely with no premonitory signs within a period of 21 days. During the period of Harris hawk mortality, there were also two 1-year-old red kites (Milvus milvus) dying in the same collection. Those deaths were also acute with no premonitory signs. On necropsy those two red kites as well as the rst three dead Harris hawks were in good body condition and showed petechial haemorrhages and inammation of the ventricular wall and the myocardium, pancreatic congestion and haemorrhages, and moderate splenomegaly. Histologic ndings of the kites and the rst three dead Harris hawks were non-specic with severe acute multifocal to coalescent myocardial necrosis and haemorrhage, and moderate to severe acute lymphoid depletion in the bursa and spleen. Inclusion bodies were not detected. Those histologic ndings were consistent with any acute bacterial or viral infection. However, based on the similar presentation on necropsy and the close timing of the deaths, adenoviral infection as cause of death in the initially affected Harris hawks and the red kites is highly suspicious. The clinical course of this outbreak with non-specic initial necropsy ndings such as haemorrhages and inammation of internal organs, and intranuclear inclusion bodies becoming more apparent during the course of infection is consistent with a previous report (FORBES et al. 1997). Experimental infections of broilers with adenovirus showed that the frequency of intranuclear inclusions was greatest after ve days post-inoculation and that histologic lesions such as ventricular erosions appeared seven to nine days after inoculation (NAKAMURA et al. 2002).

132

This progression reiterates how difcult it can be to make an adenovirus diagnosis when only a single bird is affected and dies following a peracute or acute infection. It demonstrates the potential need of serial necropsy and histologic examinations of subsequent losses within an outbreak to elucidate the aetiology of an infection within a collection. It is notoriously difcult to isolate adenovirus infecting raptors on chicken embryo liver cells (GOUGH; GERLACH, personal communication, 2004). Atadenoviruses and Siadenoviruses show generally poor in vitro replication ability. The frog Siadenovirus could only be propagated on turtle heart cells, while Turkey adenovirus type 3 can be propagated in embryos or young birds, but not in conventional cell lines (BENK et al. 2004). Electron microscopy requires a virus particle concentration of greater than one million virus particles per millilitre of sample to give positive results (RITCHIE 1994). Therefore, negative results of virus isolation and electron microscopy do not exclude adenoviral infection. PCR is a rapid and sensitive way to screen clinical samples for the presence of microbial DNA. It also facilitates the acquisition of DNA templates for sequencing. Hexon is the major component of the adenovirus capsid. Although providing several highly conserved regions, there is considerate variation in members of the different genera. For conventional Aviadenoviruses, several hexonspecic PCR primers have been described (RAUE and HESS 1998), which gave negative results in the present cases. Consensus, highly degenerated primers for the detection of a short fragment of the adenoviral DNA-polymerase gene as well as an even more sensitive, nested PCR system, targeting the neighbouring DNA-polymerase gene, have been described (WELLEHAN et al. 2004). In the Budapest laboratory, this nested PCR system was used to generate DNA fragments for sequencing. The taxonomic place of this likely new adenovirus type is as yet preliminary, and needs further conrmation with the use of Siadenovirus-specic primers. As inclusion bodies of circovirus, polyomavirus and herpesvirus can resemble adenoviral inclusion bodies (RITCHIE 1995), further PCR was performed on the tissue samples to exclude those viruses. It is interesting that adenovirus was identied in Harris hawks and eagle owls, representing species that are often considered as less susceptible to a number of bacterial or viral diseases, compared with less hardy species such as American kestrels or Mauritius kestrels. One report states a high susceptibility of Mauritius kestrels to infection with adenovirus group I (Aviadenovirus) (FORBES et al. 1997). However, during the outbreaks there were no deaths in any kestrel species that were also kept in the collection. It is known that different serotypes of adenovirus express different pathogenicity and can pose a threat to different host-species (RITCHIE 1995, FORBES et al. 1997). Certain adenoviruses, especially the members of the genera atadenovirus and siadenovirus, have proved to be highly pathogenic, being capable of infecting multiple host species and causing experimentally reproducible specic diseases (BENK et al. 2004). The supposedly higher pathogenicity of Siadenovirus involved in these cases compared to Aviadenovirus serotypes might explain the development of disease in these new host species. It is also possible that the endoparasite infections found in some of the birds may have immunocompromised them such that the adenovirus infection could become clinically apparent.

133

Free-ranging pigeons and waterfowl may present a reservoir for adenovirus (RITCHIE 1995). However, the two collections are 78 km apart from each other and to our knowledge there were no reports about other deaths in birds due to adenovirus infection in proximity of those collections. Day-old chicks infected with adenovirus were found responsible for fatal adenovirus outbreaks in Mauritius kestrels (FORBES et al. 1997). The two collections described in this report were using the same food source to purchase their day-old-chicks. No further birds died in either collection after both were advised to feed mammalian-derived food items to their birds for the next few weeks. Prevention of adenovirus infection is difcult. Simply avoiding feeding avian-derived food is relatively reliable, but this is difcult for larger collections with respect to practicability and costs. However, when confronted with unexplained deaths within a collection of raptors, the immediate halt of feeding all avian-derived food is recommended. As wild pigeons or waterfowl can also serve as source of adenovirus infection, strict measurements of hygiene including the prohibition of any contact with wild birds or their faeces are necessary to help prevent disease outbreak in collection of birds of prey. To control adenoviral disease molecular biological techniques such as PCR and serology on avian-derived food items can be considered. However, the extensive variety of different genera and serotypes is likely to complicate matters. If a sufciently common adenoviral antigen could be found, then vaccination of raptors might be an interesting strategy.

CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. 9. BENK M, HARRACH B, BOTH GW, et al (eds): Virus taxonomy, VIIIth report of the International Committee on Taxonomy of Viruses., London: Elsevier/Academic Press 2004; 213 - 228. FORBES NA, SIMPSON GN, HIGGINS RJ and GOUGH RE. Adenovirus infection in Mauritius kestrels (Falco punctatus). J Avian Med Surg 1997; 11(1): 31 - 33. NAKAMURA K, OHYAMA T, YAMADA M, et al. Experimental gizzard erosions in specic-pathogen-free chicks by serotype 1 group I avian adenoviruses from broilers. Avian Dis 2002; 46(4): 893 - 900. RAUE R and HESS M. Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus. J Virological Methods 1998; 249(2): 37 - 315. REECE RL and PASS DA. Inclusion body hepatitis in a tawny frogmouth (Podargus strigoides). Aust Vet J 1985; 62: 426. RITCHIE BW. Adenoviridae. In: RITCHIE BW (ed): Avian viruses Function and control. Lake Worth: Wingers Publishing 1995; 313 - 334. SCHELLING SH, GARLICK DS and ALROY J. Adenoviral hepatitis in a Merlin (Falco columbarius). Vet Pathol 1989; 26: 529 - 530. SILEO L, FRANSON JC and GRAHAM DL. Hemorrhagic enteritis in captive American kestrels (Falco sparverius). J Wildl Dis 1983; 19: 244 - 247. STEHLE S. Krankheiten bei Greifvgeln (Accipitres) und bei Eulen (Strges)

134

mit Ausnahme der parasitren Erkrankungen. Tiermedizinische Dissertation. Tierrztliche Hochschule Hannover, Germany. 1965. Cited by GERLACH H. Viruses. In: RITCHIE BW, HARRISON GJ and HARRISON LR (eds): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing 1994; 862 - 948. 10. WELLEHAN JFX, JOHNSON AJ, HARRACH B, et al. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the Atadenoviruses. J Virol 2004; 78(23): 13366 - 13369.

AUTHORS ADDRESS
P. Zsivanovits DMedVet MRCVS Great Western Referrals, Avian and Exotic Department, Unit 10, Berkshire House, County Park Estate, Shrivenham Road, Swindon, SN1 2NR, Great Britain Email: P.Zsivanovits@gwreferrals.co.uk

135

Schubot Exotic Bird Health Center1, Department of Pathobiology, Texas A&M University, College Station, Department of Pathobiology2, Texas A&M University, United States of America

DIAGNOSIS, PREVENTION AND TREATMENT OF IRON STORAGE DISEASE USING THE EUROPEAN STARLING AS A MODEL
D. N. Phalen1, DVM, PhD, G. OLSEN1, BA, K. Russell2, DVM, PhD

KEYWORDS
Iron storage disease - Birds - Treatment - Prevention - Diagnosis - Diet - Deferoxamine - Phlebotomy - Liver iron

ABSTRACT
Iron absorption was compared in European starlings fed diets containing high iron, high iron with inositol and tannic acid, low iron, low iron with a meat-based dog food, or low iron with vitamin C. An iron concentration of 32 ppm in the diet was found to be inadequate to meet the physiological demands of the starlings, even when vitamin C or a meat-based dog food was added. The high iron diet caused increased liver iron concentrations to concentrations similar to those found birds that have died of iron storage disease (ISD). The addition of inositol and tannic acid to the high iron diet prevented an increase in liver iron. In a second experiment, starlings were fed an iron loading diet to induce high liver iron concentrations. Birds were then switched to low iron diets and treated with the low iron diet only, or low iron diets and either deferoxamine, phlebotomy, or the addition of inositol and tannic acid to the low iron diet. Phlebotomy and deferoxamine treatments reduced nonheme liver iron concentrations to safe levels in 16 weeks of treatment at similar rates. The low iron diet alone reduced nonheme liver iron levels at a much slower rate. The addition of inositol and tannic acid to the low iron diet had no impact on nonheme liver iron concentrations. These results suggest that both phlebotomy and treatment with deferoxamine are effective treatment options for birds with ISD. Total plasma iron, iron binding capacity, percent iron saturation and staining for iron in bone marrow aspirates were not found to be effective predictors of liver iron stores. However, hepatocytes obtained from liver core biopsies, when stained for iron, were found to accurately predict liver iron concentrations.

136

1 INTRODUCTION
Iron storage disease (ISD) is a life threatening disease of captivity that occurs in several species of toucan (Rhamphastidae), mynahs (Sturnidae), birds of paradise (Paradisaeidae) and quetzals (Pharomachrus spp.) (WARD et al. 1991, CRISSEY et al. 2000, DORRESTEIN et al. 2000). Iron storage disease is characterized by a massive accumulation of iron in the liver (ROSSANDER-HULTH and HALLBERG 1996). The pathogenesis of ISD is poorly understood but ultimately appears to result in heart failure and may in some cases cause liver disease. Left untreated ISD is fatal. ISD appears to occur in these species of birds because they are very efcient at absorbing iron from their diets, they do not down regulate iron absorption when iron replete, and they are fed diets containing excess iron in captivity (WARD et al. 1991, CRISSEY et al. 2000, DORRESTEIN et al. 2000, METE et al. 2001). The actual safe and adequate concentration of iron in these birds diets is not known, but appears to be less than 135 ppm. It is possible that addition of phytates and/or tannins to these birds diets would reduce iron absorption (SEIBELS et al. 2003, GAFFNEY et al. 2004). It is also possible that vitamin C and meat may enhance iron absorption (ROSSANDER-HULTH and HALLBERG 1996). The rst part of this study was designed to determine what the minimum dietary concentration of iron is and what effect supplementation would have on iron absorption. The second phase of the experiment was to look at the effectiveness of treating birds with with high liver iron concentrations with low iron diets, low iron diets containing a phytate and tannin, and low iron diets in combination with deferoxamine administration or phlebotomy. Finally, we examined the usefulness of plasma chemistry values, liver aspirate, and bone marrow aspirates for predicting liver iron concentrations.

2 MATERIALS AND METHODS

Effect of supplements on liver iron concentrations Iron absorption was compared in European starlings (Sturnus vulgaris) fed diets containing high iron (1585 ppm), high iron with inositol (a phytate) and tannic acid, low iron (32-34 ppm), low iron with a meat-based dog food, or low iron with vitamin C. Control starlings were euthanased immediately. Starlings on the high iron diet and high iron diet supplemented with inositol and tannic acid were euthanased at 6 and 16 weeks. The remaining birds were euthanased at 16 weeks (OLSEN et al. 2004a). Effect of treatment on liver iron concentrations European starlings were fed an iron loading diet (3235 ppm) for 31 days to induce nonheme liver iron concentrations approaching those in birds with that died with iron storage disease. Once high liver iron concentrations were obtained, all birds were switched to low iron diets (32 to 48 ppm) and divided into four treatment groups.

137

One treatment group was treated with the low iron diet only. Other treatments groups were treated with either deferoxamine (100mg/kg, SQ, q 24 hr, Desferal, Novartis Pharmaceuticals, East Hanover, NJ, USA), phlebotomy (1% of body q 7 d), or the addition of a phytate (inositol) and tannic acid to the low iron diet. Starlings were treated for 16 weeks (OLSEN et al. 2004b). Nonheme liver iron concentrations for both the supplement and treatment experiments were determined using a modication of the Torrance-Bothwell nonheme method and 0.5 g of liver obtained at necropsy from each starling (OLSEN et al. 2004a). Prediction of liver iron concentrations in the live bird Prior to euthanasia, birds were anesthetized and blood was collected from the right jugular vein, bone marrow was aspirated from the tibiotarsus, and liver core biopsies were obtained by repeated transabdominal insertions of a 23 gauge needle. Core biopsies of the liver were expressed onto glass slides by forcing air through the needle. Total plasma iron, iron binding capacity, and percent saturation was determined. Bone marrow and liver samples were stained with Prussian blue for iron and the number of iron containing cells and the intensity of their iron staining was recorded (RUSSELL et al. 2005). Statistical analysis Mean liver nonheme iron concentrations and mean weights for controls and each treatment group were compared using the two tailed t-test (P<0.05).

3 RESULTS
Effect of supplements on liver iron concentrations The diet with low iron concentration (32 ppm) was found to be inadequate to meet the physiological demands of the starlings, even when vitamin C or a meat-based dog food was added and liver iron concentrations decreased in all birds on these three diets. The high iron diet caused increased nonheme liver iron concentrations to concentrations similar to those found birds that have died of ISD. The addition of inositol and tannic acid to the high iron diet prevented an increase in nonheme liver iron (OLSEN et al. 2004a). Effect of treatment on liver iron concentrations Phlebotomy and deferoxamine treatments reduced nonheme liver iron concentrations to safe levels in 16 weeks of treatment at similar rates (190 ppm/week and 163 ppm/ week respectively). The low iron diet alone reduced nonheme liver iron levels at a slower rate (45ppm/week). The addition of inositol and tannic acid to the low iron diet had no impact on nonheme liver iron concentrations (OLSEN et al. 2004b).

138

Prediction of liver iron concentrations in the live bird Plasma iron, iron binding capacity and percent saturation where of no value in predicting the nonheme liver iron concentrations. Bone marrow aspirates contained very few iron positive cells and also could not be used to predict liver iron concentrations. Core biopsy of the starlings liver was found to be safe and in the majority of attempts resulted in a sample adequate for analysis. Iron stained preparations of the biopsy material consistently allowed the differentiation between birds will low to normal liver iron concentrations, birds with moderate liver iron concentrations, and birds with high iron concentrations (RUSSELL et al. 2005).

4 DISCUSSION
The results of the additive experiments suggest that the minimum and safe dietary iron concentration for birds susceptible to ISD is close to 50 ppm. They also suggest that the addition of a phytate or tannic acid or both to commercial diets may be an inexpensive way to prevent ISD in captive birds. These results also suggest that supplementation of diets that contain adequate iron with small amounts of fruit or meat will not signicantly impact iron absorption. The treatment experiment suggests that both phlebotomy and treatment with deferoxamine are effective treatment options for birds with ISD. It is possible that a combination of these treatments or an increased frequency of phlebotomy would result in a more rapid decline in liver iron concentrations and still be safe for the bird. The low iron diet was considered a useful adjunct to the treatment of ISD by other methods, but was considered to be an inadequate by itself. Although a combination of phytates and tannins prevented excess iron absorption, these additives did not accelerate a decline in liver iron concentrations. Fine needle core biopsies of the liver were found to be the only effective means of approximating the liver iron concentrations in the live bird. Based on necropsy ndings in birds that had this procedure, this technique appeared to be safe.

5 ACKNOWLEDGEMENTS
This work was funded by a generous donation from the Morris Animal Foundation and the Schubot Exotic Bird Health Center.

6 CITATION INDEX
1. 2. CRISSEY S, WARD A, BLOCK S and MASLANKA M. Hepatic iron accumulation over time in European starlings (Sturnus vulgaris) fed two levels of iron. J Zoo Wildl Med 2000; 31: 491-496. DORRESTEIN G, METE A, LEMMENS I and BEYNEN A. Hemochromatosis/ iron storage: new developments. Proc Conf Assoc Avian Vet, Portland 2000;233-238.

139

GAFFNEY S, WILLIAMS V, FLYNN P, et al. Tannin/polyphenol effects on iron solubilization in vitro. Bios 2004; 75: 43 - 53. 4. METE A, DORRESTEIN G, MARX J, et al. A comparative study of iron retention in mynahs, doves, and rats. Avian Pathol 2001; 30: 479 - 486. 5. OLSEN GP, RUSSELL K, DIERENFELD E, et al. Impact of supplements on iron absorption from diets containing high and low iron concentrations in the European starling (Sturnus vulgaris). Submitted: J Avian Med Surg 2004. 6. OLSEN GP, RUSSELL K, DIERENFELD E and PHALEN DN. A comparison of four treatment regimens for iron storage disease using the European starling (Sturnus vulgaris) as a Model. Submitted: J Avian Med Surg 2004. 7. RUSSELL K, OLSEN GP, DIERENFLED E, and PHALEN DN. Manuscript in preparation. 2005. 8. ROSSANDER-HULTH L and HALLBERG L. Dietary factors inuencing iron absorption - an overview. In: Iron Nutrition in Health and Disease. London: John Libbey & Company 1996; 105 - 115. 9. SEIBELS B, LAMBERSKI N, GREGORY C, et al. Effective use of tea to limit dietary iron available to starlings (Sturnus vulgaris). J Zoo and Wildl Med 2003; 34: 314 - 316. 10. WARD R, SMITH T, HENDERSON G and PETERS T. Investigation of the aetiology of haemosiderosis in the starling (Sturnus vulgaris). Avian Pathol 1991; 20: 225 - 232.

3.

AUTHORS ADDRESS
David N. Phalen, DVM, PhD, Dipl. ABVP (Avian) Schubot Exotic Bird Health Center, Texas A&M University, College Station, TX 77843-4467, United States of America Email: dphalen@cvm.tamu.edu

140

Department of Animal Science1, University of Camerino, Private Practitioners2, Italy

BIO-MOLECULAR STUDY OF A PROGRESSION OF HAEMOCROMATOSIS IN HILL MYNAHS (GRACULA RELIGIOSA)

G. Rossi1, S. Pesaro1, C. Peccati2, R. Ceccherelli2, P. Petroselli2

KEYWORDS
Haemochromatosis - Hill mynahs (Gracula Religiosa) - Cirrhosis - Hepatocarcinoma - Tumour markers.

ABSTRACT
Haemochromatosis is a disease characterised by an excessive enteric absorption of iron, subsequently stored in different organs, especially in liver. This pathology is rarely shown in animals, with the exception of some species of birds. In fact this disease has been most commonly seen in captive soft billed birds such as mynahs, toucans and birds of paradise and is rarely seen in these same species in the wild. It seems to occur more commonly in frugivores and insectivores. There is growing evidence to indicate that lories may also be a species vulnerable to this disorder. Studies show that hepatic iron overload may be one of the most important causes that induce the progression to liver brosis and hepatocarcinoma. The present study tries to demonstrate the different steps in the disease progression, by showing the expression of different and specic marchers of brosis, DNA alteration, cellular proliferation and neo-plastic transformation of the hepatocytes involved in different degrees of iron overload. The study demonstrates the feasibility of an experimental animal model for the evaluation of the progression to the hepato-cellular carcinoma in the human genetic disease by using the mynah birds.

1 INTRODUCTION
In human beings haemochromatosis is characterised by excessive accumulation of body iron, most of which is deposited in the parenchymal organs such as liver, pancreas, kidney and heart (BACON and HARRISON 2003). Because humans do not have a major excretory pathway for iron, haemochromatosis results either from a genetic defect causing excessive iron absorption or as consequence of parental administration of iron (BACON et al. 2001). Genetic haemochromatosis is an autosomal recessive disorder in which the gene of the disease is located on the short arm of chromosome 6, close to HLA gene complex (BACON and HARRISON 2003). In genetic

141

haemochromatosis there seems to be a primary defect in the intestinal absorption of dietary iron, leading to net iron accumulation. Excessive iron seems to be directly toxic to tissues by different mechanisms as: lipid peroxidation by iron-catalyzed free radicals reactions (BACON and BRITTON 1990); stimulation of collagen formation, and direct interactions of iron with DNA, leading to lethal injury or predisposition to hepato-cellular carcinoma (ELMBERG et al. 2003). Haemochromatosis in animal is infrequent conditions with the exception for some species of birds (DIERENFELD et al. 1994, STYLES 1999). In fact this disease has been most commonly seen in captive soft billed birds such as mynahs, toucans and birds of paradise (DIERENFELD et al. 1994) and is rarely seen in these same species in the wild. It seems to occur more commonly in frugivores and insectivores. There is growing evidence to indicate that lories may also be a species vulnerable to this disorder. In mynahs the clinical features of the disease are very similar to those observed in human genetic or primary forms. In chronic cases the principal manifestations include hepatomegaly, abdominal dilation, hypoalbuminaemia, ascites and, in terminal phases, dyspnoea and cardiac dysfunction (arrhythmia and cardiomyopathy). In our experience, some birds with severe cirrhosis or hepatocelluar carcinoma showed high degree of iron stored into hepatocytes. The aim of the study is the evaluation of the levels of expression of different and specic marchers of brosis, DNA alteration, cellular proliferation and neoplastic transformation in the hepatocytes, related to different degrees of iron stored into hepatocytes and other cellular types.

2 MATERIALS AND METHODS

During a period of three years, 35 hill mynahs (Gracula religiosa) and 1 blackbird (Turdus merula) were necropsied in our Laboratory (Department of Veterinary Sciences, University of Camerino Italy). Two mynahs and a blackbird that died from trauma without any signs of hepatic disease and normal biochemical parameters were used as control birds. The other mynahs examined, showed alterations in biochemical values and different degrees of hepatomegaly, liver brosis, nodularity of the parenchyma and, sometimes, ascites at necropsy. In four animals, a moderate cardiomegaly was also seen. Samples of liver, heart, spleen, lung, kidney, proventriculus, ventriculus, small intestine, pancreas, bursa and bone-marrow were collected from all birds, placed in 10% buffered formalin, and parafn embedded. For morphological and histochemical evaluations, histological sections, mounted on positively charged glass slides (Superfrost Plus, Fisher, Pittsburgh, PA), were routinely deparafnised and then stained with hematoxylin and eosin, Prussians blue stain (specyc to dye Fe+++ into the cells), and Van Gieson trichrome stain for evaluation of various degrees of brosis. For immunohistochemical tests, the same deparafnised sections were immersed in distilled water containing 15% H2O2 for 30 min at room temperature in order to inactivate endogenous peroxidases. The slides were rinsed in phosphate-buffered saline (PBS) three times for 2 min each, followed by a proteinase K (DAKO Corporation, Carpinteria, CA) treatment at room temperature for 6 min. In order to minimize nonspecic background staining, the sections were incubated in a serum blocking solution (10% non-immune horse serum in 1% bovine serum albumin dissolved in PBS) for 1 hr at 37 C. The sections were then stained with primary anti mutate-P53 (monoclonal antibody, Novocastra, London ), anti PCNA (monoclonal, Santa Cruz, California), anti CEA (monoclonal antibody, Novocastra, London), anti CA19.9 (monoclonal antibody, Novocastra, London), anti AFP (monoclonal antibody, Dako Co., Burlingame, UK), and anti Collagen type IV (polyclonal rabbit anti chicken;

142

Santa Cruz, CA). All antibodies were diluted 1:50 in PBS and incubated overnight in a 4 C moist chamber, rinsed in PBS and incubated with biotinylated secondary antibody (biotinylated anti-mouse or anti-rabbit immunoglobulins) for 45 min at room temperature. After three rinses in PBS, sections were incubated with enzyme conjugate (ABC-peroxydase complex, Vector, Burlingame, UK) for 45 min at room temperature, followed by incubation with 3,3-diaminobenzidine solution containing H2O2 as substrate-chromagen for 5 min. The slides were rinsed in distilled water, rapidly counterstained with haematoxylin and examined microscopically. Negative controls included apparently normal mynahs in which Prussians Blue (PB) reaction gave negative results in livers sections and tissues of black bird that represent a birds species not involved in pathological iron overload. Optical quantication of different degrees of intracellular iron stored were performed by scoring the Prussians Blue dye using the following scores: score 0= absence of iron in hepatocytes; score 1= groups of hepatocytes with weak PB stain; score 2= diffuse PB stain of hepatocytes; score 3= diffuse PB stain of hepatocytes with strong stain in groups of perivascular macrophages and Kupffer cells presence of extrahepatic hemochromatosis (PB stain of myocardiocytes, renal epithelium and septal macrophages of the lungs). Similar scale was used for quantication of different degrees of brosis in Van Gieson (VG) and Collagen type IV stained slides: score 0= absence of brosis; score 1= mild degree of perivascular brosis; score 2= moderate degree of perivascular brosis with irradiation of collagen between hepatocytes; score 3= cirrhosis with severe degree of perivascular and intercellular brosis, accompanied by nodules of hepatocytes regeneration. For immunohistochemical evaluation, ten 40x randomly selected elds were analysed for each sample and the number of positive cells/eld was recorded. The value of expression of each marker per sample was expressed as the mean value obtained from these ten values. All values obtained were statistically analysed and compared by using the T Student test.

3 RESULTS
Macroscopic examination of necropsied birds revealed various degrees of morphological alterations of the liver in 30 of 36 (83.4%) examined animals. In some cases the most evident alterations were represented by dark colouring and enlargement of the liver. In almost the 36% of analysed animals, varying degrees of ascites were observed and only in three cases a moderate cardiac dilation was also present. Results of necropsies are summarised in table 1. Table 1: Macroscopic appearance of the liver in necropsied birds
Examined Normal Hepatomegaly Liver Cirrhosis Cirrhosis with birds liver fibrosis neoplastic lesions 36 (100%) 6 (16,6%) 11 (30,5%) 9 (25%) 4 (11,1%) 2 (5,5%) Hepatocarcinoma 4 (11,1%)

Table 2 reports the data regarding the morphological aspect of the livers, the degree of Fe+++ stored in the hepatocytes, the degree of brosis express as degree of VG and collagen IV stains, and the mean of cells positive for PCNA, P53, AFP, CEA and CA19.9 per sample.

143

Table 2: Morphology and score of different parameters evaluated in birds liver.

Case 2687/3 Control Turdus merula 2681/a control normal mynah 2190/A control normal mynah 2233/c apparently normal mynah 632/1 apparently normal mynah 528/B apparently normal mynah 369 hepatomegaly 571 hepatomegaly 2494B hepatomegaly 2231 hepatomegaly 188/v hepatomegaly 670 hepatomegaly B2494/1 hepatomegaly B2494/2 hepatomegaly dark color B2494/3 hepatomegaly 805/B hepatomegaly congested B2156 hepatomegaly, discoloured B1504/A hepatomegaly, spotted in color 6439/B Hepatomegaly, congested B2982/A hepatomegaly Hematoxylin and eosin Normal Normal Normal Normal Normal Normal Normal Normal Groups of hypertrophic hepatocytes. Acute hepatitis with severe heterophilic inltrate. Chronic-active hepatitis with perivascular inltrate Chronic-active hepatitis with perivascular inltrate Normal Chronic hepatitis with diffuse perivascular accumulation of macrophages. Chronic hepatitis Perivascular brosis with congested liver. Different areas of fat degeneration. Fat degeneration Congested with fatty spot liver. Moderate degree of congestion Van Perl Gieson score score 0 0 0 1 0 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 PCNA P53 3 7 2 1 4 0 3 5 8 23 0 2 0 1 0 0 1 0 2 5 A F P 1 2 0 0 1 0 4 0 5 7 C E A 0 1 0 0 2 0 1 0 2 32 CA Collagen 19.9 IV 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0

30

11

27

1 2 3 1 1 1 0 1 3* 2

1 0 1 0 2 1 0 0

12 45 51 12 5 3 0 7 12 3

0 53 39 7 0 2 0 1

3 15 9 11 0 4 0 0 10

18 33 42 17 7 0 0 5 6

0 5 8 0 0 0 0 0 2 2

1 1 1 0 3 1 0 1

144

B2222B Morphological alteration. 832/1 Mild atrophy. 342/F Atrophy of right lobe 589/3 Normal B/2345 Atrophy 2122 Normal 1532/M Mild atrophy 783 Mild atrophy 3338 Atrophy of the left lobe B1928 Cirrhosis 2156/1 Cirrhosis B 3345 Cirrhosis B 2521 Cirrhosis B2156 Cirrhosis with umbilicate nodules B2222A Carcinomatous nodules 669A(A) Carcinoma. 669A(B) Carcinoma

Moderate degree of brosis Mild brosis Moderate degree of brosis associated to severe hepatocellular atrophy Mild brosis. Severe and diffuse brosis. Mild brosis Mild brosis Moderate and diffuse form of brosis. Mild brosis Regenerative nodules and brosis with severe morphological alterations Morphological alterations Regenerative nodules Marked morphological alterations Macronodular cirrhosis with hepatocarcinoma and chronic active hepatitis. Cholangiocellular carcinoma with severe brosis. hepatocellular carcinoma Severe morphological alterations with high degree of hepatocytic dysplasia Nodules of hepatocarcinoma.

1 2

2 1

6 22

0 5

13 7

11 9

0 0

3 2

11

12

2 3 1 2 3 2

1 3 1 1 2 1

13 10 5 11 21 4

1 13 0 2 11 7

3 18 0 12 18 23

0 26 0 17 29 21

0 3 0 1 0 2

2 3 1 1 3 2

27

22

28

35

12

2 3 3*

3 3 3

31 41 27

19 12 21

17 32 45

29 43 18

7 1 11

3 3 3

65

93

105 99

31

3 3

3 2

57 42

102 123 75 148 97 112

143 3 32 3

37

93

128 81

15

669A(C) 3 1 115 111 132 121 86 2 Carcinoma B2453 Cholangiocarcinoma 3 Carcinoma 3 81 103 201 85 167 3 with cirrhosis. with brosis *Extrahepatic haemochromatosis characterized by myocarditis, renal epithelium and alveolar and interstitial lung macrophages staining by PB.

145

Statistical analysis revealed a positive good correlation (p<0.05) between the following parameters: PB stain and VG/collagen type IV stain; PB stain and PCNA, P53 and AFP expression by hepatocytes. For which concern the correlation between PB stain and CEA or CA 19.9 antigen expression, a positive correlation between PB stain and CEA was observed only in hepatocellular carcinoma , while in cholangiocarcinoma a correlation was observed between PB stain and CA19.9 expression.

4 DISCUSSION
Iron overload is a pathology not frequent in animals but, in species in which the pathology is described, a genetic predisposition is suggested. The mynah represents the most commonly reported bird group with susceptibility for iron overload and it was recently shown that the cause of this patho-physiological condition is high uptake and retention of dietary iron (DIERENFELD et al. 1994, GOSSELIN and KRAMER 1983, STYLES 1999). Enterocytes isolated from minahs shows that Fe+++ upatke is much lower than Fe++; although liver iron, present only in hepatocytes (typical nding that characterized primary forms of overload), was at least 10-fold higher in mynahs than in other species as chickens, quails or doves (DORRESTEIN et al. 2001). Calculated values for the uptake kinetics of the probable membrane transporter suggest that mynah bird enterocytes have a signicantly higher limiting uptake rate, due to possible increase in the number of transporters when compared with non susceptible species. In human beings, the accumulation of iron in the liver may induce brosis, cirrhosis and, in some cases, to promote the neoplastic transformation of hepatocytes (BACON and BRITTON 1990, ELMBERG et al. 2003). In this study, we have demonstrated that in mynahs, the high degrees of hepatocellular iron storage correlate positively with the expression of some antigens as mutate form of P53, AFP, PCNA and Collagen type IV. P53, a product of the p53 gene, acts in the nucleus and has the ability to inhibit the cell cycle; this protein is called in apply emergency brakes when the DNA is damaged by exposure to mutagenic chemicals as iron or free radicals (BACON and BRITTON 1990). In view of these activities, P53 in normal wild form has been rightfully called a guardian of the genome. -fetoprotein (AFP) is a well-established cancer marker encountered during cancer arising principally in the liver; as with CEA, hepatitis may cause modest elevations of this marker but the marker may still provide the evidence of hepatocellular carcinoma. PCNA is a 36kD protein that operates as DNA polymerase during phase S of cell cycle and cooperates during DNA synthesis. An increase of PCNA expression indicates an high mitotic index rates in hepatocytes population. Finally collagen IV, an amorphous-non brillar form of interstitial collagen that increases during brosis and cirrhosis, indicates this transformation directly related to iron overload. Interesting, the study demonstrates a good correlation between the expression of CEA and hepatocellular carcinoma and CA.19.9 in carcinoma of cholangial origin (HAGLUND et al. 1991). These ndings suggest that mynahs are a good model for the study of human idiopathic form of haemochromatosis and for evaluations of different factors that induce the progression of the disease into brosis, cirrhosis and hepatocarcinoma.

146

5 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. 9. BACON B.R. and BRITTON R.S. The pathology of hepatic iron overload: a free radical mediated process. Hepatology 1990; 11: 127 137. BACON B.R. and HARRISON S.A. Hereditary hemochromatosis. J Hepat 2003; 38 14 23. BACON B.R., PARKKILA S., NIEMEL O., et al. Molecular aspects of iron absorption and HFE expression. Gastroenterology 2001; 121; 6: 1489 1496. DIERENFELD E.S., PIOIS M.T. and SHEPPARD C.D. Hemosiderosis and dietary iron in birds. J Clin 1994; 124: 26858 26868. DORRESTEIN G.M., METE A., MARX J.J.M., et al. A comparatives study of iron retention in Mynahs, Doves and Rats. Avian Pathol 2001; 30: 479 486. ELMBERG M., HULTCRANTZ R., EKBOM A., et al. Cancer risk in patients with hereditary hemochromatosis and in their rst-degree relatives. Gastroenterology 2003; 125; 6: 1733 1741. GOSSELIN S.J. and KRAMER L.W. Pathophysiology of excessive iron storage in mynah birds. J Am Vet Med Assoc 1983; 183: 11. HAGLUND C, LINDGREN J. and ROBERTS P.J. Difference in tissue expression of tumour markers CA 19.9 and CA 50 in hepatocellular carcinoma and cholangiocarcinoma. Br J Cancer 1991; 63: 386 389. STYLES D.K. Hemochromatosis: a metabolic disease of softbills. Proceedings of the Exotic Bird Breeders Symposium. Foothill Bird Society 1999; 86 88.

AUTHORS ADRESS
G. Rossi, DVM, PhD Department of Animal Science, University of Camerino, Via della circonvallazione 93/95 62014 Matelica MC, Italy Email: giacomo.rossi@unicam.it

147

Hospital Zoologic Badalona1 SL, Badalona, Veterinary practitioner2 Departamento de Medicina y Cirugia Animal3, Facultat de Veterinria, Universitat Autnoma de Barcelona, Barcelona, Spain

CIRCOVIRUS INFECTION IN A CANARY BREEDING FLOCK

J. Grfols1 DVM, MS, F. Bargall1 DVM, D. Perpin2 DVM MS, A. Ramis3 DVM, PhD, Dipl ECVP

KEYWORDS
Canary - Circovirus - Secondary infections - Immunosuppression

ABSTRACT
Circovirus infection was identied in a breeding canary ock with an outbreak of high morbidity and mortality. The birds died with signs of conjunctivitis, progressive weight loss, diarrhoea, apathy and nervous signs just before death. Some animals died suddenly without previous signs. Necropsy and histopathology ndings were consistent with immunospression and secondary infections. Circovirus was detected in tissues by DNA in situ hybridization and PCR.

1 INTRODUCTION
Circoviruses are the smallest pathogenic DNA animal viruses that have been described (WOODS and LATIMER 2000, RITCHIE, 1995). Members of the genus Circovirus (family Circoviridae) that infect birds include psittacine beak and feather disease virus (PBFD), pigeon or columbid circovirus (CoCV), goose circovirus (GCV) and canary circovirus (CaCV) (WOODS and LATIMER 2000, PHENIX et al. 2001, TODD et al. 2001). Circovirus infections are commonly associated with immunodeciency-related diseases that can be fatal (PHENIX et al. 2001). In canaries, circoviral infection has been reported both in adults (TODD et al. 2001) and neonates (GOLDSMITH 1995), but data regarding clinical signs, mortality and morbidity rates and infection progression are scarce. This paper documents the occurrence of circoviral infection in a canary breeding ock and describes clinical history and signs, post-mortem analysis and diagnostic tests results.

148

2. CASE REPORT
In August 2003 a canary breeder reported high mortality and morbidity in his breeding ock. Signs observed varied among individuals and included conjunctivitis, progressive weight loss, dehydration, diarrhoea, apathy, and nervous signs before death. Some animals died without previous clinical signs. The breeder had been treating affected animals with metronidazole and reported a slight improvement for some days, after which mortality remained high. Laboratory studies included fecal analysis, postmortem examinations and blood work of some live and dead animals. Faecal analyses were negative in 7 live canaries tested. Blood analyses were performed in 2 live birds with different results. One animal showed severe immunosuppression with leucopenia, heteropenia, lymphocytosis and monocytosis. The other bird showed leucocytosis with monocytosis. Gross necropsy ndings were inconsistent, but histological lesions included hepatitis, purulent meningitis, lymphocytic depletion and focal necrosis in the spleen. In one animal Pseudomonas aeruginosa and Escherichia coli was culture from kidney, lung, and liver. DNA in situ hybridization was positive for Circovirus in one of two animals, and PCR was positive for Circovirus in other two of two animals. Results of several PCRs and DNA in situ hybridisation are still going on at the time of writing this manuscript and will be discussed on the presentation. Mortality continued for two months, but the owner refused performing more tests.

3 DISCUSSION
Circoviral infection was rst reported in canaries in 1995, as a condition called Black Spot disease (GOLDSMITH 1995). This condition included signs like abdominal enlargement, gallbladder congestion, and failure to thrive (GOLDSMITH 1995). Subsequently this virus have been cloned, sequenced and named as canary circovirus (CaCV) (PHENIX et al. 2001; TODD et al. 2001). Currently, the genus Circovirus of the family Circoviridae comprises porcine circoviruses types 1 and 2 (PCV1 and PCV2), psittacine beak and feather disease virus (BFDV), and pigeon or columbid circovirus (CoCV), with goose circovirus (GCV) and canary circovirus (CaCV) being tentatively added to this genus (PHENIX et al. 2001, TODD et al. 2001). Circovirus-like infection has also been describe in a southern black-backed gull (Larus dominicanus), but the virus was not sequenced (TWENTYMAN et al. 1999). Circovirus infections are commonly associated with immunodeciency-related diseases that are potentially fatal (PHENIX et al. 2001, TODD 2000). Canary circovirus seems to induce immunodeciency by depletion or necrosis of the lymphoid organs, mainly spleen and bursa of Fabricius (GOLDSMITH 1995). Birds die of secondary infections, so clinical signs, necropsy ndings, and morbidity and mortality can show a wide range of variation depending on the agent or agents involved. The description of GOLDSMITH (1995) of a typical condition for canary circovirus called Black Spot should be discarded as these signs are probably produced by secondary infections, and posterior description of infections (TODD et al. 2001), including the present paper, did not report a similar syndrome. Finally, canary circovirus should be suspected in cases of mortality affecting several animals, especially when histopathology shows depletion or necrosis of the lymphoid organs.

149

4 REFERENCES
1. 2. GOLDSMITH TL. Documentation of passerine circoviral infection. Proc Assoc Avian Vet, Nashville 1995, 349 - 350. PHENIX KV, WESTON JH, YPELAAR I, et al. Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae. J Gen Virol 2001; 82: 2805 - 2809. RITCHIE BW. Avian Viruses: Function and Control. Lake Worth: Wingers Publishing Inc; 1995. TODD D. Circoviruses: immunosuppressive threats to avian species: a review. Avian Pathol 29: 373 - 394. TODD D, WESTON J, BALL NW, et al. Nucleotide sequence-based identication of a novel circovirus of canaries. Avian Pathol 2001; 30: 321 - 325. TWENTYMAN CM, ALLEY MR, MEERS J, et al. Circovirus-like infection in a southern black-backed gull (Larus dominicanus). Avian Pathol 28: 513 - 516. WOODS LW and LATIMER, KS. Circovirus infection of nonpsittacine birds. J Avian Med Surg 2000; 14(3): 54 - 163.

3. 4. 5.

6.

7.

AUTHORS ADDRESS
Jordi Grfols Zoolgic Badalona Veterinria Conquesta 74, 08912 Badalona (Barcelona), Spain Email: jordi.grifols@hzb.es

150

Free University of Berlin, Faculty of Veterinary Medicine Institute of Poultry Diseases Knigsweg 63, 14163 Berlin Germany

CENTRAL NERVOUS SYMPTOMS CAUSED BY THIAMINE DEFICIENCY IN JUVENILE GOSHAWKS (ACCIPITER GENTILIS)
M. Carnarius, H. M. Hafez, A. Henning, H. J. Henning, M. Lierz

KEYWORDS
Vitamin B - Thiamine deciency Beriberi Nutrition Raptors - Birds of prey Goshawk

ABSTRACT
In the last 2 years an increased number of juvenile goshawks (Accipiter gentilis) with central nervous symptoms were presented to the Institute of Poultry Diseases of the Free University Berlin, Germany. The birds were between 45-55 days old and were fed with thawed day-old chicks. As a diagnosis a thiamine deciency was suspected. The last case, a 45 day old female goshawk was admitted with irregular attacks of central nervous signs like opisthotonus, rotations and somersaults especially when the bird was handled or stressed. Comparable to the goshawks presented before the bird always preened after an attack. The goshawk was wild caught at the age of approximately 20 days and received a diet of thawed day-old chicks. In spite of these central nervous attacks the bird showed a normal appetite. The bird demonstrated a thiamine content of 2.2g/l in plasma while all other blood values examined revealed normal results. As reference values for thiamine level in goshawks are not presented in the available literature. A study was performed to establish comparable values for goshawks. 6 healthy captive bred goshawks at the age of 45-55 days showed an average of 113g/l (95-120g/l). 4 adult captive goshawks at the age between 10-23 years showed an average of 97.25g/l (44-149g/l). Two healthy free ranging juvenile goshawks showed a level of 133g/l and 162 g/l and 3 starved free ranging juvenile goshawks showed results of 51g/l, 91g/l and 123g/l. The therapy of the bird with the suspected thiamine deciency was an initial intramuscular injection of 4 mg/kg thiamine hydrochloride, continued by an oral gavage of 2mg/kg twice daily. Already after the rst injection of thiamine the symptoms resolved. After 5 days treatment the blood value of thiamine raised to 476g/l. Therefore a continuous therapy of 2mg/kg thiamine hydrochloride once daily for a few days and a change of the diet are recommended as treatment.

151

1 INTRODUCTION
Thiamine is a water soluble and heat-labile vitamin out of the group of B vitamins. Thiamine (vitamin B1) is mainly existent in its active form thiamine pyrophosphate (TPP, also called thiamine diphosphate, ThDP) (VOET, VOET 1995). The thiamine pyrophosphate is an essential cofactor of decarboxylation reactions in many metabolic pathways, for example in the citric acid cycle. It is also a coenzyme of the transketolase. Furthermore TPP plays a role in the release of the neurotransmitter acetylcholine (PARKHOMENKO et al. 1996). It is involved in the synthesis of collagen, improving cognitive function, and maintaining memory (HALLIDAY et al. 1994). This vitamin is a factor in the normal intestinal function and in the health of cardiovascular and nervous systems (WORLD HEALTH ORGANIZATION 1999). Thiamine is neither synthesized nor stored in signicant amounts by the tissues of most vertebrates; consequently it is required in their diets (VOET and VOET 1995). Thiamine is found in nearly every animal and herbal aliment but generally only in few amounts. The highest concentrations of thiamine are found in un-grinded cereals, yeasts, legumes, in liver, heart and kidney. Day-old chicks are commonly used as feed in captive raptors. Whole day-old chicks contain an average of 0.065mg thiamine per 100 g. In comparison, quails contain about 0.18 mg/100g. On the other hand juvenile rats contain about 0.31mg/100g, adult rats 1.33mg/100g and mice 0.02mg thiamine per 100g (FORBES and FLINT 2000). A healthy adult human being needs an intake of 5.6 mol (1.7 mg) thiamine per day or 0.30-0.36 mol (0.10-0.12 mg) thiamine per 1000kJ and day (LFFLER 1998). At the moment there are only a few studies about the required intake of thiamine in birds. BRUE (1994) state a recommended allowance of thiamine for maintenance about 5ppm per day for most psittacines and the commonly kept passerines. OLKOWSKI and CLASSEN (1996) explored in chickens the thiamine status in blood and tissues in response to a wide range of dietary supplementation of thiamine. Blood thiamine concentration in those birds supplemented with 8 mg/kg body weight thiamine increased at day 7 and remained relatively constant. A thiamine deciency can arise from decreased intake or absorption, from increased turnover or from loss of thiamine by enteritis or coccidiosis (MANORE 2000). Furthermore a thiamine deciency can occur from an increased amount of enzymes that require thiamine or from interference by other ingredients of nutrition, for example aatoxin (VOIGT et al. 1980), or by drugs like amprolium (ANJALI et al. 2000) or tannin (BEGOVIC et al. 1980). The requirements of thiamine are increased when carbohydrates are taken in large amounts and during periods of increased metabolism, for example, fever, muscular activity, growth and hyperthyroidism (WORLD HEALTH ORGANIZATION 1999). In birds fed large amounts of raw sh or sh that has had an opportunity to sit without inactivation of the enzyme thiaminase, thiamine deciency may occur. The probability of a reduction in thiamine as a result of thiaminase activity is dependent upon a number of factors, for example the species of sh fed (ROUDYBUSH 1997). Birds, above all chickens, turkeys and quails, were often used to study the pathogenesis and the symptoms of beriberi for human medicine (DJOENAIDI et al. 1995) as well as the histological signs of thiamine deciency (ANJALI et al. 2000). In chicken, histologically the deciency of vitamin B1 demonstrates a polyneuropathy

152

with axonal degeneration, minimal segmental demyelination (DJOENAIDI et al. 1995), congestion and oedema of brain (ANJALI et al. 2000). Furthermore, a thiamine deciency leads to a degeneration of the cells lining the duodenal crypts of Lieberkuhn, with dilatation and lling of the crypts with cellular debris and necrotic cells, and vacuolation of the pancreatic acinar cells with hyaline body formation (GRIES et al. 1972). Moreover degeneration of testes and ovaries and atrophy of liver can be observed (ANJALI et al. 2000). In psittacines a thiamine deciency may lead to loss of appetite, opisthotonus, seizures and death. But a deciency of thiamine is uncommon in birds on a seed diet because seeds and grains generelly contain a sufcient amount of thiamine. Free ranging honey-eaters in urban areas of southern Australia may develop thiamine deciency during winter (MACWHITER 1994). In a single case of feeding polished rice to adult cockatiels, head tremors occurred before the experiment was discontinued. These signs were consistent with the polyneurotic signs observed in poultry (ROUDYBUSH 1997). Reports about a suspected thiamine deciency in birds of prey are rare and un-detailed. For example, WARD already described in 1971 a case of a peregrine falcon with opisthotonus. He suspected a thiamine deciency after a long period of unsuccessful treatments and a sudden improvement of the symptoms after a treatment with 2 days primidone (125 mg once daily) and 5 days intramuscular injections of thiamine hydrochloride (1 mg/day). Furthermore the diet of this peregrine falcon was changed from young cockerels into whole pigeons and quails. PERICARD and ANDRAL (1993) described several cases of nervous disorders called staggers in free ranging rescued griffon vultures (Gyps fulvus) of South Central France and the French Pyrenees. They were fed with dead sheep (including viscera). The affected birds turned round quickly, one wing half extended, the neck stretched out and the beak half open, for one to three minutes. During one year 11 birds died. Chlamydophila psittacii, a pathogenic E. coli, Salmonella, Nematodes and viral encephalitis were found in some of the birds respectively. The only treatment the remaining birds got was an injection of vitamin B1 and vitamin B6. In addition levamisole and niclosamide base were applied orally followed by a vitamin and mineral complement added to the feed. Hereupon the affected vultures improved. Because of this improving a thiamine deciency was suspected. After all in none of the cases the diagnosis was conrmed. For a diagnosis of a thiamine deciency in human medicine, the decrease of the enzyme transketolase in the erythrocytes is used. Consequently the pentose phosphates in the erythrocytes are increased. This can be measured before clinical symptoms appear (LFFLER 1998). The present paper describes central nervous symptoms in juvenile goshawks due to thiamine deciency and laboratory approach to determine the plasma thiamine level in goshawks.

Case report
In the last 2 years an increased number of juvenile goshawks (Accipiter gentilis) with central nervous symptoms were presented to the Institute of Poultry Diseases of the Free University Berlin, Germany. Typically the birds showed these disorders at the age of 45-55 days and were fed with thawed day-old chicks. As the birds improved after a thiamine supplementation a thiamine deciency was suspected. The therapy

153

of such birds was an initial intramuscular injection of 4 mg/kg thiamine hydrochloride, continued by an oral gavage of 2mg/kg twice daily. Already after the rst injection of thiamine the symptoms resolved. After 5 days treatment the blood value of thiamine raised to 476g/l. Therefore a continuous therapy of 2mg/kg thiamine hydrochloride once daily for a few days and a change of the diet are recommended as treatment. The present case, a 45-days-old, female goshawk, showed severe central nervous disorders with opisthotonus and rotations, similar to the birds presented before. This bird was wild caught at the age of approximately 20 days and fed with thawed dayold chicks. Typically this goshawk showed attacks with central nervous symptoms when the bird was handled or stressed. Immediately after such an attack the bird started to preen and showed a normal appetite. After a general, neurological and radiographic examination a thiamine deciency was suspected due to the experience with the goshawks presented before. Blood was taken and tested for aspartate aminotransferase, bile acids, total protein, albumin, cholinesterase, uric acid, creatine kinase, lactate dehydrogenase, phosphate, calcium, potassium and thiamine. Thiamine level was 2.2g/l while all other blood values were within reference ranges. For a clear diagnosis of a thiamine deciency comparable reference values of thiamine in blood are missing. Therefore it was tried to establish those.

2 MATERIAL AND METHODS

In total 11 juvenile and 4 adult goshawks were used to determine the level of vitamin B1 in blood-plasma. Six juvenile birds of both genders were captive bred from a private breeder and at the time of blood sampling 45-55 days old. They were fed with a mixed diet of fresh killed rats, pigeons and thawed day-old chicks. The 4 adult goshawks came from the same breeder and they receive only thawed day-old chicks. 5 juvenile goshawks of both genders were found in the wild and brought to the institute by private persons. 2 birds were generally healthy, found freshly injured, 3 birds were starved. All birds received a detailed clinical examination, and the birds from the wild in addition a radiological and, after blood sampling, an endoscopic examination. A blood sample was taken from every bird from the V. metatarsalis plantaris supercialis medialis into a Lithium-Heparin tube. The blood was centrifuged 5 minutes, 5000 U/min. The plasma was examined for aspartate aminotransferase, bile acids, total protein, albumin, cholinesterase, uric acid, creatine kinase, lactate dehydrogenase, phosphate, calcium, potassium and thiamine. For testing thiamine values a sample of 250 l from EDTA plasma is used. First proteins were denatured and afterwards the enzymatic hydrolysis is accomplished with acidic phosphatase. The whole vitamin B1 (consists of mono-, di-, triphosphate derivates of thiamine and the free thiamine) is oxidised into bluish coloured thiochrome and detected with HPLC/uorescence method (HPLC = High Performance Liquid Chromatography). The HPLC analysis is carried through a HPLC system LC 6 apparatus via Lichrosphere 100 C18-columns, 5 m as well as citrate buffer-methanol-alloy (VET MED LABOR 2001).

154

3 RESULTS

Table 1. Results of the plasma thiamine content of 15 goshawks in comparison to a case with CNS symptoms and a suspected thiamine deciency. Thiamine in plasma Goshawk No Sex Age Characteristics (g/l) Wild caught at the age of 20 days, Case f 45 days 2.2 CNS disorders 50 days 50 days 55 days 55 days 55 days 60 days 5 years 10 years 15 years 20 years 70 days 75 days 65 days 70 days 75 days Captive bred, healthy Free ranging, healthy Captive bred, healthy Captive bred, healthy Captive bred, healthy Captive bred, healthy Captive bred, healthy Captive bred, healthy Captive bred, healthy 116.0 120.0 116.0 95.0 149.0 106.0 90.0 44.0 162.0 133.0 91.0 123.0 Free ranging, starved, anaemia 51.0 Captive bred, healthy 117.0 Captive bred, healthy 114.0

The examination of all above mentioned blood parameters (except thiamine) revealed results within usual ranges. The obtained results of thiamine and of the clinical examination are presented in table 1.

155
Free ranging, healthy Free ranging, humerus fracture, starved Free ranging, starved

10

11

12

13

14

15

4 DISCUSSION A juvenile goshawk was presented with central nervous symptoms. As all examinations revealed no pathological ndings a vitamin B1 deciency was suspected. This especially due to the experience that similar symptoms in juvenile goshawks were observed in the past and responded to thiamine supplementation. The bird had a thiamine content in plasma of 2.2g/l. As reference values for vitamin B1 in goshawks are missing a deciency was not proved in this case. Therefore other goshawks were used to detect the usual range of thiamine in plasma for comparing with the presented case. The blood thiamine value (2.2g/l) of the presented bird was much lower compared to the other juvenile goshawks tested, indicates a thiamine deciency. The six juvenile, captive bred and healthy goshawks showed thiamine contents in blood between 95-120 g/l. Even the three free ranging, juvenile but starved goshawks had thiamine values in blood of 51g/l, 91g/l and 123g/l. interestingly the two free ranging and healthy juvenile goshawks showed thiamine contents of 133g/l and 162g/l. The inhomogeneous constitution of the tested group of birds, juvenile and adult ones, received different feed, does not allow the establishment of statistical proved reference values for thiamine. Additionally only 15 goshawks were tested for thiamine in plasma in this study. Therefore the given values are just for orientation and reference values have to be established in further studies. Based on the results of the 12 healthy birds the average of the plasma thiamine values is 113.5g/l and therefore much higher than the value of the presented bird. Even one of the starved goshawks showed a result of 51g/l thiamine in plasma and showed no clinical symptoms of a thiamine deciency. Another proof of the deciency of vitamin B1 in the presented bird is the successful therapy with thiamine hydrochloride only. Determination of thiamine blood values is easy and appropriate to a diagnosis of thiamine deciency. Additionally this method is easier than the one used in human medicine where not the thiamine in plasma or serum but the increased amount of pentose phosphates in the erythrocytes were measured. Moreover this method has not been used for birds so far. Seeing the regular onset of such disorders in juvenile goshawks around 45-55 days on a thawed day-old chicks diet it is suggested that the feed and its thiamine content plays an important role at the development of a thiamine deciency. Maybe these day-old chicks did not have enough amount of thiamine because of the inadequate supply of thiamine to the parents. It was found that maternal supplementation of thiamine increased content in the heart of the offsprings. Also the TPP content in the heart increased in response to both maternal and offspring supplementation. Additionally the thiamine supplementation in the offspring diets increased blood TPP (OLKOWSKI, CLASSEN 1999). FORBES and FLINT (2000) showed that the vitamin B1 content in day-old chicks is not as high as in other diets such as rats. Furthermore the kind of freezing and the storage time can also have an effect on the thiamine content of day-old chicks. Food kept for protracted periods in domestic and commercial freezers deteriorates in nutritional quality, particularly in terms of water soluble vitamins and vitamin E. Freezing is a drying process and long-term storage (unless sealed) can reduce the water content of feed. Thats why feed should not be kept stored frozen for more than 3 months (FORBES and FLINT 2000). It might

156

also play a role that partial defrosting of the day-old chicks during transport from the breeder to a dealer and to the falconers is responsible for their reduced vitamin B1 content. Based on all these facts a pure diet with thawed day-old chicks cannot be recommended for goshawks. Rodents (above all rats) including their livers seem to be more adequate at least as part of the diet. Slight clinical symptoms might resolve by changing the diet. It is suggested that the high incidence of thiamine deciency in young birds compared to adults is connected with their increased requirement of thiamine because of their growth. Also it can be possible that the enteral enzymes of juvenile birds are not yet fully developed. So the enzymes cannot convert enough TPP into thiamine which can be absorbed from the intestine, but this seems not very likely. Goshawks present CNS symptoms in a typical way. They show irregular attacks with opisthotonus, rotations and somersaults above all when the goshawks were handled or stressed. It is conspicuous that the diseased birds always showed a normal appetite and preened after such an attack with central nervous disorders. As therapy for the presented bird it was given an initial intramuscular injection of 4mg/kg thiamine hydrochloride, continued by an oral gavage of 2mg/kg twice daily. After 5 days of treatment the blood value of thiamine increased to 476g/l. This excessive high level in the blood value shows that this thiamine application rate was overdone. Therefore half of the dose should be far enough but further studies are necessary to establish a recommendable treatment. On the other hand vitamin B1 as a water soluble vitamin can quickly be excreted and overdose might not be a big problem. According to the available literature it is the rst time that a vitamin B1 deciency in goshawks was proven and blood values of vitamin B1 for orientation were provided.

5 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. ANJALI et al. Sequential pathological changes in experimental thiamine deciency in Japanese quail (Coturnix coturnix japonica). Indian J Vet Path 2000; 24:1; 16 - 18. BEGOVIC S et al. Effects of tannin and pyrogallol on the course of experimental B-avitaminosis in fowls, with particular reference to nervous disorders. Veterinaria, Yugoslavia 1980; 29: 1 - 2, 177 - 179. BRUE RN. Nutrition. In RITCHIE BW, HARRISON GJ and HARRISON LR (ed): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing, Inc. 1994; 71. DJOENAIDI W et al. Experimentally induced beriberi polyneuropathy in chickens. Electromyogr Clin Neurophysiol 1995; 35(1): 53 - 60. FORBES NA and FLINT CG. Raptor Nutrition. Honeybrook Farm Animal Foods 2000; 23 - 24, 29 - 30. GRIES CL et al. The pathology of thiamine, pantothenic acid and niacin deciencies in the chick. J Nutrit 1972; 102(10): 1269 - 1285. HALLIDAY G, CULLEN K and HARDING A. Neuropathological correlates of memory dysfunction in the Wernicke-Korsakoff syndrome. Alcohol Alcohol Suppl 1994; 2: 245 - 251.

157

8.

LFFLER G. Stoffwechsel der Kohlenhydrate. In Lfer G, Petrides PE. Biochemie und Pathobiochemie. 6th edition. Berlin: Springer-Verlag 1998; :387-9 9. LFFLER G. Vitamine. In LFFLER G and PETRIDES PE. Biochemie und Pathobiochemie. 6th edition. Berlin: Springer-Verlag 1998; 649 - 651, 664 - 665. 10. MACWHITER P. Malnutrition. In RITCHIE BW, HARRISON GJ and HARRISON LR (editors). Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing, Inc., 1994; 856.

The full list of references is available from the author

AUTHORS ADRESS
M. Carnarius Free University of Berlin, Faculty of Veterinary Medicine, Institute of Poultry Diseases, Knigsweg 63, 14163 Berlin, Germany Email: geuegelkrankheiten@vetmed.fu-berlin.de

158

Clinic for Birds and Reptiles1, Department of Small Animal Medicine, Faculty of Veterinary Medicine, University of Leipzig; Institute for Virology2, Faculty of Veterinary Medicine, University of Leipzig, Germany

A COMPREHENSIVE STUDY ON A DISEASE COMPLEX ASSOCIATED WITH PIGEON CIRCOVIRUS INFECTION, YOUNG PIGEON DISEASE
V. Schmidt1, R. Raue2 and M-E. Krautwald-Junghanns1

KEYWORDS
Young pigeon disease - YPD - Pathology - Immunosuppression - PiCV - E. coli

ABSTRACT
A disease in young racing pigeons, associated with high morbidity and mortality rates, was designated as young pigeon disease (YPD) by pigeon breeders. The aetiological agent remained unknown. In order to collect more convincing data, a comprehensive study was performed with pigeons present in German lofts with or without clinical outbreaks of YPD. Clinical, pathological, histopathological, haematological, parasitological and microbiological examinations were performed. Pigeons in their 4th to 12th week of life exhibited those clinical signs more frequently and more severely than pigeons of other ages. Numerous pathological and histopathological ndings were observed, most probably caused by the multitude of pathogens which were isolated. Intranuclear inclusion bodies were observed in various organs especially in the bursa of Fabricius. In contrast to other viral pathogens, the presence of the genome of pigeon circovirus (PiCV) was demonstrated in lymphoid tissues from all pigeons with YPD. The results of this comprehensive study indicate that YPD is a multifactorial disease in which PiCV might be a crucial factor, possibly by inducing immunosuppression in infected birds.

1 INTRODUCTION
A disease in young racing pigeons associated with high morbidity and mortality rates reported from parts of Central Europe, mainly Germany, was designated as young pigeon disease (YPD) or young bird sickness; designations such as swollen gut syndrome describing pathological observations were also used by pigeon fanciers and veterinarians. Although rst observed more than two decades ago, the etiological agent of this disease still remained unknown.

159

In order to get more insight into the epidemiological situation of YPD, a questionnaire was developed and distributed to German breeders of racing pigeons. Among others, questions referred to the number of pigeons, the type of the pigeon loft, the feeding and drinking habits, the occurrence of YPD and the clinical signs observed. Various clinical and pathological pictures for YPD have been described. The analysis of more than 1,600 questionnaires revealed that pigeons were affected by YPD mainly between 4 to 12 weeks post weaning. Clinical signs observed were unspecic: anorexia, depression, rufed feathers, vomiting, diarrhoea, polyuria and uid lled crop. It was reported that generally less than 20% of the young pigeons were affected and that mortality rates were about 20%. In individual cases, however, mortality rates of more than 50% were also reported. Furthermore, it became evident that stress conditions such as long transports or hot weather during the young pigeon races might play an important role in the induction of YPD. Various non-infectious causes as well as infectious pathogens, such as Spironucleus columbae, Escherichia coli (E. coli) or avian adenoviruses, were considered to contribute to the pathogenesis of YPD (DORRESTEIN and VAN DER HAGE 1992, DE HERDT and VAN GINNEKEN 1994). In order to collect more convincing data, a comprehensive study was performed with pigeons present in 25 German pigeon lofts with clinical outbreaks of YPD in 2003 or 2004 compared with pigeons of 4 lofts without clinical outbreak of YPD in 2004. The aim of this study was to get knowledge on pathological and histological alterations in pigeons with YPD and to compare those with the results gained from haematological, parasitological and microbiological examinations. Due to the results gained, a second part of this study was performed thereafter including examinations for Chlamydophila spp. and viral agents.

2 MATERIALS AND METHODS

Seventy six juvenile racing pigeons of both sexes obtained from 29 lofts located in various parts of Germany were investigated. Out of these, 67 pigeons were from 25 lofts with clinical outbreaks of YPD diagnosed by veterinarians according to the following criteria: (i) increased mortality rates among pigeons in their 3rd to 20th week of life; (ii) poor racing performance. For comparison 9 young racing pigeons, grown up in 4 lofts without YPD, were included in the study. All pigeons hatched in 2003 or 2004, and all lofts were tested negative for salmonellosis. A detailed history of each pigeons husbandry and the course of the disease were reported by the owners. Clinical examinations including white blood cell counts (WBC) and differentiation of white blood cells were performed and evaluated according to standard procedures and compared to describe reference haematological values for racing pigeons (VOGEL 1992, RUPIPER and EHRENBERG 1994). Pigeons were humanely killed with potassium chloride (2mmol/kg body weight, intravenously) after an initial anaesthesia with ketamine (40mg/kg body weight, intramuscularly) and diazepam (5mg/kg body weight, intramuscularly); the birds were necropsied according to standard procedures (LATIMER and RAKICH 1994).

160

Impression smears prepared from liver, spleen, lung, bone marrow, small and large intestines were examined cytological (DiffQuik; Dade Behring, Marburg, Germany) at 100x magnication using oil. Liver, lung, heart, spleen, kidneys, bursa of Fabricius, thymus, crop, proventriculus, ventriculus, small and large intestines, brain, thyroid, parathyroid, adrenal gland, testes or ovary and bone marrow were collected under sterile conditions. Parts of these organs were xed in 10% neutral buffered formalin for at least 24h. Formalinxed samples were dehydrated, embedded in parafn wax and sectioned at 4m for light microscopy examinations. All sections were stained with haematoxylin and eosin and evaluated. Samples from crop and small and large intestines were collected immediately after opening of the carcasses and investigated microscopically for agellates using 200x and 400x magnication, respectively. Ingesta from small and large intestines were mixed with saturated sodium chloride solution at a 1:1 ratio and oated for 20 minutes. A semi-quantitative evaluation of 5 microscopic elds was performed at 200x magnication. For bacteriological and mycological investigation, swabs were taken from liver, lung, heart, kidneys, small and large intestines. Cultivation of bacteria was performed at 38C under aerobic, microaerobic (AnaerocultC; Merck, Darmstadt, Germany) as well as anaerobic conditions (AnaerocultA; Merck, Darmstadt, Germany) for 24 and 72h, respectively, using Columbia agar supplemented with debrinated sheep blood and brilliant green agar (Oxoid, Wesel, Germany). Differentiation of bacteria was carried out using Chrystal Tube (BD, Heidelberg, Germany). Resistance screening of the isolated bacteria by agar diffusion test and determination of the minimum inhibitory concentration ensued. For mycological examinations, Sabourauds dextrose agar (Oxoid, Wesel, Germany) was inoculated and incubated at 38C for 72-120h. For detection of salmonella, samples taken from liver and the contents of gut were cultivated in selenite lactose medium (Oxoid, Wesel, Germany) at 38C for 48h. Drop volumes taken from these media were incubated on selective culture plates (XLT4 agar; Oxoid, Wesel, Germany) at 38C for 24h. Suspicious colonies were screened with polyvalent antibody of Salmonella A-E (Enterocolon anti-Salmonella I (A-E); SIFIN, Berlin, Germany) in an agglutination test as described. Statistical analysis was performed using the program SPSS, version 11.5.1 (SPSS Inc., Chicago, IL, USA). In the case of WBC, mean values and standard deviations were also calculated. Deviations from normal ndings were veried for signicance using cross tables and Pearsons Chi2 test.

3 RESULTS
Most of the pigeons (63 out of 76) were sent to examination alive and during the hot summer month (14 out of 29 lofts). In 32 out of 57 pigeons (56%) from YPD-affected lofts clinical signs were observed. Clinical signs were greenish to black diarrhoea (36.8%, 21 out of 57 pigeons), vomitus, anorexia, polyuria, yellow urates (10.5% each, 6 out of 57 pigeons), torticollis (5.3%, 3 out of 57 pigeons), cachexia, dyspnoea and splayed leg (1.8% each, 1 out of 57 pigeons). 10 pigeons from affected lofts died during transport. Clinical signs were observed in none of the pigeons from lofts without

161

history of YPD. Pigeons in their 4th to 12th week of life exhibited the signs mentioned above more frequently and more severely (17 out of 23 pigeons representing 73.9%) than pigeons of other ages. Clinical signs were observed more often in pigeons who were examined in summer (45.6%, 26 out of 57 pigeons, p<0.008). Many of the dissected birds from lofts with signs of YPD (n = 67) showed a poor (34.3%) to bad (17.9%) nutritional condition, whereas pigeons from YPD-free lofts were of good nutritional status. Findings of greenish liquid in crop, proventriculus and ventriculus had a signicantly higher prevalence in diseased birds (p<0.001) than in birds without clinical signs. Yellow uid in small intestine was found in 18 out of 67 pigeons with YPD (26.9%) as well as 2 out of 9 pigeons without YPD (22.2%). Gross lesions of lung and air sacs (12 out of 67 pigeons, 17.9%), liver and spleen (11 out of 67 pigeons each, 16.4%), kidney (7 out of 67 pigeons, 10.4%), intestines (4 out of 67 pigeons, 6.0%), heart and pancreas (3 out of 67 pigeons each, 4.5%), and bursa of Fabricius (1 out of 67 pigeons, 1.5%) were seen only in pigeons from lofts with signs of YPD. In none of the pigeons cytological alterations such as necrosis or inclusion bodies were detected in smears taken from the bone marrow, and no inclusion bodies were detected in erythrocytes or leukocytes of blood smears. Blood smears of 50 out of 67 pigeons from lofts with YPD and 6 out of 9 pigeons from lofts without YPD were evaluated. Pigeons showing leucocytosis (8 out of 50 pigeons) originated from lofts with YPD, but were more than 12 weeks old. Leucopenia was seen in 7 out of 50 pigeons from lofts with YPD (14%, [8.570.66x103/l]a). In contrast, Leucopenia was seen in 3 out of 6 pigeons from lofts without YPD (50%, [8.50.69x103/l]). Lymphocytic depletion and lymphocellular necrosis was observed in the spleen of 13 out of 67 (19.4%) pigeons and in the bursa of Fabricius of 9 out of 57 (15.8%) pigeons. Multiglobular basophilic intranuclear and intracytoplasmatic inclusion bodies were observed in lymphocytes and macrophages of the bursa of Fabricius in 21 of 57 (36.8%) pigeons from lofts with YPD. Furthermore, they were present in lymphocytes and macrophages of the gut-associated lymphoid tissue of 3 pigeons, of the kidney of 2 pigeons, of the bronchus-associated lymphoid tissue and of the spleen in one case each as well as of the liver of 5 out of these 67 pigeons, respectively. Multiglobular basophilic inclusion bodies were also found in the bursa of Fabricius of one pigeon without YPD. Inclusion bodies were observed with a statistically signicant higher incidence in pigeons with uid lled small intestine (p<0.035). In none of the pigeons histological alterations such as necrosis or inclusion bodies were detected in the bone marrow. Pigeons with uid lled small intestine (p<0.085), green uid in crop, proventriculus and ventriculus (p<0.001) or inclusion bodies in the bursa of Fabricius (p<0.079) were affected by haemosiderosis signicantly more often. In contrast, none of the pigeons from YPD-free lofts showed haemosiderosis in liver, kidneys or spleen. In small and large intestines, histological changes were grouped as follows: mild to severe catarrhalic duodenitis (6 out of 67 pigeons, 9.0%), mild acute catarrhalicpurulent enteritis (3 out of 67 pigeons, 4.5%), severe multifocal granulomatous enteritis (4 out of 67 pigeons, 6.0%). Enteritis was found more often in pigeons who were dissected in summer (p<0.087), in pigeons with clinical signs of YPD (p<0.033) and in pigeons with depletion of spleen lymphocytes (p<0.053). Other histological changes were induced by septicaemia or mold-infection.

162

As expected, no salmonella were isolated from liver and intestines of all pigeons. However, E. coli was isolated from 69.7% (53 out of 76) of the examined pigeons. Although E. coli was observed in both, pigeons from YPD free lofts (8 out of 9 pigeons, 88.9%) and from those with YPD (45 out of 67 pigeons, 67.2%), it was detected statistically more frequently in pigeons with clinical signs (p<0.008), greenish liquid in crop, proventriculus and ventriculus (p<0.05) and uid lled small intestine (p<0.019). Main site of isolation was the large intestine. However, in pigeons with clinical signs (32 pigeons) E. coli was isolated more often from small intestine additionally (59.3%, 19 out of 32 pigeons) contrary to E. coli isolation from the large intestine only (21.9%, 7 out of 32 pigeons). In 10 out of 67 pigeons with E. coli isolation from the small intestine, the above mentioned histopathological alterations of the gut were detected (p<0.019). Furthermore, numerous bacteria were isolated from pigeons with YPD and without YPD. Yeast or molds were only detected in pigeons with YPD. In these yeast (p<0.017) as well as molds (p<0.02) were isolated more frequently from pigeons with inclusion bodies in the bursa of Fabricius. Spironucleus columbae was present in the intestines of 15 out of 76 (19.7%) pigeons. Statistical signicance for the presence of this parasite was proven for pigeons with clinical signs (p<0.051). Trichomonas gallinae affected 11 out of 76 (14.5%) pigeons, from both, YPDfree lofts and lofts with YPD. Eimeria spp. were demonstrated in 3 out of 76 pigeons (3.9%) and helminths (Ascaridia spp. and Capillaria spp.) were observed in 9 out of 76 birds (11.8%). However, 30 out of 76 (39.5%) pigeons carried pigeon lice (Columbicola columbae columbae), and 19 out of 76 (25.0%) pigeons feather mites (Falculifer rostratus, Megnina columbae).

4 DISCUSSION
YPD is characterised by unspecic clinical signs and pigeons are affected in their 4th to 12th week of life more severely and frequently. Comparable results were obtained from the clinical examination in this study. As expected, numerous pathological and histopathological ndings were observed, most certainly caused by numerous (secondary) pathogens. Interestingly, Spironucleus columbae, previously discussed as the causative agent, was observed in only 19.7% of the pigeons. However, facultative pathogenic E. coli isolated from 53 of 67 pigeons affected with YPD and other facultative pathogens were identied in the majority of cases, indicating that the immune system of these pigeons might be affected. This hypothesis is supported by the observation that lymphoid depletion and lymphocellular necrosis were observed in spleen and bursa of Fabricius and that intranuclear and intracytoplasmatic inclusion bodies were demonstrated most frequently in the bursa of Fabricius but also in lymphocytes and macrophages of the liver, spleen, intestines, kidneys and lung. Intranuclear inclusion bodies as observed here are mostly caused by DNA viruses. Recently such inclusion bodies have been reported from the bursa of Fabricius of pigeons infected with the pigeon circovirus (PiCV) (WOODS 1994, SHIVAPRASAD 1994). Having this in mind, PCR protocols for the detection of pigeon DNA viruses were developed. These assays allowed the detection of viruses such as PiCV which, at present, can not be isolated in tissue culture systems (WOODS and SHIVAPRASAD 1997). In contrast to former studies (DORRESTEIN and VAN DER HAGE 1992)

163

avian adenovirus was not detected. Attempts to demonstrate the presence of avian polyomavirus remained negative as well. Furthermore, the low number of pigeon herpesvirus positive results (15.6%) revealed the expected prevalence rate (EF KALETA, unpublished observations). However, PiCV DNA was demonstrated in all pigeons with YPD, but only in 3 of 6 pigeons from lofts without YPD. Remarkably, the amount of PiCV DNA in the organs was reduced, these pigeons might represent persistently infected carriers and might be responsible for the spread of this virus. These results, together with published data, indicate that the prevalence rate of PiCV in Germany is generally high. Comparable observations have been reported from other countries like Czech Republic, Belgium and Northern Ireland (TODD 2002). In accordance with recently published reports (TODD 2002; HATTERMANN 2002), PiCV DNA can be detected in various organs and blood in different amounts. Lymphatic tissues, such as bursa of Fabricius and spleen, were most frequently tested positive. All of the pigeons with clinical signs of YPD were infected with PiCV; PiCV DNA was also detected in the blood of most of the birds (48.8%) indicating that they were viraemic. In contrast PiCV DNA was not detected in blood samples of pigeons from lofts without clinical outbreaks of YPD. Taken these results into account, the investigation of blood samples may be a good choice for the conformation of the diagnosis YPD in vivo. From the results of this comprehensive study it was predicted that most probably, YPD represents a multifactorial disease in young pigeons. The exposure of such pigeons to stress conditions such as racing, overcrowding, hot weather, might result in enhanced viral replication, rst of all in lymphatic tissues. Consequently, the depletion of lymphocytes and lymphocellular necrosis in the bursa of Fabricius and other lymphatic tissues lead to immunosuppression. This hypothesis is supported by the fact that pigeons with YPD are highly susceptible to secondary infections with bacteria, fungi and parasites. In particular E. coli, a facultative pathogenic bacterium belonging to the normal ora of the large intestine, may overgrow the bacterial ora of the large intestine. Furthermore E. coli was isolated more often from the small intestine of clinical ill pigeons. The high quantity of E. coli may lead to an overcome of the barrier of the gut, resulting in septicaemia. This irregular gut ora supported the spread of parasites and other secondary pathogens, especially yeast, and, therefore, the induction of systemic infections. The multitude of clinical signs observed during an outbreak of YPD reects these various secondary infections. However, most of the young racing pigeons with YPD did not show leucopenia. This might indicate that a (re)activation of the immune system has taken place due to the numerous of secondary infections commonly observed. In pigeons with YPD neither necrosis nor inclusion bodies were observed in the bone marrow; this is in contrast to the situation in circovirus infected parrots (H GERLACH, personal communication) and indicates that the immunesystem might be able to be reactivated.

5 ACKNOWLEDGEMENT
This study was supported by the Verband Deutscher Brieftaubenzchter e.V.

164

6 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. DE HERDT P and VAN GINNEKEN C. Escherichia coli infections in pigeons: Characteristics of the disease and its etiological agent. Proc 9th Symp Avian Diseases, Munich 1994: 211 - 214. DORRESTEIN G.M and VAN DER HAGE MH. Adenovirus inclusion body hepatitis: A new pigeon disease? Proc Symp Avian Diseases, Munich 1992: 7 - 14. HATTERMANN K. A method to diagnose pigeon circovirus infection in vivo. J Vir Meth 2002; 104: 55 - 58. LATIMER SL and RAKICH P.M. Necropsy examination. In: RITCHIE BW, HARRISON GJ and HARRISON LR (eds): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing, Inc. 1994; 355 - 379. RUPIPER DJ and EHRENBERG M. Introduction to pigeon medicine. Proc Assoc Avian Vet, Lake Worth 1994: 203 - 211. SHIVAPRASAD HL. Particles resembling circovirus in the bursa of Fabricius of pigeons. Avian Diseases 1994; 38(3): 635 - 641. TODD D. Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections. Vet Microbio 2002; 89: 1 - 16. VOGEL C. Tauben. Berlin: Deutscher Landwirtschaftsverlag 1992; 31. WOODS LW. A retrospective study of circovirus infection in pigeons: nine cases (1986-1993). J Vet Diag Invest 1994; 6: 156 - 164. 20. WOODS LW, SHIVAPRASAD HL. Pigeon circovirus infection. In: CALNEK BW, BARNES HJ, BEARD CW, et al. (eds): Diseases of Poultry, 10th edition. Ames: Iowa State University Press 1997; 1050 - 1053.

AUTHOR ADRESS
V. Schmidt Clinic for Birds and Reptiles, Department of Small Animal Medicine, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 17 D-04103 Leipzig, Germany Email: vschmidt@vogelklinik.uni-leipzig.de (Footnotes) a mean value and standard deviation

165

Department of Veterinary Clinical Science1, Padua; Wildlife Rehabilitation Center2, L.I.P.U., Treviso; Wildlife Rehabilitation Center3, WWF, Ancona, Italy

SERUM BIOCHEMICAL AND ELECTROPHORETIC PATTERNS IN THE EURASIAN BUZZARD (BUTEO BUTEO) : REFERENCE VALUES.
D.Gelli1,D.V.M., F. Franceschini2, D.V.M., M. Bedin3, D.V.M. V. Ferrari1, D.Biol. S., PhD, D. Bernardini1, D.V.M., S. Romagnoli1, D.V.M., PhD

KEYWORDS
Buteo buteo Biochemistry Haematology - Electrophoresis

ABSTRACT
Clinical and diagnostic usefulness of haematological and biochemical investigations in avian medicine is still a subject of debate. We collected blood samples from 20 healthy Eurasian buzzards and carried out serum biochemical and electrophoretic tests. In order to conrm health status, clinical evaluation, radiography, endoscopy and laboratory investigations (haemogram, biochemistry, bacteriology, faecal exams) were performed on each animal. The following mean values were obtained: BIOCHEMISTRY: albumin 14.6 g/l, total protein 3.84 g/l, glucose 337.1 mg/ dl, aspartate aminotransferase 330.9U/I, alanine aminotransferase 40.6U/I, lactate dehydrogenase 2008.4U/I, creatine kinase 1604.1U/I, gamma-glutamyl transferase 0.3U/I, alkaline phosphatase 89.8U/I, total bilirubin 0.03 mg/dl, cholesterol 192.2 mg/dl, triglycerides 116.4 mg/dl, lipase 26.37U/I, amylase 616.93U/I, uric acid 3.68 mg/dl, creatinine 0.15mg/dl, urea 12.72mg/dl, calcium 8.67mg/dl, phosphorus 2.22mg/dl, magnesium 2.42mg/dl; ELECTROPHORESIS: pre-albumin 3.06g/ l, albumin 14.74g/l, -globulin 4.89g/l, -globulin 5.78g/l, -globulin 13.08g/l. Electrophoresis can provide useful diagnostic information when used in conjunction with other laboratory tests, as patterns of alteration in protein electrophoretograms are commonly associated with clinical disease. Our results can be used as a reference when evaluating electrophoretograms of diseased buzzards.

1 INTRODUCTION
Many haematological and biochemical values in the Eurasian buzzard (Buteo buteo) have been published, however some biochemical parameters of this species are still not available. Electrophoretic patterns in serum or plasma protein are not available; this is probably due to the fact that wild bird medicine has little commercial interest, so it is not always possible to perform electrophoresis as a routine test, especially in a Wildlife Rehabilitation Centres. Besides, changes in plasma proteins in birds are not exclusively related to diseases, but can be also physiological, due to age, sex,

166

season and reproductive status (KURYE and GASPARSKA 1985). This lack of data related to standard values, hinders the use of electrophoresis in many species of wild birds to provide diagnostic information. In mammalian medicine electrophoretogram patterns provide information about increasing and decreasing levels of proteins with attention to single fraction and alterations in these fractions are pathognomonic of some specic diseases (e.g. a monoclonal peak in the beta fraction may occur in patients with multiple myeloma or lymphoma). Also, in the acute phase of some infectious mammal diseases, changes in serum proteins are detected early, even when serological tests fail to detect the infectious agent. In the avian patient, changes in total blood proteins and in some fractions like albumin and gamma globulins are, currently, a useful diagnostic tool to evaluate body condition (starvation, dehydration, inammatory processes of different aetiology), to indicate a diagnosis and to monitor the animal during therapy and rehabilitation (BLANCO and HOFLE 2003). To make a diagnostic tool useful in avian medicine, both physiological and pathological patterns of electrophoretograms in the different species must be established. The aim of this work was to characterise and document normal reference ranges and electrophoretograms patterns in the Eurasian buzzard.

2 MATERIAL AND METHODS

From June to July 2004 the blood of 40 Eurasian buzzards waiting to be released after medical and surgical care performed during the previous winter at the Wildlife Centres of L.I.P.U, W.W.F and the University of Padua, was collected for pre-release routine investigations. The birds were all adults of both sexes. During the rehabilitation period the birds were fed with the same diet based on dead birds and rodents. Twenty of these animals were assessed as healthy, after clinical evaluation, radiography, endoscopy and laboratory investigations. Serum was used to enable further biochemical and electrophoresis investigations to be performed. Blood samples for biochemistry and electrophoresis were collected from the metatarsal vein, 1 ml from each bird, in tubes without anticoagulant. In order to perform electrophoresis, serum was preferred because brinogen in plasma can often obscure the electrophoretogram in the - region (THOMAS 2000). The serum samples for electrophoresis were diluted 1:25 with water prior to analysis to avoid the adhesion of proteins to the capillary walls. Biochemical exams were performed by a 912 automatic analyser (Roche -Hitachi), seroproteins were obtained using P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Inc, Ca), equipped with an automatic constant volume sample injection device and temperature control for the capillary. In capillary electrophoresis separation of protein fraction occurs in a borate buffer, in a narrow-bore uncoated fused silica capillary that is exposed to high voltages. The elecrophoretic prole is obtained by direct measurement of the proteins at 200 nm. Systems control data collection and analyses were done with the software of the equipment.

3 RESULTS
3.1 Biochemistry Values of biochemical parameters found in the birds of this study are reported in Tables 1 and in Table 2

167

Table 1. Values of biochemical parameters found in buzzards. AST 330.9 U/I U/I U/I U/I U/I U/I mg/dl mg/dl mg/dl 40.6 2008.4 1604.1 0.3 89.8 0.03 192.2 116.4 26.37 U/I ALT LDH CK GGT AP TB CHOL TG LIP AMY 616.93 U/I

Parameters

Alb

TP

Glu

Mean

14.5

3.84

337.1

Units

g/l

g/l

mg/dl

Albumin (Alb), total protein (TP), glucose (Glu), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase (CK), gamma-glutamyl transferase (GGT), alkaline phosphatase (AP), total bilirubin (TB), cholesterol (CHOL), triglycerides (TG), lipase (LIP) and amylase (AMY).

Table 2. Values of biochemical parameters found in buzzards. Crea 0.15 mg/dl mg/dl mg/dl 12.72 8.67 Urea Ca P 2.22 mg/dl Mg 2.42 mg/dl

Parameters

UA

168
Albumin 14.65 g/l -globulin 4.89 g/l 5.78 g/l

Mean

3.68

Units

mg/dl

Uric acid (UA), creatinine (Crea), urea, calcium (Ca), phosphorus (P) and magnesium (Mg) 3.2 Electrophoresis

Values of serum protein are reported in Table 3.

Table 3. Values of serum protein parameters found in buzzards. -globulin -globulin 13.08 g/l

Parameters

Prealbumin

Mean

3.06

Units

g/l

4 DISCUSSION
Although some of our biochemistry results were similar to previous reports such as: total proteins, glucose, cholesterol, urea, calcium, phosphorus and magnesium; other parameters differed. Enzyme levels including creatine kinase and lactate dehydrogenase appeared to be very high despite the good physical condition of the buzzards. Both of these enzymes are sensitive to stress and physical exercise. The blood was collected a few minutes after capturing the animals in the aviaries to avoid any increase in glucose level in serum due to stress and the level of this metabolite did not appear to be inuenced by manipulation of the birds. However, all these birds were kept in large aviaries to allow them to y, so, considering the levels of glucose and of other enzymes like alanine aminotransferase and aspartate aminotransferase, which appeared to be within physiological ranges, we could hypothesise that the high levels of LDH and CK may be due to high daily exercise. Alkaline phosphatase appeared to be lower than in many other species of birds, but the variety of methods of analysis reported in literature does not allow us to evaluate the real baseline level of this enzyme. Values of metabolites such as creatinine and uric acid appeared to be much lower than those reported by other authors for the European buzzard (HERNANDEZ 1990), but within the ranges reported for avian patients (HOCHLEITHNER 1994). Values of triglycerides for Buteo buteo are not reported, and as this metabolite is also reported to increase after exercise and stressors factors (HOCHLEITHNER 1994), further studies are needed to evaluate if our results may have been inuenced by the physical manipulation of the raptors. Amylase and lipase reference values for this species were not found in literature, although the value of amylase can be considered within the ranges reported for the healthy avian patient. A low to negligible pre-albumin fraction was present in serum of the healthy buzzards, migrating at a time of 7-7.5 minutes. Pre-albumin appears in the farthest area of migration. Albumin is represented by a high peak shouldered by the fraction. As in other birds and mammals the globulins consist of a heterogenous group of proteins that have a weaker negative charge and migrate at a slower rate than albumin. In B. buteo, they migrate at a time of 4 to 6.5 minutes. Beta globulins values are slightly lower than the ones found in literature for other species of raptors. This is because we used serum instead of plasma to evaluate globulins in healthy animals without brinogen, which is an acute phase reactant of this fraction and can obscure the real value of this globulin. Gamma and beta globulins in this species of raptor appear in the electrophoretograms in a bridged peak and not as distinct fractions. Although further studies are needed to improve knowledge, both on physiological and pathological electrophoretic patterns of protein fraction in serum and plasma of the European buzzard the values obtained in our study can be considered rst reference values in this species. Electrophoresis is gaining importance in avian medicine as a diagnostic tool and for monitoring the avian patient.

169

5 CITATION INDEX
1. BLANCO JM and HOFLE U. Plasma protein electrophoresis as diagnostic and prognostic tool in raptors. Proc Euro Assoc Avian Vets, Tenerife 2003: 256 - 261. 2. CRAY C. Plasma protein electrophoresis: an update. Proc Assoc Avian Vets, Reno 1997: 209 - 212. 3. CRAY C. Applications of protein electrophoresis to avian diagnostics. J Avian Med Surg 12; 4 - 10. 4. CRAY C. Diagnostic use of protein electrophoresis in birds. In: BONAGURA JD (ed): Kirks Current Veterinary Therapy, XIII. New York: W.B. Saunders Co. 2000; 1107 - 1109. 5. HERNANDEZ M. Clinical Hematology and blood chemistry values for the common buzzard. J Rapt Res 1990; 14:113 - 119. 6. HOCHLEITHNER M. Biochemistries. In: RITCHIE BW, HARRISON GJ and HARRISON LR (eds): Avian Medicine: Principles and Applications. Lake Worth: Wingers Publishing 1994; 223 - 245. 7. LANZAROT MP. Hematological, protein electrophoresis and cholinesterase values of free-living nestling peregrine falcons in Spain. J Wild Dis 2001; 37: 172 - 177. 8. QUESENBERRY K. Plasma electrophoresis in psittacine birds. Proc Assoc Avian Vet, Chicago 1991: 112 - 118. 9. TATUM LM. Protein electrophoresis as a diagnostic and prognostic tool in raptor medicine. J Zoo Wild Med 2000; 31(4): 497 - 502. 10. THOMAS JS. Protein electrophoresis. In: FELDMAN BF, ZINKL JG and JAIN NC (eds): Schalms Veterinary Hematology 5th ed. Baltimore: Lippincott Williams and Wilkins 2000; 899 - 903.

AUTHOR ADRESS
Donatella Gelli Department of Veterinary Clinical Science Viale delluniversit 16, 35020 Legnaro, Padova, Italy Email: donatella.gelli@unipd.it

170

Versele-Laga R&D Department, Deinze, Belgium

HEPATIC HAEMOSIDEROSIS IN BIRDS : NOT ONLY TOTAL DIETARY IRON LEVEL, BUT SEVERAL DIETARY FACTORS AND BIRD-SPECIFIC DEFENSE MECHANISMS CONTRIBUTE TO THE DEVELOPMENT OF THE DISEASE
G.J.D.L. Werquin DVM and K.J.S. De Cock (Food engineer)

KEYWORDS
Birds - Haemosiderosis - Iron storage disease - Iron availability - Tannin - Phytic acid - Immune mechanisms - Transferrin

ABSTRACT
Although the exact pathogenesis of haemosiderosis in frugivorous birds has not been elucidated, there is a common consensus that dietary iron load should be restricted. However, current recommendations for total dietary iron are very empirical and disregard differences in bio-availability. This paper describes the different chemical forms of iron and their biological availability. Other dietary components (tannins, phytic acid, bers, ascorbic acid, organic acids and minerals) signicantly inuence the bio-availability of the iron in the food and are at least as important. Also stress reactions and defense mechanisms may induce iron accumulation in the liver: in contrast to mammals, stress stimulates iron absorption from the gut and may initiate haemosiderosis in birds.

1 INTRODUCTION
Haemosiderosis is a common nding in frugivorous birds kept in captivity. The most common group of birds to suffer from haemochromatosis are toucans, toucanettes and aracari (family Ramphastidae). The disease is also frequently found in mynahs, birds of paradise (Sturnidae), quetzals (Caprimuligiformes), tanagers and starlings (Passeriformes), curassows (Struthioniformes), and even some parrots (Psittaciformes). In general, frugivorous, insectivorous and omnivorous birds tend to accumulate more iron in their liver and, thus, are more prone to the disease than carnivorous, piscivorous and granivorous birds. A higher intestinal iron uptake by mynah enterocytes compared to chicken enterocytes was demonstrated (METE 2003). This higher intestinal iron uptake could be due to a physiological adaptation to the low iron in the natural habitat of these birds, where the duodenal epithelia are

171

programmed to take up all iron available. These ndings still do not explain why the mucosal block does not function (when body iron status is adequate, the iron in the enterocytes is normally bound to ferritin and not transferred to the blood, but shedded in the lumen of the gastrointestinal tract). With the current knowledge, it is generally accepted that genetic predisposition and dietary iron intake play an important role in the pathogenesis of iron storage disease. There is a common consensus that dietary iron load should be limited in birds prone to haemosiderosis. However, the recommendations for dietary iron levels vary by author and seem to be set up rather empirically. They are often not the result of controlled feeding trials and disregard the variability in iron bio-availibility. Furthermore, it was observed that low iron diets do not protect birds from haemosiderosis, indicating that other factors may also play an important role in the development of iron storage disease. Surveys of captive birds have produced arguments that there is a direct correlation between immunological or nutritional stress and haemosiderosis. This paper inventarises factors that inuence dietary iron load and describes how defense mechanisms may contribute to iron storage disease in birds.

2 DIETARY IRON LOAD AND HAEMOSIDEROSIS

The amount of iron absorbed from the food is determined by iron status of the bird, chemical form of iron present in the diet and the balance of dietary components enhancing or inhibiting the absorption of dietary iron. It is well known that the variation in dietary iron absorption is due more to differences in the bioavailability, which can lead to a > 10-fold variation in iron absorption, than to a variation in iron content. Therefore, the evaluation of dietary iron intake should not be limited to the absolute iron level of the diet, but should also take the chemical form of dietary iron and factors inuencing dietary iron absorption into consideration: 2.1 Dietary iron level Restriction of total dietary iron is the most obvious measure: Empirically based data suggest a maximum iron level of 100 ppm. When formulating food for frugivorous birds, raw materials with low iron levels should be selected. Also, special premixes without iron containing additifs should be used. As most available raw materials contain signicant amounts of iron, restriction of total dietary iron is limited. Iron levels below 60 ppm are often difcult to realise without compromising dietary calcium supply, as mineral sources are often rich in iron. The iron content of vegetable materials is often very variable and depends on soil conditions as iron content, chemical form of iron, pH, and levels of other minerals. Raw materials used for the production of feeds for frugivorous birds should be analysed systematically on iron content. Manufacturers of feed for frugivorous birds should monitor iron content in nished products for each production batch. Own research showed important variations in iron content of nished products. Further investigations of raw materials demonstrated that these variations where mainly due to the large variations of the iron content in

172

mineral sources. The use of special chalk types with guaranteed low iron level proved to be essential in controlling the iron content. Concerning the labeling of iron content on the packaging of bird feed, it is important to realise that the European animal feed legislation normally only allows the labeling of the supplemented trace elements. The amount of iron mentioned under the food additives stands for the added iron: to obtain the total iron content, this amount must be increased with the iron content of the raw materials. Some birdfeed companies however obtained derogations on this legislation and are allowed to mention the total iron content for feed destinated for frugivorous and insectivorous birds. As food consumption is determined by the energy content of the food, which is very variable for bird food, recommendations for iron levels should be expressed on energy basis in stead of on weight basis.On energy basis, an iron content < 5 mg / MJ ME has been proposed. Table 1: Iron content in raw materials (mg/kg) chalk - normal 2000-5000 calciumphosphate 800-1400 Meat 480 Spirulina 400 chalk - low iron <300 alfalfa 300 oystershell 290 peas 263 corn gluten 200 brewers yeast 175 sorghum 170 ax 120 rape seed 90 wheat 40-75 corn 40-75 milocorn 40-75 paddy rice 50 g 35 sunower seed 30 walnut 25 peanut 20 honey 15 rose hip 5 juniper berry 5 raisin 5

173

2.2 Sources of dietary iron Dietary iron is available in two valence states, Fe2+ (ferrous) and Fe3+ (ferric). The majority of ferrous iron is found in haem iron and the majority of ferric iron is found in non-haem iron. Haem and non-haem iron are absorbed differently.

dietary iron

inorganic

organic

Fe3+
Ferric Fe insoluble poorly absorbable

Fe2+
Ferrous Fe soluble absorbable

= iron porfyrines (haem) = better availability (different absorption mechanisms) = less influenced by dietary factors

iron bio-availability
Iron oxide < Iron(II)sulfate < iron chloride < iron citrate < iron amino acid complex < haem iron

Fig 1: Bio-availability of different chemical compounds of iron Haem iron is present in the haemoglobin and myoglobin of animal products. This form of iron is relatively available: about 30% of haem iron is absorbed from the diet. The level of haem iron absorption is relatively unaffected by other dietary factors. Non-haem iron is found in materials of vegetable origin such as cereals, vegetables, pulses and dried fruit. Compared to haem iron it is relatively poorly absorbed, typically less than 10% and often under 5%. The absorption of non-haem iron is much more inuenced by the iron status of the animal and several components in the diet that can either inhibit or enhance its absorption. Also the chemical composition of non-haem iron is important: e.g. iron oxide is less absorbed than iron chloride or iron citrate. 2.3 Factors inuencing iron absorption Several dietary factors signicantly inhibit iron absorption, whereas other factors enhance iron absorption. The presence of iron chelating agents in the food reduces the bio-availability of non-haem iron. Plant polyphenols such as tannins present in tea, bayberries, nuts and sorghum reduce the absorption of non-haem iron due to the formation of insoluble iron tannate complexes. In vitro studies demonstrated that tannic acid is a very potent inhibitor of non-haem iron uptake, as maximal inhibition (97.5%) of iron uptake occurred at a ratio of 1:1 or less Fe to tannic acid (GLAHN 2002). In wild-caught starlings, supplementation of black tea leaves prevented iron absorption after feeding an iron-enriched diet (SEIBELS 2003). Phytate, present in the bran of wheat and other wholegrain cereals, pulses (peas, beans and lentils) and nuts, strongly inhibits non-haem iron absorption by interacting

174

with it, rendering it less soluble. At a level of 1000ppm, phytic acid decreases iron absorption by about 60% (HALLBERG 1989). Also oxalic acid in green leafy vegetables may bind iron to form salts known as oxalates, preventing iron absorption. Sources of oxalates are most berries, beans, bell peppers, rhubarb, tea, plantains, ginger, soybeans, mango and lentils. Also calcium and soil clay interfere with iron absorption. A strong dose-effect relation between the amount of calcium and the reduction in nonhaem iron absorption has been observed in humans (HALLBERG 1991). Moreover, calcium also inhibited the absorption of haem iron similarly, suggesting a common step in the transport of these 2 kinds of iron. Therefore, the effect was not located in the intestinal lumen but suggested competitive binding at a receptor of the mucosal cell. Table 2. Factors That Inuence Iron Absorption Physical State (bioavailability) Inhibitors Competitors Facilitators haem > Fe2+ > Fe3+ phytates, tannins, oxalates, soil clay, iron overload lead, cobalt, copper, strontium, manganese, zinc ascorbate, citrate, amino acids, iron deciency

Several other metals are taken up by the same absorption mechanism, and secondarily block iron through competitive inhibition. Especially zinc supplementation revealed to reduce iron absorption: in vitro studies demonstrated that zinc decreases iron uptake by 57 to 80% (GLAHN 2002).

Fig 2:

Inuence of different dietary components on iron absorption ratio. (a)

175

Inuence of ascorbic acid, (b) Inuence of tannic acid, (c) Inuence of phytate phosphorus, (d) Inuence of calcium. Derived from the algorithm of HALLBERG AND HULTHEN (2000). Tannic acid is clearly the most potent inhibitor of iron uptake. The tannic acid level is of more importance for the iron absorption than the total dietary iron: a sorghum based diet containing 350 mg tannic acid and 250 ppm iron gives less available iron than a low iron diet containing 60 ppm iron, but formulated without tannins Ascorbic acid, organic acids or a low dietary pH enhance iron absorption by reducing the ferric iron to the more readily absorbed ferrous form. Particularly ascorbic acid is a strong promoter of iron absorption and has a counteracting effect on phytate and polyphenols. The evaluation of the dietary iron load should not be limited to the iron content of the food ration, but should also attempt to take all other important dietary factors into consideration. Until now, the evaluation of commercial feed for frugivorous food focused on the total iron content of these foods (SCHOEMAKER 2001). However, analysing only total iron content is not sufcient to assess available dietary iron in a reliable way. Due to the strong inhibitary effect of tannins and phytates, the amounts of these nutrients may be much more important than the iron content itself. When evaluating dietary iron load, attempts should be made to take the factors that inuence iron absorption (chemical form of iron, levels of tannic acid, phytic acid, ascorbic acid) into consideration. HALLBERG AND HULTHEN (2000) developed an algorithm (g. 2) for predicting the effects of factors known to inuence haem and non-haem iron absorption in humans. It may be interesting to develop a similar algorithm for bird nutrition. Another approach may be to assess available iron in bird diets by the use of in vitro methods. For practical feeding recommendations, it is important not to focus only on the iron content of the food, and to make sure that sufcient nutrients that inhibit iron absorption are present in the diet. Diet should also contain ingredients rich in tannins (sorghum, black tea) and phytic acid (wheat bran). Adding tea to the drinking water may be helpful. Of course, also the iron content of drinking water should be low. Animal products should be avoided due to the highly available haem iron. Fruits rich in ascorbic acid (citrus) should not be fed to birds prone to haemosiderosis.

3 DEFENSE MECHANISMS AND HAEMOSIDEROSIS

Besides dietary factors, there are more and more indications that also other mechanisms play an important role in the pathogenesis of haemosiderosis. The incidence of haemosiderosis is higher in birds which are kept in suboptimal, stressful conditions. Also in birds suffering from viral, bacterial or fungal infections, more iron deposition in the liver has been observed. It has been suggested that these observations are the result of the iron withholding defense system. Biochemical

176

evidence indicates that immunologic stress raises the level of iron-binding proteins that function in iron transport and that synthesis of these proteins is regulated by stress hormones. As in mammals, also in birds, withholding iron from potential pathogens has been described as a primitive host defense mechanism. Iron-binding proteins such as transferrin, ferritin, and lactoferrin (latest only in mammals) have a central role in ferrokinetics. These iron-binding proteins also participate in the process of decreasing iron availability for the microorganisms. In mammals, blood iron is regulated by 2 proteins: lactoferrin and transferrin. Both proteins have a high afnity for iron and are bacteriostatic in vitro for a number of bacteria. In birds, blood iron is regulated by transferrin only. During stress or infections, transferrin is released in birds (HALLQUIST AND KLASING 1994, XIE 2002, MAZUR-GONKOWSKA 2004), which binds the blood iron but also increases intestinal iron absorption and iron ux to the liver. This mechanism may be more important than the dietary iron load in the pathogenesis of haemosiderosis. With these new insights, avoiding stress and infections are probably key factors in the prevention of haemosiderosis in birds. Stress can be reduced by optimal housing in spacious aviaries and avoiding overcrowding. Also transport stress should be limited as much as possible by the use of comfortable, large transport cages and appropriate feeding during transport. Optimal veterinary care with a disease control program, quarantine measures for new birds and preventive anti-parasitic treatments are also important. Regular, consistent feeding of an equilibrated diet at xed feeding hours may further reduce stress.

4 CITATION INDEX
1. 2. 3. 4. 5. 6. GLAHN R, WORTLEY G, SOUTH P and MILLER D. Inhibition of iron uptake by phytic acid, tannic acid and ZnCl2: studies using an in vitro digestion/ Caco-2 cell model. J Agric Food Chem 2002; 50(2): 390 - 395. HALLBERG L, BRUNE M and ROSSANDER L. Iron absorption in man: ascorbic acid and dose-dependent inhibition by phytate. Am J Clin Nutr 1989; 49: 140 - 144. HALLBERG L, BRUNE M, ERLANDSSON M, et al. Calcium: effect of different amounts on nonheme- and heme-iron absorption in man. Am J Clin Nutr 1991; 53: 112 - 119. HALLBERG L and HULTHEN L. Prediction of dietary iron absorption: an algorithm for calculating absorption and bioavailability of dietary iron. Am J Clin Nutr 2000; 71: 1147 - 1160. HALLQUIST N and KLASING K. Serotransferrin, ovotransferrin and metallothionein levels during an immune response in chickens. Com Biochem Phys B, Biochem Mol Biol 1994; 108 (3): 375 - 384. MAZUR-GONKOWKA B, KONCICKI A, KRASNOD and BSKA-DEPTA A. Assessment of acute phase response in turkeys experimentally infected with Escherichia coli or haemorrhagic enteritis virus. Bull Vet Inst Pulawy 2004; 48, 19 - 23. METE G, HENDRIKS H, KLAREN P, et al. Iron metabolism in mynah birds

7.

177

(Gracula religiosa) resembles human hereditary haemochromatosis. Avian Pathol 2003; 32(6): 625 - 632. 8. SCHOEMAKER N and BEYNEN A. Composition, and in particular the iron content, of commercial feeds for mynah birds. Tijdschr Diergeneeskd 2001; 126: 620 - 623. 9. SEIBELS B, LAMBERSKI N, GREGORY C, et al. Effective use of tea to limit dietary iron available to starlings (Sturnus vulgaris). J Zoo Wild Med 2003; 34 (3): 314 - 316. 10. XIE H, HUFF G, HUFF W, et al. Identication of ovotransferrin as an acute phase protein in chickens. Poult Sci. 2002; 81(1): 112 - 120. AUTHORS ADRESS G. WERQUIN, DVM R & D Department, Versele-Laga, Kapellestraat 70, B9800 Deinze, Belgium Email: guy.werquin@verla.be

178

Section of Pet Avian, Exotic Animals and Wildlife Division, Faculty of Veterinary Medicine, Utrecht University, The Netherlands

EXOCRINE PANCREATIC INSUFFICIENCY IN A RACING PIGEON (Columba livia domestica)

O. Amann, M.J.M. Visschers, G.M. Dorrestein, N.J. Schoemaker, I. Westerhof, J.T. Lumeij

KEYWORDS
Pancreas- Birds- Amylase- Lipase- Coagulopathy

ABSTRACT
A six-year-old female racing pigeon (Columba livia domestica) was examined because of an 8-month-history of weight loss, reproductive problems, polyphagia and production of voluminous sand-coloured faeces. Faecal examination revealed large amounts of fat and starch and lack of fatty acids. Faecal trypsin and amylase activities were decreased in comparison with reference values obtained from faeces of 24 racing pigeons. While supplementing the pigeons diet with pancreatic enzymes, food intake and faeces production decreased and the pigeon started gaining weight. After 21 days of hospitalisation the pigeon died following an acute onset of haemorrhagic nasal discharge. Histology of the pancreas showed lymphocytic inltration and almost a total loss of tubuloacinar tissue. Aetiology, diagnosis and treatment of exocrine pancreatic insufciency and the pathophysiological relation between the haemorrhagic syndrome and the underlying disease will be discussed.

1 INTRODUCTION
In domestic animals progressive loss of exocrine pancreatic acinar cell function leads to failure of digestion due to inadequate production of pancreatic enzymes. The most common cause of such severe loss of exocrine tissue in the dog is pancreatic acinar atrophy (PAA). Pancreatic insufciency is caused less commonly by chronic pancreatitis and rarely pancreatic neoplasia (WILLIAMS 1989). Chronic pancreatitis resulting in progressive destruction of pancreatic tissue is also a common cause of EPI in adult human beings.

179

In birds a decrease in acinar tissue or brosis can occur after a chronic inammatory process and cause clinical symptoms suggestive of pancreatic exocrine insufciency (GRAHAM 1992). EPI, the disorder studied here was previously described in a yellow naped Amazon parrot (Amazona ochrocephala) caused by an adenocarcinoma (RITCHEY et al. 1997), also a case of pancreatic atrophy in a peregrine Falcon (Falco peregrinus) (SAMOUR and NALDO 2002) and in a blue and gold macaw (Ara ararauna) (QUESENBERRY and LIU 1986).

2 MATERIALS AND METHODS

This paper has been submitted for publication, therefore no data can be provided.

3 RESULTS
This paper has been submitted for publication, therefore no data can be provided.

4 CASE REPORT
A 7-year-old female racing pigeon (Columba livia domestica) was presented to the avian and exotic animal division of the Faculty of Veterinary Medicine at Utrecht University with an 8 months history of weight loss (especially during the breeding season), polyphagia and voluminous sand coloured faeces. She was vaccinated once per year against Paramyxovirus I (Colombovac, Fort Dodge, inactivated adjuvant vaccine). On physical examination, the bird was alert but very lean weighing 372 g. The bird was hospitalised for diagnostic tests. Preliminary diagnostic work-up included faecal microscopical examination, haematology and plasma biochemistry. There was a marked leucopoenia 1.1x109 /L (reference 13-22.3x109 /L, RITCHIE et al. 1987) and the protein spectrum showed an increase of the gamma globulins; 12.3g/L (reference 1-3g/L; LUMEIJ 1985) and subsequent decrease in the albumin/globulin ratio; 0.01 (reference 1.5-3.6; LUMEIJ 1987) suggesting a chronic inammatory process. Amylase and lipase where also measured, but they were normal (493 and 3 U/L respectively) compared to the reference values reported in this study. Further tests were done to evaluate exocrine pancreatic function: faecal examination revealed the presence of starch and fat in absence of fatty acids. Amylase and trypsin in faeces were also determined (0 and 2U/L respectively) and were below the reference values. The pigeon was hospitalised and fed with Harrison food (Juvenile FormulaTM, Harrisons Bird Food, Brentwood, TN, USA) mixed with pancreatic enzyme powder (Zymoral powder, AST farma B.V., Oudewater, The Netherlands), 5 gram per

180

100 gram Harrison food. After one day of treatment an increase in the weight was observed, while the amount of faeces was smaller and was normal in colour. After a few days of therapy, it was decided to challenge the pigeon and only Harrison baby bird food, without supplementation of pancreatic enzymes, was fed. Within 24 hours a decrease in bodyweight (10 %) and an increase in food intake (14 g more than the previous day) and faecal production were seen. The faeces returned to be voluminous and sand-coloured. Returning the pigeon on its original diet of Harrison and pancreatic enzymes once again resulted in an increase of bodyweight and a decrease in food intake and faecal production. Once again the appearance of the faeces went back to normal. After 21 days of hospitalisation the pigeon showed an acute onset of dyspnoea and haemorrhagic nasal discharge and it was placed in an oxygen cage. A few hours later it was found dead in its cage and was submitted for necropsy. On macroscopic examination a pale and skinny carcass was seen. Blood was found in the beak, trachea and lateral parts of both lungs. Severe haemorrhage was seen on the left leg from just above the tarsal joint to the left groin. Microscopically the lungs showed blood in the parabronchial lumen and capillaries and interstitial oedema. Microscopy of the pancreas revealed a large lymphocytic inltration of the exocrine pancreas and an almost total loss of tubuloacinar tissue. Langerhans islets from the exocrine part of the pancreas were not affected. There was also a proliferation of the pancreatic ducts found. Microscopy of the spleen showed proliferation of the white pulp. The nal conclusion in the pathology report was pancreatitis, nephritis and bleeding due to trauma in combination with coagulation disorder.

5 DISCUSSION
In this report, the occurrence of exocrine pancreatic insufciency in a racing pigeon is described. All of the symptoms the pigeon showed were similar to those seen in dogs and cats with exocrine pancreatic insufciency: a chronic history of weight loss despite a vigorous appetite, coprophagia and sand-coloured voluminous faeces. In dogs, results of history and physical examination differentiate primary small intestinal disease and malabsorption from exocrine pancreatic insufciency. Also, qualitative faecal analysis for the presence of trypsin and amylase activities and undigested food particles can be used to arrive at a presumptive diagnosis. Some dogs with exocrine pancreatic insufciency have high serum ALT activity because of disrupted small intestinal barriers and resultant hepatic inammation. In dogs, a tentative diagnosis of exocrine pancreatic insufciency is conrmed by measuring serum trypsin-like immunoreactivity (TLI) after a 12-hour fast. Subnormal values indicate inadequate pancreatic exocrine secretion. The low faecal trypsin and amylase activities found is this pigeon, together with high amounts of fat and starch and lack of fatty acids, indicate a problem with the production and/or secretion of pancreatic enzymes. Amylase and lipase values of the blood were normal, but this would not exclude pancreatic disease in dogs where increases in amylase and lipase occur during acute pancreatitis or acute inammatory crisis during chronic pancreatitis.

181

Unfortunately, TLI measuring techniques are not available for the racing pigeon and therefore the diagnosis could not be conrmed with this technique. Necropsy revealed inammation and loss of exocrine pancreatic tissue, consistent with the existence of exocrine pancreatic insufciency due to chronic pancreatic inammation and consistent with the results of blood analysis, whereby indications for a chronic inammatory process were found due to a leucopoenia and an increase in gamma- globulins. Furthermore, microscopy of the spleen revealed comprehensive proliferation of the white pulp, also consistent with the presence of a chronic inammation. Necropsy indicated that the pigeon had died of haemorrhage, based on the macroscopical nding of severe haemorrhage in the area of the left leg and the airways. This could be explained if the pigeon had a coagulopathy, which, in combination with trauma to the left leg, could cause excessive haemorrhage, which could extend to the lungs via the air sacs. The development of a coagulopathy in the course of exocrine pancreatic insufciency can be explained by the inability of digesting and therefore absorbing food in the intestinal tract. In EPI, lipase deciency prevents breakdown of dietary triglycerides into monoglycerides and fatty acids. This step in lipid digestion is needed for adequate solubilisation and absorption of fat-soluble vitamins including vitamin K, and subsequently a vitamin K deciency can develop as described in cats and humans (EDWARS et al. 1987; DUTTA 1982). This can result in a coagulopathy since vitamin K is essential in the pathways of secondary coagulation. After developing such a coagulopathy, minor trauma can lead to excessive bleeding and eventually death. Unfortunately the pigeon died before all diagnostic testing could be concluded. Further research is therefore recommended in order to assess the possibilities of diagnosing EPI in vivo and the relationship between EPI and coagulopathies in avian species.

6 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. DUTTA SK. Deciencies of fat-soluble vitamins in treated patients with pancreatic insufciency. Ann Intern Med 1982; 97: 549 - 52. EDWARDS DF. Probable vitamin K-decient bleeding in two cats with malabsorption syndrome secondary to lymphocytic-plasmocytic enteritis. J Vet Intern Med 1987; 1: 97 - 101. GRAHAM DL. Diseases of the exocrine pancreas in pet, exotic, and wild birds: a pathologist perspective. Proc Assoc Avian Vet, New Orleans 1992: 190 - 193. LUMEIJ JT. The diagnostic value of plasma proteins and non-protein nitrogen substances in birds. The Vet Quarterly 1987; 9: 262 - 268. LUMEIJ JT. Blood chemistry reference values in racing pigeons (Columba livia domestica). Avian Pathol 1985; 14: 401 - 408. QUESENBERRY KE and LIU SK. Pancreatic atrophy in a blue and bold macaw. J Am Vet Med Assoc 1986; 189: 1107 - 1108. RANDALL C J. and REECE RL. Color atlas of avian histopathology. 1996; 60 - 64. RITCHEY JW. Exocrine pancreatic insufciency in a yellow-naped Amazon (Amazona ochrocephala) with pancreatic adenocarcinoma. Vet Pathol 1997; 34: 55 - 57.

182

9.

SAMOUR JH and NALDO J. Pancreatic atrophy in a peregrine falcon (Falco peregrinus). Vet Rec 2002; 151: 124 - 125. 10. WILLIAMS DA. Exocrine pancreatic disease. In: ETTINGER SJ (ed): Textbook of veterinary internal medicine. 3rd edition. Philadelphia: WB Saunders 1989; 1528 - 1554.

AUTHORS ADDRESS
Olga Amann, DVM. Division of Avian and Exotic Animal Medicine Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Universiteit Utrecht, Yalelaan 8, 3584 CM Utrecht, The Netherlands. Email: o.amann@vet.uu.nl

183

Institute for Poultry Diseases Free University of Berlin, Germany

ENDOSCOPY GUIDED MULTIPLE ENTRY SURGERY IN BIRDS

M. Lierz, Dr med vet and H.M. Hafez, Prof Dr med vet

KEYWORDS
Castration - Sterilisation - Avian Endoscopy-guided surgery - Minimally invasive surgery

ABSTRACT
Endoscopic guided surgery is a routine procedure in human medicine and commonly used in small animal practice. In avian medicine endoscopic guided surgery is in its infancy and therefore seldom described. Apart from surgical interventions through the working channel of an endoscope, multiple entry surgical techniques can be applied in birds. So far, double and triple entry surgical techniques have been used. Special equipment of certain size and design (connectable to electro surgery) for use in avian medicine is commercially available. Using these techniques it is also possible to sterilise and castrate birds of both genders. This is very important as the Government of certain European countries are discussing this as a routine procedure in hybrid falcons. They are named as a potential genetic risk for the wild falcon populations (especially peregrines) in the case of mating with wild birds after escaping from captivity. In addition this procedure is important in female pet birds with chronic egg laying. Also imprinted aggressive parrots might be successfully treated with castration, reducing their sexual behaviour, despite the fact that imprinting parrots should always be avoided. The different techniques for endoscope guided multiple entry surgery in birds are described using sterilisation and castration as a model. In addition risks and problems using these techniques are discussed.

1 INTRODUCTION
Avian endoscopy was developed in the 70s for sexing monomorphic species. Despite establishment of DNA-methods for sexing, endoscopic sexing has still advantages. One example is the visual assessment of the gonads to determine anatomical breeding functionality. In addition, endoscopy is a very valuable diagnostic tool within avian practice. As in human medicine, endoscopy-guided surgery is a routine procedure and it is more and more used in small animal medicine, but rarely used in avian medicine. Only endoscopy-guided tissue biopsies are routinely performed in avian medicine. This is surprising, as the bird with its large air-sac system is ideal for

184

such procedures and insufations pumps are not necessary. Recently endoscopyguided surgery in birds has been developed (HERNANDEZ-DIVERS 2004, LIERZ 2004), as it represents a lower impact to the bird compared to a laparotomy. Basically different techniques can be distinguished using endoscope guided surgery. Well known and regular used is a single-entry technique. A single instrument is directed through a working channel into the visual eld of the endoscope. Therefore the instrument cannot be manipulated independently of the endoscope. Using a double entry technique an additional cannula with trocar (as channel for an instrument) is placed, enabling the surgeon to work with two different instruments. Last but not least, in triple entry techniques two cannulae are placed in addition to the centrally placed endoscope. The approaches to the avian coelom are chosen according to the procedure performed. Sterilisation and castration of birds are regular requested procedures and can ideally be performed using endosurgery. The present paper describes the use of endosurgery techniques in sterilisation and castration of birds as an example. These should be used as practical model for introducing endosurgery into avian medicine. With the use of these techniques, other surgeries will be indicated such as tumour or granuloma resection. This has already been performed by the authors successfully with an air sac tumour in a hybrid falcon.

2 STERILISATION AND CASTRATION

Sterilisation and castration are routine procedures in veterinary medicine. Beside the quality improvement of meat (castration of bulls and boars), the modication of behaviour (castration of male dogs), the control of populations (sterilisation of feral cats) or the adaptation to the owner for an easier keeping (castration of female dogs) are reasons for such interventions. In avian medicine, just the castration of male chickens (caponization) is routinely performed to modify the meat quality. But comparable to small animal medicine also bird owners require such procedures for their animals for the following reasons: Castration of hand reared parrots of both sexes to avoid a sexual related aggression against the owner. (Comm.: Therefore hand- rearing should always be avoided.). - Castration and sterilisation of female pet birds to avoid a permanent egglaying. - Castration and sterilisation to control free ranging populations (feral pigeons, geese) (CONVERSE and KENELLY 1994). - Sterilisation of male birds to use them as stimulation partners for females (used for articial insemination with sperm of other males) (SAMOUR and MARHAM 1987). Examples are: Production of falcon hybrids and species conservation projects (A female bird of a rare species is stimulated by a partner of a common species but inseminated with sperm of the rare species, therefore one rare male can be used for several females).

Recently a governmental request has been made for castration and/or sterilisation of birds. Hybrids species (birds of prey, nches, waterfowl) are often produced but there

185

is a possibility that they may escape. Within the wild they might be able to reproduce with the nominate species introducing foreign genetic material. As prohibition of producing such hybrids is not always possible or useful to castrate or sterilise such hybrids is in discussion. Within the USA hybrid falcons have to be imprinted or sterilised prior their use in falconry. As imprinting leads to unwanted behaviour modication in the birds the sterilisation becomes important (JONES and REDIG 2003). Castration and sterilisation of birds by endosurgery seem to be ideal. It is minimally invasive and the gonads located at the cranial pole of the kidney are easy to reach. Larger surgical intervention can be avoided and the risks, as described for castration of chickens (COLLIGNON 1949), can be minimised.

3. SURGICAL TECHNIQUE
3.1. Equipment The main problem using endosurgery is the maintenance of haemostasis. Therefore a radiosurgery unit for the connection to the endosurgical instruments is vital. Also, laser units can be used but they are almost not routinely available in veterinary practice. The development of 2 and 3mm human paediatric laparoscopy equipment (Karl Storz Inc.) has forced the endosurgery in birds. In addition to the basic endoscopic equipment (2.7mm 30 telescope with a working channel, exible biopsy and grasping forceps as well as scissors) a variety of instruments are available and necessary. This includes dissection forceps, scissors, grasping forceps from which parts should be monopolar and therefore connectable to the radiosurgery unit. In addition a bipolar forceps and a monopolar sling are essential. The instruments are introduced into the surgical eld using 2.5mm or 3.5mm (according to the instrument size) cannulae with trocars. After inserting, the trocar is removed holding the cannula in place. The material of such a unit is stainless steel, plastic or graphite. Advanced surgeons might extend their equipment to palpation probes, needle holders and knot tiers. 3.2. Surgical Procedure Preparation for sterilisation or castration by endosurgery (single or double entry technique) is similar to a routine endoscopic examination pulling the left pelvic limb caudal (LIERZ 2004). The approach is from the left side (female birds) or from both sides (male birds). Firstly, the approach to introduce the endoscope is chosen caudal to the last rib. Using this approach nearly all organs can be evaluated. The additional cannula using the double entry technique is placed cranioventral to the endoscope between the last two ribs. Using the triple entry technique the same approaches can be used, placing the second trocar ventrocaudal to the endoscope. Because of the advantageous angle to the gonad and the larger surgical eld the following approaches are recommended, which can also be used for the other above described techniques. Placing the birds identical the pelvic limb is pulled cranial. Ventral to the m. exor cruris medialis the endoscope is introduced caudal to the last rib. One cannula is inserted caudal to the endoscope through the obliquus abdominis externus muscle just caudal to the mid-point of the pubis. This double entry technique can be extended by introducing a second cannula cranial to the endoscope into the last or second last

186

intercostals space. By using a multiple entry technique it is very important to keep the approaches of the instrument ports and the endoscope as far apart from each other as possible. Therefore the triple entry technique is only possible in birds larger than 400g. For sterilisation and in most cases castration the single or double entry techniques are sufcient. The endoscopist controls the endoscope and the instrument by using the single or double entry technique. Performing a triple entry technique the endoscope is placed on a sand bag or handled by an assistant while the endoscopist controls both instruments. The radiosurgery unit is activated by a foot- pedal. 3.2.1. Sterilisation and castration of female birds The ovary is a very fragile organ and its endoscopic removal represents a high risk of lethal bleeding. In addition the total removal of all hormone-producing tissue is very difcult. Therefore female birds are sterilised rather than castrated. In juvenile birds a grasping forceps can be introduced through the endoscopes working channel (single entry technique). The infundibulum can be grasped and the oviduct can be removed from underlying tissue by gently pulling. By this way it is possible to remove nearly the entire oviduct without greater bleedings. Such females are developing uneventful (incl. ovary) and are hormonal active, as it was shown in cockatiels (PYE et al. 2001). Ovulation was not noted in such birds. According to our experience the ovary stay juvenile in some of those birds. As an alternative the oviduct can be obliterated by a monopolar or bipolar grasping forceps. In some cases it was noticed that the oviduct lls with egg albumin during sexual activity and this might lead to problems. Therefore the complete removal of the oviduct is recommended. In adult birds, the oviduct can only be removed using a double or triple entry technique. The oviduct is xed by a grasping forceps and obliterated as far cranial as possible using a mono or bipolar grasping forceps through a second entry port. A second obliteration is performed slightly behind the rst one. Now the oviduct is cut between both obliterations using a monopolar scissor. The same is performed at the most caudal part of the oviduct which can be reached. 3.2.2. Sterilisation of male birds Sterilisation of male birds is described using a laparotomy (BIRKHEAD and PELLAT 1989). The endosurgery technique is similar to those of female birds. In juvenile birds the deferent duct can be grasped and removed by pulling using a grasping forceps through the endoscope working channel. Using this method in Japanese quail it was demonstrated that the testicles developing uneventful and that the testosterone blood levels increasing with sexual maturity similar to un-sterilised males. In addition the sexual behaviour of such males was unchanged (JONES and REDIG 2003). As an alternative the deferent duct can also be obliterated by electrosurgery, not expecting any disadvantages as observed in female birds. In adult birds endoscope guided sterilisation is only possible using a double or triple entry technique. Using a grasping forceps the testicle is elevated from the underlying tissue, leading also to an elevation of the deferent duct. Using a second entry port a scissor is introduced cutting the deferent duct. It is of advantage to obliterate the deferent duct in two places and to cut it in between. In addition it is recommended to remove at least 1 cm of the duct to avoid a re-establishment of functionality if both ends reunies, as described in human medicine.

187

3.2.3. Castration of male birds The castration of male birds is only possible using a double or triple entry technique. Especially in adult birds, the triple entry is of advantage as it allows a better view to the surgical eld. The testicle is elevated from the kidney using a grasping forceps through the working channel of the endoscope (double entry technique) or an extra entry port (triple entry technique). The mesorchium can be cut using a monopolar coagulation scissor. Afterwards the testicle can be removed. In adult birds, the blood supply of the testicle is increased making an additional obliteration of the blood vessels by monopolar or bipolar coagulation forceps necessary. The risk of lethal bleedings is very high in male birds during sexual activity and therefore castration must be avoided during that time. In juvenile birds the testicle can be grasped and removed by a radiosurgical sling, obliterating the vessels when closing the sling.

4. RISKS
The risk of uncontrolled lethal bleeding is always present during endosurgery. Therefore sterilisation and castration of juvenile birds is always preferable as the blood supply of the genital tract is low. On the other hand, the structures of the genital tract are sometimes difcult to distinguish from other tissues (adrenal gland, kidney, ureter, blood vessels) or attached to other organs (depending on the species) leading to further difculties. This requires more experience and training. Using coagulation techniques it is vital to avoid any damage to underlying tissues especially the kidneys and ureters.

CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. BIRKHEAD TR and PELLATT JE: Vasectomy in small passerine birds. Vet Rec 1989; 125: 646. COLLIGNON P: Das Kapaunisieren Die Kastration der Junghhne und anderen Gegels. In: ROEMER R GEFLGELZUCHT BCHEREI. Heft 7; Stuttgart: Eugen-Ulmer Verlag, 1949. CONVERSE K.A. and KENELLY JJ: Evaluation of Canada goose sterilisation for population control. Wildl Soc Bull 1994; 22: 112 - 117 HERNANDEZ-DIVERS S. Personal communication, 2004. JONES R. and REDIG PT: Endoscopy guided vasectomy in the immature japanese quail (Coturnix coturnix japonica). Proc Euro Assoc Avian Vet, Tenerife 2003; 117 - 123. LIERZ M: Endoskopie. In: Pees M (ed.): Leitsymptome bei Papageien und Sittichen. ISBN: 3-8304-1023-9; Enke Verlag, Stuttgart 2004; 185 - 194. PYE G.W., BENNETT RA, PLUNSKE R and DAVIDSON J: Endoscopic salpingohysterectomy of juvenile cockatiels (Nymphicus hollandicus). J Avian Med Surg 2001; 15: 90 - 94. SAMOUR J.H. and MARKHAM JA: Vasectomy in budgerigars (Melopsittacus undulatus). Vet Rec 1987; 120: 115.

188

AUTHORS ADRESS
Michael Lierz Institute for Poultry Diseases, Free University of Berlin, Knigsweg 63, 14163 Berlin, Germany Email: lierz.michael@vetmed.fu-berlin.de

189

The Wildlife Center, National Veterinary School of Nantes, France

TWO CASES OF FEMORAL HEAD LUXATIONS IN CORMORANTS (PHALACROCORAX CARBO) TREATED WITH A MODIFIED MEIJ-HAZEWINKEL-NAP TECHNIQUE
E. Risi DVM, D. Ordonneau DVM, O. Gauthier DVM, PhD

KEYWORDS
Bird - Surgery - Coxofemoral luxation - Cormorant

ABSTRACT
(Phalacrocorax carbo) were two captive cormorants presented with a femoral head dislocation. The injury occurred during a show in a zoo. A rst treatment with capsulorraphy failed. It was decided to try a modied Meij-Hazewinkel-Nap technique to treat these dislocations. The technique consists of reducing the dislocation and xing the greater trochanter to the hip using a synthetic prosthesis. The femoral head is conserved to improve mechanical functions of the hip. We will present the clinical and radiological aspects of the dislocation, the anaesthetic and surgical techniques used and the good results obtained using this surgical technique. One bird died a few weeks post-surgery from probable non-related causes; the femoral head was in a good position. The other bird recovered over several months.

1 INTRODUCTION
Two cormorants (Phalacrocorax carbo) were seen due to a lack of activity and reluctance to move for 4 days. The two birds were adult and weighed 1.8 and 2kg respectively. They lived in a zoo and participated every day in a bird show. Between each performance, the birds were kept on a beam, 1.8mt from the oor, with one leg restrained with a rope. Signs developed after they had fallen from the beam with their leg caught.

190

2 MATERIAL AND METHODS

Clinical examination At the clinical examination, the two birds were in sterno-abdominal recumbency and refused to move. By forcing them, they could stand up with difculty for only a few seconds on one leg, before falling down again. A severe unilateral non-weight-bearing lameness was present in the two cormorants. The birds were in a good condition and no other abnormality was found. The clinical exam of the musculoskeletal system revealed some abnormalities on the right leg of one bird and on the left one of the second bird. In the two cases, the following signs were present: - Leg paresis. - Pain at coxofemoral mobilization - Negative proprioception test. - Positive nociception test: strong reaction of the bird but difcult movement of the leg. - Slight amyotrophy of the damaged leg. - Lack of tonicity of the damaged leg. - Abnormal latero-lateral movements of the damaged leg when the bird was held over the oor. - Asymmetric position of the two legs in extension, the injured leg appeared internally rotated and longer than the healthy one. Differential diagnoses The differential diagnosis included: Ventral luxation of the femoral head. Ventral luxation of the femoral head associated with a proximal fracture of the femur. Proximal fracture of the femur. Femoral head or neck fracture. Ventral luxation of the femoral head associated with nervous damaged. Ventral luxation of the femoral head associated with acetabular fracture. Other examinations Radiographs in ventrodorsal and lateral recumbency conrmed the ventral coxofemoral luxation of the right leg and the left one respectively. No other lesions were visible.

3 TREATMENT
A surgical treatment was made 7 days after the accident. An open reduction technique with capsular suture and non-absorbable sutures between the periosteum of the greater trochanter and the acetabular rim was performed. The rst cormorant was anaesthetized using an injection of midazolam (Hypnovel, 0.5 mg/kg IM), followed by ketamine (Chlorketam, 10mg/kg IV) via a catheter in the axillary vein. Anaesthesia

191

was then maintained with isourane after intubation. Analgesia was provided by nalbuphine (Nubain, 0.5mg/kg IM). The bird was placed in a lateral recumbency on the healthy side with the wings pulled dorsally. The damaged leg was plucked and disinfected with iodine povidone (Vetedine). A curved skin incision was made over the greater trochanter. The gluteal muscles were torn, caudally and dorsally to the hip articulation. In order not to damage the healthy cranial soft tissue, it was decided to use a caudal approach to the hip, between the already damaged muscles. The articular cavity was gently cleaned by removing all the coagulated blood and necrotic tissue. The ventral dislocation was then reduced by internal rotation, abduction and traction of the leg. The capsular lesions were too severe to allow a capsulorrhaphy. Five sutures with a non-absorbable suture material (Flexiden) were made through the gluteal muscle tendons on their trochanteric attachments and the acetabular rim periosteum. Necrotic gluteal muscles borders were debrided before suturing these muscles with an absorbable material (Sal). Skin was then closed with U points. The good reduction of the luxation was conrmed by a radiograph. The leg was then maintained in a bent position using a bandage under the body. The day after surgery, the leg was held abnormally. Radiography revealed a new ventral luxation. The day after, surgery was performed. A modied Meij-HazewinkelNap technique was attempted (MEIJ et al. 1992, MEHEUST et al. 1999). This technique consists of stabilizing the hip by passing a prosthetic ligament, made with a non-absorbable material, between the greater trochanter and the ilium by perforating them. This technique had to be modied, because of the hip anatomy of the cormorant and the biped posture. The caudal border of the acetabulum and the caudal protuberance (present in cormorants) were gently exposed by removing the muscles around. A Kirshner pin was introduced through the acetabular protuberance and then through the greater trochanter in order to make tunnels in each of these bones. The prosthetic ligament (a 1/0 non-absorbable woven multilament suture) was then passed trough these tunnels and tied in a gure-of-eight pattern. Then, the same bandage, in a bent position for performed. The luxation of the second cormorant was treated using the same technique. Anaesthesia was induced with diazepam (Valium, 0.5mg/kg) and ketamine (10mg/kg) by intravenous injection then isourane.

4 RESULTS
The two birds were hospitalized for 3 weeks while bandaged. Regular radiographs have been taken to make sure of the good position of the articulation. Antibiotics (marbooxacin, Marbocyl, 10mg/kg bid for 10 days) and NSAID (ketoprofen, Ketofen, 4mg/kg sid IM for 7 days) were given. Bandages were removed when going back to the zoo. Cage connement and limited activity were required. A re-educational program was established: the leg was handled daily with rotating movements of the knee and the hip in order to make muscles work, reduce amyotrophy and joint ankylosis. Time and intensity of this re-education were daily increased. Moreover, the birds were allowed to swim in their pool giving further leg movements. A non-weightbearing lameness, associated with an amyotrophy and a moderate muscular brosis, was present for the 10 rst days after bandage removing. A moderate lameness was

192

then observed over the following weeks and locomotory function improved gradually with swimming and re-education sessions. Seven weeks after surgery, one of the cormorants was found dead without clinical signs. Unfortunately, no necropsy was made only an incision was made on the operative site to conrm the good position of the coxofemoral articulation and the absence of infectious or abnormal inammatory reaction around the prosthesis. The second bird gradually returned to full activity, with a slight lameness but a normal range of motion, over a 13 week period after surgery.

5 DISCUSSION
In pet mammals, hip luxations are frequently due to trauma (BRINCKER et al. 1994, FOSSUM 2002, HOLSWORTH and DECAMP 2003). Soft tissue damage always includes round ligament and articular capsule rupture (BRINCKER et al. 1994, FOSSUM 2002). Gluteal muscle damage is seen in severe cases (BRINCKER et al. 1994). Craniodorsal luxations are the most common type (70-80% of patients) (BRINCKER et al. 1994, HOLSWORTH and DECAMP 2003).Ventral luxations are rare (1.5-3.2% of patients) and frequently associated with acetabular fractures (BRINCKER et al. 1994). In birds, luxations are not a common problem and are less frequently reported than fractures (MARTIN and RITCHIE 1994, MACCOY 1989). As in mammals, they are more generally craniodorsal to the acetabulum (MARTIN and RITCHIE 1994, BENNETT 1997, MACCOY 1989) and occur with tendons afictions or more frequently with trauma by traction and rotation of the leg (HEIDENREICH 1997, BENNETT 1997). Elbow and shoulder luxations are more common than coxofemoral dislocation (HEIDENREICH 1997). In birds, the luxation must be treated to prevent periarticular brosis formation leading to joint ankylosis (BENNETT 1997), pseudarthrosis, muscle contracture and chronic deviation of the limb (MACCOY 1989). Moreover, because of the birds bipedal posture, hip luxation may rapidly cause problems in the nonluxated leg, e.g. bumblefoot, due to abnormal or compensatory weight-bearing (MACCOY 1989). Luxations in birds should be reduced as early as possible (less than 3 days) (BENNETT 1997). In our 2 cases, the luxations were ventral and associated with gluteal muscle damage. They may be considered as rare and serious luxations. The ventral position of the femoral head is here simply explained by the trauma history: a fall from a beam with a caught leg. In mammals, hip luxations must be treated quickly, by closed or open reduction (BRINCKER et al. 1994, FOSSUM 2002, HOLSWORTH and DECAMP 2003). The different techniques of stabilization include: capsule reconstruction (capsulorrhaphy), translocation of the greater trochanter, excision arthroplasty of the femoral head and neck, prosthetic capsule (modied Knowles toggle technique), prosthetic round ligament, transacetabular pin (through the great trochanter into the head of the femur across the acetabulum) or extra-articular stabilisation with a prosthetic ligament between the femoral neck and the ilium (Meij-Hazewinkel-Nap technique) (BRINCKER et al. 1994, FOSSUM 2002, HOLSWORTH and DECAMP 2003, MEIJ et al. 1992, MEHEUST et al. 1999). For ventral luxations, suture of the dorsal joint capsule alone is inadequate to maintain reduction. A prosthetic capsule technique or a toggle pin procedure is preferred (FOSSUM 2002).

193

In birds, very few techniques are described for hip luxation treatment. MACCOY describes an excision arthroplasty technique used in 3 birds (a hyacinth macaw, a Moluccan cockatoo and an African grey parrot) with a caudolateral approach to the articulation (MACCOY 1989). The birds weighed between 554g and 1355g). The results were good several years after surgery. Owing to the larger weight of the two cormorants and their poor and injured gluteal muscles masses, we decided not to use this technique to prevent gluteal muscles perforation by the femur. Few other methods are reported in birds. A manual reduction and application of a hip splint has been reported in a hyacinth macaw but the bird did not tolerate the splint and frequent recurrences occurred (MACCOY 1989). Transacetabular pinning is not recommended because of the risk of penetrating the kidney by inserting the pin too deep (leading to kidney lesions and haemorrhages) (BENNETT 1997). Moreover, this technique requires a second anaesthetic to remove the pin after a few days, in order to prevent degenerative joint disease development (BENNETT 1997). Reduction and stabilization with supporting structures (non absorbable sutures), placed from the trochanter to the dorsolateral iliac crest and to the cranial rim of the acetabulum are proposed (MARTIN and RITCHIE 1994, BENNETT 1997), but no cases have been reported, in the authors knowledge. The modied Meij-Hazewinkel-Nap technique used in our cases seems to give good results in birds. Without post-mortem examination, we cannot be sure that the cause of death was unrelated to surgery. But, due to long time between the surgery and the death (7 weeks), and the absence of gross lesions around the prosthesis, a death due to surgery seems unlikely, even if this possibility can not be excluded. The caudal approach of the articulation was made here to avoid other muscle damages and it is reported that, in birds, the caudal approach is preferred to avoid potential injury to the cranial coxal artery and to provide better exposure of the femoral head and neck (MACCOY 1989). No damage to the sciatic nerve occurred with this approach in our two cases. In spite of the recommendation to place the prosthetic ligament cranial to the articulation (between the greater trochanter and the ilium, Meij-Hazewinkel-Nap technique (MEIJ et al. 1992, MEHEUST et al. 1999), we preferred a caudal position (between the greater trochanter and the acetabular caudal protuberance) to avoid instability by pushing on the leg in the biped position. The forces applied to the hip are almost vertical in birds because of this biped position and the ligament seemed to have a better function in a caudal position, to maintain the femoral head into the acetabular cavity. In mammals, an Ehmer bandage is made to assist hip reduction (4-7 days postoperatively) (BRINCKER et al. 1994, FOSSUM 2002). Limited activity is required for an additional 3 weeks (FOSSUM 2002, HOLSWORTH and DECAMP 2003). The animal is then gradually returned to full activity over a 2-3 weeks period (BRINCKER et al. 1994, FOSSUM 2002). In our case, a real Ehmer bandage was not possible, because of the anatomy of the leg. An immobilization was made in order to hold the leg in a bending position under the body. The birds kept this bandage for 3 weeks to prevent recurrence of the luxation. This long immobilization time could have been responsible for the muscle contracture seen after removal. The recovery time to full locomotion may have been prolonged by this immobilization. The bandages should have been removed earlier and physiotherapy begun sooner to have better and quicker results.

194

6 CONCLUSION
A modied Meij-Hazewinkel-Nap technique can be used in cormorants for coxofemoral luxation treatment. A bandage must be applied after surgery and removed before 3 weeks. Physiotherapy in water is required to improve hip mechanical functions.

7 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. 9. MARTIN H and RITCHIE BW. Orthopedic surgical techniques. In: RITCHIE BW, HARRISON GJ and HARRISON LR (eds): Avian medicine, principles and application. Lake Worth: Wingers Publishing 1994; 1137 - 1169. HEIDEN REICH M. Birds of prey, medicine and management. Oxford: Blackwell Science 1997; 284. BENNETT RA. Orthopedic surgery. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM, QUESENBERRY K (eds): Avian medicine and surgery. Philadelphia: WB Saunders Co 1997; 733 - 766. BRINCKER et al. Manuel dorthopdie et de traitement des fractures des petits animaux. Maisons-Alfort, Editions du Point Vtrinaire 1994; 560. FOSSUM TW. Small Animal Surgery 2nd ed. St Louis: Mosby 2002; 1400. HOLSWORTH IG and DECAMP CE. Coxofemoral luxation. In: SLATTER I (ed): Textbook of small animal surgery 3th ed. Philadelphia: WB Saunders 2003; (2): 2002 - 2008. MACCOY DM. J Am Vet Med Ass 1989; 194(1): 95 - 97. MEIJ BP, HAZELWINKEL AW, NAP RC. Results of extra-articular stabilisation following open reduction of coxofemoral luxation in dogs and cats. J Small Anim Pract 1992; (33): 320 - 326. MEHEUST P, BOURGERON A, LEGEAR F. Stabilisation des luxations coxofmorales traumatiques par la technique de Meij-Hazewinkel-Nap modie: tude rtrospective de 63 cas. Prat Med Chir Anim Comp 1999; (34): 611 - 621.

AUTHORS ADDRESS
Emmanuel Risi, DVM Centre de Soins de la Faune Sauvage, Ecole Nationale Vtrinaire de Nantes, Route de Gachet, 44300 Nantes, France Email: Emmanuel.risi@wanadoo.fr

195

Department of Surgery and Internal Medicine1, School of Veterinary Medicine, University Complutense of Madrid, Madrid Department of Veterinary Clinical Sciences2, School of Veterinary Medicine of Lugo, University of Santiago de Compostela, Lugo, Spain

MANAGEMENT OF THE FEMOROTIBIAL LUXATION BY COAPTATION SPLINTING, INTRAMEDULLARY PINS, EXTERNAL SKELETAL FIXATOR AND TENSION BANDS: A PILOT STUDY IN DOMESTIC PIGEONS (Columba livia domestica)
S. Villaverde DVM1; J. Benito de la Vibora DVM1; R. Gonzalez DVM1; M. Freire DVM1; M. Lopez Pea DVM PhD2, F. Muoz Guzon DVM PhD2, J. Rodriguez-Quiros DVM PhD1

KEYWORDS
Avian Birds Pigeons Stie Luxation Orthopaedics

ABSTRACT
Repair of a stie luxation requires stabilisation to minimize the formation of periarticular brosis. The purpose of this study is to show comparatively different surgical techniques for luxation repair of the femorotibial joint. Domestic pigeons were selected. The initial goal consisted of providing lack of femorotibial stability. Animals were assigned to six groups. We evaluated anaesthetic and surgical duration, functional angles, mobility and normal use of the limb, bony changes and alignment of the joint. Decreased anaesthetic and surgical duration was seen with single band tension and splinting. The functional angles were similar in external skeletal xators (ESF) before and after the surgery. The alignment was restored except in the control group. The articular surfaces had a minimal damage. Animals were weight bearing two or three days later with splinting and IM pins techniques. Open surgical managements may increase morbidity. The formation of scar tissue to stabilise the joint requires 3 to 6 weeks. All the techniques performed represented a viable option to luxation repair. There were not signicant histological differences among the different groups when evaluating periarticular brosis tissue and bone reaction.

196

1 INTRODUCTION
Stie luxation studies and case reports in different species of exotic mammals and reptilian have been reported. Stie luxation is uncommon in birds (ROSENTHAL et al. 1992), but has been mainly diagnosed in psittacines and raptors (BOWLES and ZANTOP 2002, HARCOURT-BROWN 2000). The femorotibiobular joint is luxated with concomitant damage of the ligaments (Bennett 1998) and rarely meniscal damage is diagnosed (HARCOURT-BROWN 2000). Dislocation of the femorotibial joint with damage to the collateral ligaments generally results from a developmental abnormality (BENNETT 1998, CLIPSHAM 1991), spontaneous orthopaedic disease (HARCOURT-BROWN 2000) and following traumatic episodes (BOWLES and ZANTOP 2002). Findings include hyperextension of the joint, positive withdrawal sign and also medial or lateral instability on examination. Absence of palpable fracture, lack of crepitus on palpation, and the ability to partially reduce the tibiotarsus into its normal anatomic position suggested a luxation instead of a fracture. Luxation of the femorotibial joint occurs craniolaterally, craniomedially, caudolaterally and caudomedially. This luxation is diagnosed denitely radiographically (BOWLES and ZANTOP 2002). There are scanty reports in the literature showing comparatively surgical techniques for stie luxation repair in avian patients (MACCOY 1988). Repair of a stie luxation requires stabilisation of the joint by reduction and immobilisation, as early as possible to minimize the formation of periarticular brosis (BENNET 1998, BOWLES and ZANTOP 2002, MARTIN and RITCHIE 1994). Within a period of as little as 3 days, signicant brosis occurs, inhibiting reduction of the luxation and predisposing to joint ankylosis (BENNETT 1998). The purpose of this pilot study was to show comparatively ve different surgical techniques for luxation repair of the femorotibial joint in birds.

2 MATERIAL AND METHODS

Eighteen mature domestic pigeons (Columba livia domestica), with a mean body weight of 290 g were selected for the pilot study. The pigeons were housed indoors and were allowed to acclimatise for at least two weeks before the start of the experimental procedures which were performed after obtaining authorization from our Hospital Institutional Animal Care Committee. Butorphanol (1 mg/kg IM) and meloxicam (0.2 mg/kg IM) were administered preoperatively. Hydration with lactated Ringers solution injected subcutaneously was made for each pigeon before induction and at the completion of each surgery. The total uid volume was 5% weight equally divided between the two injections. The birds were induced with isourane by mask and they were maintained on a surgical plane of anaesthesia using an oxygen fresh gas ow through a modied Jackson Rees non-rebreathing circuit adjusted to 0.9 L/minute and isourane inspired concentration adjusted to 2%. Anaesthetic monitoring included electrocardiogram (ECG), relative arterial oxygen (SpO2) and pulse rate by use of a pulse oximeter, respiratory rate,

197

end tidal carbon dioxide partial pressure (ETCO2) and rectal temperature (Julian DRGER anaesthesia machine and monitoring, Lbeck, Germany). All of the surgical and radiographic procedures were performed under general anaesthesia. After aseptic preparation and draping, the initial goal of the experimental design consisted of providing lack of femorotibial stability. Before the skin incision was accomplished, lateral collateral ligament was identied. The surgical procedure was initiated with a lateral approach to the stie. A linear incision was made cranial to the lateral collateral ligament, from lateral condyle of the femur to the bular head. The joint capsule was incised, lateral collateral and femorobular ligaments were sectioned to achieve a medial patellar dislocation, and then collateral ligaments were also sectioned to allow a mediolateral stie luxation. On this way withdrawal sign was positive on palpation of the stie. Following dislocation of the femorotibial joint, pigeons were randomly assigned to six groups, consisting of three birds per group, by the management performed to stabilise the luxation: Group 1 served as control group since the capsule was incised without repairing the joint, but the closure was made with a 4-0 synthetic absorbable suture material of polyglycolic acid to suture the joint capsule incision, the subcutaneous tissue and the skin in a simple continuous pattern. The leg was wrapping with a cohesive exible material (i.e. Vetrap) in a Robert Jones bandage to protect the surgical repair. Group 2 consisted of coaption splinting. A splint was applied from femur to metatarsal bones including the digits I and III to keep the joint immobile and to maintain the pigeons leg in a proper position. The splint with a normal angle was attached by wrapping with an elastic tape (i.e. Vetrap) around the leg. Group 3 consisted of two intramedullary pins (IM). One 1.0 mm Kirschner wire was placed into the medullary cavity of the tibiotarsal bone and the second one was seated into the medullary cavity of the femur. The external portion of each pin was bent 90 form its insertion portion and then they were joined with a veterinary thermoplastic material (i.e. VTP) to maintain the stie joint placed in a functional angle of 60. Group 4 consisted of a transarticular type I ESF. Five K-wires (1.0 mm ) were xed into the lateral aspect of the femur (2) and tibiotarsus (3). The wires were bent using a veterinary thermoplastic casting material (i.e. VTP) for the connecting bars. It was applied with the joint placed in a functional angle of 60. Group 5 consisted of a screw and tension band with a single strand of nylon suture. A pin of 1.2 mm was used to drill a hole through the condyles of the femur. Through the hole a 7 mm long, 1.5 mm bone screw was placed into the femoral condyle. A second hole was drilled through the bular crest with a 3-0 nylon suture needle. The luxation was reduced with a tension band of a single strand of 3-0 nylon suture that extended from the screw in the lateral condyle of femur to the predrilled hole into the cnemial crest.

198

Finally, group 6 consisted of a cortical bone screw and double tension band. The luxation was reduced similar to group 5 and a second strand of nylon suture was xed from lateral condyle of femur to the space between the bula and tibiotarsus, ventral to the bular head.

Animals recovered from anaesthesia. The postoperative care was the same for all the groups. Postoperative radiographs were taken to conrm that the reduction was made and remained intact. Enrooxacin (10 mg/kg IM q12h) was administered prophylactically for a minimum of 5 days following surgery. Analgesia was provided with buthorphanol (1.0 mg/kg IM q6-8h) or meloxicam (0.2 mg/kg orally q24h, for three or four days). This study evaluated the anaesthetic and surgical duration of the different managements; the functional angles of the limb exing and extending the knee, before and after the surgery; the bony changes and alignment of the joint radiographically; morbidity and normal use of the limb putting weight, bearing the foot and ambulating. The stie was taken out for histophatological study at 6 weeks post surgery, in order to value some aspects such as periarticular brosis tissue and bone reaction and to compare denitely the different surgical techniques. Samples were xed in 10% buffered formalin, decalcied, processed by routine histological methods and embedded in parafn. Four m sections were stained with haematoxylin and eosin for histopathology.

3 RESULTS
All the eighteen procedures were performed safely and easily. Healing of the joint was assessed before removal of the orthopaedic material. Decreased anaesthetic and surgical duration was seen in management with single band tension and splinting compared with other techniques. The functional angles of the exing limb were similar in ESF technique before and after the surgery. The joint was observed radiographically without evidence of potential complications or bony changes in the groups. The stie alignment was restored except in the control group. The return to normal function of the limb putting weight after surgery suggests that the articular surfaces of the stie had a minimal damage. Animals were weight bearing on the limb two or three days later with splinting and IM pins techniques. There were not signicant histological differences among the different groups when evaluating some aspects such as periarticular brosis tissue and bone reaction. Histological changes were observed only in groups treated with IM pins and ESF. In these groups some cases of articular ankylosis and calcications were observed. No inammatory changes were presented.

199

4 DISCUSSION
It is very important to reduce the luxation as early as possible to minimize the formation of brosis, which inhibits the reduction of luxation predisposing to joint ankylosis since it is assumed that joint luxation carries a poor prognosis (BENNETT 1998). Stie exploration in avian species may be difcult because of the vulnerability of the neurovascular bundle to damage (BOWLES and ZANTOP 2002). The procedure used to repair the dislocation depends on the size of the patient and the nature of the injury (BENNETT 1998). Reduction and stabilisation of the femorotibial joint by surgical xation is considered the best option because of the patients small size, weight, and age (BOWLES and ZANTOP 2002, ROSENTHAL et al. 1992). The femorotibial joint can be stabilised by using close or open techniques. The main disadvantage of close managements such as coaptation by bandages, casts and splinting, is the poor anatomic reduction to maintain stabilisation of the joint, compromising its function and range of motion. Open surgical management and internal or external xation techniques have been described (ROSENTHAL et al. 1992). Although these techniques save the limb, sequelaes include increased morbidity and prolonged recovery, post-luxation bumblefoot, degenerative changes and abnormal weight-bearing (BENNETT 1998, HARCOURT-BROWN 2000). The repair of damaged ligaments in large birds using suture material may be attempted (HARCOURT-BROWN 2000). Arthrotomy has been used to repair femorotibial joint injuries in birds (BOWLES and ZANTOP 2002). ESF with connecting bars with polymethylmethacrylate or stainless steel may be used in avian patients (BENNETT 1998, REDIG 2001, ROSENTHAL et al. 1992, SATERFIELD and OROURKE 1981). Complications of ESF include pathological fracture of the femur or tibiotarsus, with increased anaesthetic and surgical duration and increased soft tissue trauma. ESF do not allow limb growth in paediatric patients (BOWLES and ZANTOP 2002). Recovery may be adversely affected by excessive weight of the xator and decreased patient compliance (BOWLES and ZANTOP 2002, HARCOURT-BROWN 2000, ROSENTHAL et al. 1992). The use of methacrylates in birds instead of stainless steel connecting bars of ESF, allows readjustment until the proper anatomic position is achieved (ROSENTHAL et al. 1992). Techniques describing IM pins joined with acrylic to rigidly stabilise the knee in its normal anatomic position have been reported (BOWLES and ZANTOP 2002). The signicant disadvantage of these techniques is that the pins could interfere with normal stie joint function (MACCOY 1988). The formation of sufcient scar tissue to stabilise the joint requires 3 to 6 weeks, which is the interval before the pins are removed (BENNET 1998). Procedures with band tension have been reported with cerclage wire and other different suture and different splinting techniques (HOLZ 1992). Cortical bone screws with band tensions have a signicant disadvantage because once the screw is placed, it is impossible to readjust the angle in which the stie become xed (ROSENTHAL et al. 1992). Evidence of the return to normal function of birds after stabilisation is by its ability to use the limb to bear weight, to ambulate and to balance on the other limb (MACCOY 1988).

200

The results of this preliminary study indicate that all of these techniques performed represent a viable option to femorotibial luxation repair in avian species. We conclude that stie luxation repair in small birds might be accomplished with surgical techniques whichever stabilise the joint and permit a good clinical evolution.

5 ACKNOWLEDGEMENTS
The authors gratefully acknowledge Pilar Llorens, chief of the radiological service, and especially to Isabel Garcia, Carmen Osorno and Sonia Pavon, radiology technicians of Imaging Diagnostic Section of the Veterinary Teaching Hospital. We want to express our gratitude to all the students workers, especially to Ruben Mota and Alicia of the Avian, Exotic and Wild Animal Surgery Group, in the Veterinary Teaching Hospital, for their help. Without their competence and persistence in the technical and nursing care of the patients, the success rate of orthopaedic procedures we enjoy would not be realised.

6 CITATION INDEX
BENNETT RA. Management of joint luxations in birds. In: REYNOLDS MD (ed): From Science to Reality - A bridge to the 21st Century (Proceedings of the International Wildlife Rehabilitation Council Conference, Concord, California, (1997). International Wildlife Rehabilitation Council (IWRC), Suisun City, California, 1998; 11 - 15. 2. BOWLES HL and ZANTOP DW. A novel surgical technique for luxation repair of the femorotibial joint in a monk parakeet (Myiopsitta monachus). J Avian Med Surg 2002; 16(1): 34 - 38. 3. CLIPSHAM R. Correction of pediatric leg disorders. Proc Assoc Avian Vet, Chicago 1991: 200 - 204. 4. HARCOURT-BROWN NH. Birds of Prey: Anatomy, radiology and clinical conditions of the pelvic limb. CD-Rom, Zoological Education Network, Inc. Lake Worth, Florida, 2000. 5. HOLZ P. Luxation of the stie joint in a Major Mitchell cockatoo. Vet Rec 1992; 130(2): 34. 6. MACCOY DM. Management of coxofemoral luxations by femoral head ostectomy and long bone fractures by K-E splint, pinning, plating, and coaptation splinting. Proc Eastern States Vet Med Assoc 1988: 166 - 167. 7. MARTIN HD and RITCHIE BW. Orthopedic surgical techniques. In: RITCHIE, BW, HARRISON, GJ and HARRISON LD (eds): Avian Medicine: Principles and Application. Wingers Publishing Inc., Lake Worth: 1994; 1137 - 1169. 8. REDIG PT. Effective methods for management of avian fractures and other orthopaedic problems. Proc Eur Assoc Avian Vet, Munich 2001: 26 - 42. 9. ROSENTHAL K, HYLLYER E and MATHIESSEN D. Stie luxation repair in a moluccan cockatoo and a barn owl. J Assoc Avian Vet 1992; 6(4): 235 - 238. 10. SATTERFIELD WC and OROURKE KI. External skeletal xation in avian orthopedics using a modied through-and-through Kirschner-Ehmer splint technique (the Boston technique). J Am Anim Hosp 1981; 17: 635 - 637. 1.

201

AUTHORSS ADRESS
S. Villaverde ESVA, Asistencia Veterinaria s.l., C.E.R.I. Centro de Estudios de Rapaces Ibricas, Sevilleja de la Jara, 45671 Toledo, Spain Email: cerito@jccm.es

202

The Louisiana State University, School of Veterinary Medicine Department Veterinary Clinical Sciences, Baton Rouge, Louisiana, United States of America

USE OF A DENTAL COMPOSITE TO CORRECT BEAK DEVIATION IN PSITTACINE SPECIES

T. N. Tully, Jr. DVM, MS, Diplomate ABVP (avian), Dip ECAMS, A. G. Stevens, DVM, O. Diaz - Figaroa, DVM, T. Zachariah DVM, M. A. Mitchell DVM, MS, PhD

KEYWORDS
Birds - Beak deviation - Psittacine - Dental composite - Mandibular prognathism Scissor beak

ABSTRACT
Psittacine species have 2 common beak deviation presentations that present during their growth phase. The 2 beak deviation presentations have been classied as scissor beak, where the rostrum maxillare (upper beak) grows to the side of the rostrum mandibulare (lower beak) and mandibular prognathism, where the lower beak is longer than the upper beak causing the upper beak to grow into the lower beak (CLIPSHAM 1989 and 1992, SPEER 1995 and 2003). There have been a number of corrective devices promoted to correct these beak deviation conditions in parrot species (CLIPSHAM 1992, MARTIN and RITCHIE 1994). The advantage of this technique is that it uses a cold rapid curing human dental composite and the birds natural beak dynamics to support the corrective device. In supporting the corrective device the birds natural beak dynamics allow for greater force and quicker manipulation of the beak back into its normal position without the consequences often involved with previously published techniques.

INTRODUCTION
Scissor beak and mandibular prognathism are common beak deviation presentations in captive raised psittacine species (CLIPSHAM 1992, SPEER 1995). Scissor beak most often affects macaw species while mandibular prognathism is diagnosed commonly in cockatoos, although other psittacine species may be affected by either of these conditions. In severe cases the birds ability to eat may be compromised, but at the very least, continued beak trimming is required due to the malocclusion and

203

continued growth of the keratin surface of the beak. Birds with deviated beaks also are difcult to sell or bring a reduced price because of their undesirable appearance. Previously published techniques have used ultraviolet cured dental acrylic extensions either placed directly over the beak surface or onto a wire/plate foundation to push the beak back into place (CLIPSHAM 1989) and pins/wires placed through the beak and frontal bone using rubber bands to pull the beak back into place (MARTIN and RITCHIE 1994). The problems that are often encountered with previously published techniques is the dental acrylic having difculty adhering to the beak surface, the pins pulling through the beak and/or frontal bone, the acrylic foundation breaking off from the beak with pieces of the beak attached and iatrogenic beak trauma from the forces applied leaving permanent beak scarring. Another disadvantage of previously published beak correction techniques is the use of general anaesthesia to apply the acrylic or hardware. With this new technique the bird is placed in an avian restraint board and the corrective composite is applied while the bird is awake. The other advantages include rapid, cool curing and the use of the birds natural beak anatomy for support of the composite mass. By using the birds natural beak anatomy to support the composite mass the chance of iatrogenic beak damage is reduced and gives more support for correction as pressure is place to move the beak into normal position. A disadvantage with this new technique is similar to other corrective hardware placements, the patient may have to be hand or tube fed during the treatment period.

MATERIALS AND METHODS

The patient and owner are initially assessed on compatibility for this long term (3 to 6 month) intensive beak correcting process. Once there is an understanding of the primary goal: function not perfection and acceptance of this goal then the process of correction can begin. The bird is placed in an avian restraint board (Miami Vice, Veterinary Specialty Products, Boca Raton, Florida, USA) and held in a standing position. An avian beak speculum (Lafeber Products, Cornell, Illinois, USA) is used to open the beak and tongue depressors are used to manipulate l the dental acrylic. The dental acrylic (Protemp 3 Garant, 3M ESPE AG, Dental Products, D-82229 Seefeld Germany) is then placed on a beak that has been scored using a Dremel tool (Dremel, Robert Bosch Tool Corp., Mount Prospect, Illinois, USA) tted with a sandstone bur. The dental acrylic is layered on the tip of the upper beak for mandibular prognathism and the lower beak for birds presenting with scissors beak. The beak speculum and tongue depressors are left in place as the acrylic cures, usually 5 to 7 minutes. Once there is a substantial amount of dental composite applied to the beak the Dremel tool with the sandstone bur is used to modify the mass into the corrective shape. This may also include forming a trough in the composite to manipulate the beak toward centre for scissor beak cases. The composite will fall off the beak but appears to remain longer on the beak than the ultraviolet cured acrylic products. The composite can be cut off the beak in pieces and if the material does not fall off can be pulled off by using a slight twisting motion. For most cases weekly revisits over the rst 6 weeks of treatment are required to assess the patients condition and response to the corrective device. Modications are made to the composite by applying new layers of material over the cured appliance. Reforming the composite must take place after the material cures.

204

RESULTS
This beak correction technique has been tried on approximately 10 birds, the majority being macaw species presenting with scissor beak. A few cockatoos with mandibular prognathism have also been treated using the dental composite. We have found that this technique offers a quick correction in the scissor beak cases while reducing the number of iatrogenic damage associated with previously published beak corrective techniques. Problems that have been encountered with this technique include, hand feeding the patient, perfection of deviation correction not obtained, composite falling off prior to visit and owner compliance problems with follow up visits.

DISCUSSION
This technique is not for every beak deviation case but it can provide many advantages to previously published techniques. The success we have noted with this dental composite technique gives clients hope that their animals will not end up with permanent scarring associated with pins, wires and dental bar foundations. If an owner is willing to hand feed a bird, possibly throughout the corrective period, make routine appointments and call if there is a change in position of the device then they are good candidates for having their bird treated. There should be minimal if any pain associated with this procedure and the bird is awake during the application of the composite appliance. By using the natural physics and anatomy of the beak the dental composite correction gives a rapid, secure adjustment to the malformed beak.

CITATION INDEX
1. 2. 3. 4. 5. 6. CLIPSHAM R. Surgical beak restoration and correction. Proc Ass Avian Vet, Seattle 1989: 164 - 176. CLIPSHAM R. Non-infectious disease of pediatric psittacines. Semin Avian Exotic Pet Med 1992; 1: 22 - 33. MARTIN H and RITCHIE BW. Orthopedic surgical techniques. In: RITCHIE BW, HARRISON GJ, HARRISON LR (eds): Avian Medicine: Principles and Application. Lake Worth: Wingers 1994; 1165 - 1169. SPEER BL. Non-infectious diseases. In: ABRAMSON J, SPEER BL, THOMSEN JB (eds): The Large Macaws. Fort Bragg, California: Raintree 1995; 323 - 324. SPEER BL. Trans-sinus pinning technique for the correction of chronic mandibular prognathism in psittacines. Proc Euro Assoc Avian Vet, Tenerife 2003: 3. SPEER BL. A technique for correction of chronic mandibular prognathism. Proc Assoc Avian Vet, Pittsburgh 2003: 45 - 49.

205

AUTHORS ADDRESS
Thomas N. Tully, Jr., DVM, MS, Diplomate ABVP (avian), Dip ECAMS Louisiana State University, School of Veterinary Medicine, Department Veterinary Clinical Sciences, Skip Bertman Dr., Baton Rouge, Louisiana 70803, United States of America Email: ttully@vetmed.lsu.edu

206

Avian and Exotic Animal Clinic of Indianapolis Indianapolis, United States of America Loro Parque-Clinica Veterinaria Puerto de la Cruz, Tenerife, Spain

THE EFFECTS OF ISOFLURANE EFFECTS OF ISOFLURANE ANAESTHESIA ON GASTROINTESTINAL TRANSIT TIME

A.M. Lennox, DVM, Dipl. ABVP-Avian, L. Crosta, DVM

KEYWORDS
Isourane - Gastrointestinal transit time - Anaesthesia

ABSTRACT
Oral contrast media is frequently used in psittacine radiology to evaluate the structure and motility of the gastrointestinal tract, and to distinguish the gastrointestinal tract from other celomic structures. Most textbooks and references describing the use of contrast advise against the use of anaesthetics during a contrast study. The purpose of this study is to determine if the use of brief isourane anaesthesia for the collection of radiographs during a contrast study slows the progression of a contrast media when compared to the use of manual restraint alone.

1 INTRODUCTION
Nearly all avian medicine and surgery textbooks advice against the use of any anaesthesia in the collection of radiographs during a contrast study, stating that anaesthesia may slow gastrointestinal transit time. Not one of four leading avian textbooks offers a specic reference to support this claim (SMITH BJ and SMITH SA 1997, HOWLETT 2000, KRAUTWALD-JUNGHANNS 1996, MCMILLIAN 1994). It is assumed authors of radiology sections of these books base the recommendations against anaesthesia on the current understanding of the effects of anaesthesia on the gastrointestinal transit time of dogs and cats. Several small animal radiology texts mentioning a transit time decrease with the use of anaesthetics in dogs refer to a single study in 1973 using 2 dogs. Pentobarbital sodium, halothane, methoxyurane, meperidine, fentanyl, droperidol, promazine hydrochloride, acetylpromazine meleate and triupromazine hydrochloride were studied using uoroscopy. Most drugs decreased gastrointestinal motility, with

207

halothane and methoxyuorane producing profound depression. Of interest is the comment that even minor psychic disturbances of the subjects caused at least some delay in gastrointestinal transit time (ZONTINE 1973). A study using cats in 1988 investigated the effects of a number of injectable anaesthetics on gastrointestinal transit time. Ketamine/diazepam combinations produced the least effect on transit time, while ketamine and ketamine/acepromazine, signicantly decreased transit time. Another study in cats demonstrated that IV diazepam also decreased gastrointestinal transit time (HOGAN and ARONSON 1988, STEYN et al. 1997). The use of manual restraint alone to collect radiographs involved some degree of difculty. Psittacines resent being secured to a radiograph cassette, even with the use of sophisticated avian restraint devices. Non-anesthetized birds cannot be intubated, and regurgitation of contrast material is a risk, especially in birds with gastrointestinal disease, especially crop or upper GI stasis. Non-anesthetized birds may struggle at the moment the radiograph is collected, producing poor-quality radiographs. Numerous factors are known to inuence gastrointestinal motility and GI transit time in mammals and birds. Studies in poultry indicate that crop motility is signicantly inhibited by excitement, fear, and struggling, while the presence of food (GI distention) and increased length of fasting increases motility. Contractions of the proventriculus, ventriculus and duodenum are then coordinated in sequence. The content of food items also affects motility. For example, foods of high fat and protein content cause a slowing of gastrointestinal time. Certain antibiotics have been documented to reduce gastrointestinal transit time in poultry. Diurnal rhythms affect motility, with a decrease normally seen at night (DUKE 1986). Isourane as a sole agent is the most commonly used anaesthetic in avian medicine. As inhalant anaesthetics are rarely used as a single anaesthetic agent in dogs and cats, it is unlikely a study on the effect of isourane alone on gastrointestinal transit exists. The authors are unaware of a similar study utilizing psittacines. Manually restraining a psittacine for radiographs is very likely to decrease gastric motility, as the procedure produces at least some degree of excitement, fear and struggling. It should also be considered that some excitement and struggling occurs during administration of isourane as well. Evaluation of the gastric transit time alone in psittacines is likely best accomplished in a non-anesthetized, non-restrained and acclimated bird utilizing uoroscopy, or in a similar situation with a perching patient using standard radiographs collected via a lateral beam. A rough estimation of gastrointestinal transit time might also be without radiography by measuring the time it takes for coloured markers, such as vegetable dye, to appear in the stool (SILVERMAN S, personal communication). The purpose of this study is to compare sets of radiographs using contrast media with isourane and with manual restraint alone. If the use of isourane does not signicantly slow gastrointestinal transit time when compared to manual restraint, then avian practitioners should no longer be advised to avoid anaesthesia for that reason alone.

208

2 MATERIALS AND METHODS

Fifteen birds of varying age, sex, species and physical health were used in the study (table 1). Barium sulphate was administered into the crop at a dosage of 25ml/kg, and radiographs were collected without anaesthesia at set intervals using either a commercial manual restraint device, or simply taped to the cassette. After several days, radiographs were again collected using the same amount of barium and at the same time intervals, with isourane administered via facemask for the duration of the brief radiographic procedure. Each individual birds radiographs with and without isourane were compared. No attempt was made to compare one bird to another bird. Radiographs were subjectively examined for a signicant difference in the level of barium in the lower gastrointestinal tract, and the amount remaining in the crop, proventriculus, and ventriculus at the different time intervals. Table 1. Summary of birds used to compare gastrointestinal time with manual restraint vs. isourane anaesthesia. Species Sex Age Comments App. healthy 1 Red fronted macaw Ara rubrogenys M 3y on PE 2 Red fronted macaw Ara rubrogenys F 2y Same 3 4 5 6 7 8 9 10 11 12 13 14 15 Blue and gold macaw Ara ararauna Blue and gold macaw Ara ararauna Green winged macaw Ara chloroptera Green winged macaw Ara chloroptera Little corella Cocatua sanguinea Little corella Cocatua sanguinea Sulphur crested cockatoo C. g. galerita Sulphur crested cockatoo C. g. galerita Cockatiel Nymphicus hollandicus Cockatiel Nymphicus hollandicus Cockatiel Nymphicus hollandicus Cockatiel Nymphicus hollandicus Cockatiel Nymphicus hollandicus M F M F M F M F F M M F F Unknown Unknown Unknown Unknown 9y 11y Unknown Unknown Unknown Unknown Unknown Unknown Unknown Same Same Same Same Same Same Same Same Same from mixed viary with PDD Same Same Same

3 RESULTS No signicant differences were detected in the progression of barium between radiographs collected with isourane and manual restraint alone. It is assumed that stress and excitement during manual restraint produces the same effect on gastrointestinal transit time, as it does isourane anaesthesia. Therefore, practitioners should no longer avoid anaesthesia during these procedures for that reason alone. Rather, decisions on whether or not to use anaesthesia should be based on other pertinent factors, such as patient condition and perceived risk.

209

4 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. 9. SMITH BJ and SMITH SA. Radiology. In: ALTMAN R, CLUB S, DORRENSTEIN G and QUESENBERRY K (eds): Avian Medicine and Surgery. Philadelphia: WB Saunders 1997; 170 - 199. HOWLETT JC. Clinical and diagnostic procedures. In: SAMOUR J, (ed): Avian Medicine. London: Mosby Co 2000; 28 - 79. KRAUTWALD-JUNGHANNS ME. Avian radiology. In: ROSSKOPF WJ and WOERPEL RW (eds): Diseases of cage and aviary birds. Baltimore: Williams and Wilkins 1996; 630 - 663. MCMILLIAN MC. Imaging techniques. In: RITCHIE BW, HARRISON GJ and HARRISON LR (eds): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing1994; 246 - 326. ZONTINE WJ. Effect of chemical restraint drugs on the passage of barium sulfate through the stomach and duodenum of dogs. J Am Vet Med Assoc 1973; 162: 878: 878 - 884. HOGAN PM and ARONSON E. Effect of sedation on transit time of feline gastrointestinal contrast studies. Vet Radiol Ultrasound 1988; 29(2): 85 - 88. STEYN PF, TWEDT D and TOOMBS W. The effect of intravenous diazepam on solid phase gastric emptying in normal cats. Vet Radiol 1997; 28(6): 469 - 473. DUKE GE. Alimentary canal: anatomy, regulation of feeding and motility. In: STURKIE PD (ed): Avian Physiology. New York, NY: Springer-Verlag 1986; 268 - 288. Personal communication: SILVERMAN S, DVM, PhD, Dipl. American Academy of Veterinary Radiology.

AUTHOR ADDRESS
M. Lennox Avian and Exotic Animal Clinic of Indianapolis, Indianapolis, United States of America Email: BirdDr@aol.com

210

Service de mdecine zoologique, Facult de mdecine vtrinaire, Universit de Montral, Qubec, Canada

MONITORING OF END-EXPIRED PARTIAL PRESSURES OF CARBON DIOXIDE IN ANAESTHETIZED BIRDS OF PREY

M. Desmarchelier, DVM, IPSAV, Y. Rondenay, DMV, IPSAV, Cert. Rs. (anaesthesiology), G. Fitzgerald, DMV, MSc and S. Lair, DMV, DES, DVSc, Diplomate ACZM

KEYWORDS
Anaesthesia - Avian - Birds of prey - Capnometry - Monitoring

ABSTRACT
We compared the value of end-expired partial pressure of carbon dioxide (PETCO2) measured with a handheld capnograph (NPB-75, Nellcor Puritan Bennett, Plesanton, CA, USA) and the arterial partial pressure of carbon dioxide (PaCO2) in isourane-anaesthetized birds of prey. Nine birds, ranging from 416 g to 2062 g, were used for this study. Each bird was anaesthetized once via face mask, intubated and manually ventilated. Respiratory rates were controlled to achieve ve levels of PETCO2 from 20 to 60 mm Hg. Concomitantly measured values of PaCO2 were determined for each level of PETCO2 obtained. Arterial blood pressure, heart rate and body temperature were monitored during the whole procedure. Strong correlations (p < 0.0001) were observed between PETCO2 and PaCO2, as well as between PETCO2 and arterial pH. These results suggest that the Nellcor NPB-75 capnograph could be a useful non-invasive monitoring tool for the evaluation of CO2 dynamic in birds over 400 g.

1 INTRODUCTION
Isourane is the most commonly used inhalant anaesthetic in birds. Despite numerous qualities, such as limited cardiovascular depression, low hepatotoxicity, and fast inductions and recoveries, this agent induces potentially signicant respiratory depression (ABOU-MADI 2001). Birds are especially sensitive to the effects of this respiratory depression since both inspiration and expiration are active processes. Thus, assisted ventilation is recommended to prevent hypercapnia in bird anaesthetized with isourane (FORBES 1999). Analysis of blood gas in arterial

211

samples is the gold standard to assess the balance between CO2 production and elimination. Unfortunately, arterial blood sampling is not practical in most bird species. In mammals, end-expired partial pressure of carbon dioxide (PETCO2) is widely used as an indirect non-invasive alternative to monitor the ventilatory status during general anaesthesia (OFLAHERTY 1994). Very few studies have explored the usefulness of PETCO2 as a parameter to evaluate arterial CO2 dynamic in birds (PETTIFER et al. 2002, EDLING et al. 2001). The signicant dissimilarities between the avian and mammalian respiratory physiologies prevent cross-species extrapolations. For instance, the pulmonary parenchyma of birds is not formed of alveoli as in mammals but of parabronchi, surrounded by air capillaries. Parabronchic air ow is unidirectional during both expiration and inspiration. Gas exchange occurs in a cross-current system that increases O2 uptake and CO2 release, in such a way that the partial pressure of carbon dioxide of the air exiting the pulmonary parenchyma is higher than the PaCO2 (SELLER 2000) Obviously, the relation between PaCO2 and PETCO2 will be affected by this physiological feature. Furthermore, since most species of birds seen by veterinarians have relatively small tidal volumes and high respiratory rates, measurement of PETCO2 in these species with traditional high ow (150 ml/min) side-stream capnometers designed to be used in humans or in domestic mammals will most likely result in erroneous readings. Microstream capnometers, such as the NBP-75 (Nellcor Puritan Bennett, Plesanton, CA, USA), have a much lower aspiration rate (50 ml/min). Therefore, PETCO2 measured with these monitors might provide a sufciently accurate estimation of PaCO2 (CASATI et al. 2000). The objective of our study was to evaluate the usefulness of a handheld Microstream capnograph (NPB-75, Nellcor Puritan Bennett, Plesanton, CA, USA) to assess the PaCO2 and PETCO2 relationship in different species of birds of prey anaesthetized with isourane using a Bain non-rebreathing system.

2 MATERIAL AND METHODS

Three red-tailed hawks (Buteo jamaicensis), two rough-legged hawks (Buteo lagopus), a Coopers hawk (Accipiter cooperii), two barred owls (Strix varia) and a great-horned owl (Bubo virginianus) were used for this study. These birds, which weighed from 416 g to 2062 g (mean SD, 1059 459 g), were admitted in the rehabilitation program of the Raptor clinic of the Facult de mdecine vtrinaire, Universit de Montral. Due to permanent injuries that prevented their release, these birds were euthanased at the end of the experiment. This project was carried out according to animal utilization protocols approved by the Facult de mdicine vtrinaire animal care committee which operate under the auspices of the Canadian Council on Animal Care. To reproduce anaesthetic protocols used for orthopaedic procedures commonly performed in raptors, 0.5mg/kg of butorphanol (Torbugesic, Wyeth Animal Health, Guelph, ON, Canada) was administered IM prior to mask induction with isourane (AErrane, Baxter, Toronto, ON, Canada) delivered in oxygen. Each bird was intubated with a Magill uncuffed endotracheal tube (diameter: 3 to 4.5mm) connected to the semi-open non-rebreathing Mapleson D modied Bain circuit. Heart rate was monitored with an ultrasonic Doppler (Ultrasonic Doppler Flow Detector, model 811-

212

B, Parks Medical Electronics, Inc., Aloha, OR, USA) applied on the palatine artery. The sampling tube of the capnograph (Handheld capnograph, NPB-75, Nellcor, Pleasanton, CA, USA) was connected to a paediatric T-piece adapter located between the endotracheal tube and the Bain circuit. An intraosseous catheter was inserted into the tibiotarsus using a proximal approach. Isourane was delivered in oxygen at a concentration ranging from 2.5% to 4% with a fresh gas ow of 0.8 L/min. Birds were manually ventilated with a peak inspiratory pressure varying between 15 and 20cm H2O. Ventilation rates were modied to reach different levels of PETCO2. Due to spontaneous breathing, ve of the nine birds had to be paralyzed with an intravenous or intraosseous injection of 0.3 mg/kg atracurium (atracurium besylate, Sabex, Boucherville, QC, Canada) (NICHOLSON et al. 1992). A 24-Gauge catheter was inserted in the supercial ulnar artery to facilitate repeated arterial blood collections. The arterial catheter was connected to a three way valve which allowed blood pressure monitoring between arterial blood collections. Arterial blood was sampled for ve levels of PETCO2 : 20, 30, 40, 50 and 60 mm Hg. PETCO2 levels were rst stabilized near each target level for ve minutes, then 0.5 ml of arterial blood was collected from the arterial catheter and injected back into the intraosseous catheter. Approximately 0.4 ml of arterial blood was then sampled from the arterial catheter in a heparinised 1 ml syringe. Blood gas analysis was performed within 5 minutes of sample collection with values normalized to body temperature according to human normograms associated with the blood gas analyzer (Stat Prole M, Nova Biomedical, Whaltam, MA, USA) (KILEY 1979). Birds were euthanased at the end of the procedure with an intravenous injection of T-61 (Euthanasia solution, Intervet, Whitby, ON, Canada). Statistical analysis were performed using a mixed multiple regression model with individual as a random factor to correlate PETCO2, PaCO2, arterial pH, weight, arterial blood pressure, and the setting of the isourane vaporiser.

3 RESULTS AND DISCUSSION

We collected 38 analyzable samples from the nine birds used for this study. Strong correlations were observed between PETCO2 and PaCO2 (r2 = 0,97 ; p < 0,0001) (Fig.1) and between PETCO2 and arterial pH (r2 = 0,95 ; p < 0,0001). However, individual differences between measured PETCO2 and PaCO2 were quite variable. Fig. 1: Scatterplot of PaCO2 against PETCO2 in mmHg (n=38). Continuous line represents the best-t linear trend line. Dashed line is the line of equality for comparison. PETCO2 values below and above the dashed line respectively overestimate and underestimate the concomitantly measured values of PaCO2.

213

80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 10 15 20 25 30 35 40 45 50 55 60 65 70 PETCO2 (mmHg)

All but one of the PETCO2 levels measured below 39 mm Hg exceed the concomitantly measured values of PaCO2 by 3 to 11 mm of Hg (mean SD, 5,7 3,0 mm of Hg). This result is similar to what was reported for African grey parrots (Psittacus erithacus timnus) anaesthetized with isourane using a modied Norman elbow non-rebreathing system and receiving intermittent mechanical positive pressure ventilation. This overestimation of PaCO2 by PETCO2 is in agreement with the effect of the cross-current exchange system in the pulmonary parenchyma of birds. Indeed, the parabronchi and aerial capillaries arrangement enables a cumulative transfer of CO2 from the venous blood to the parabronchic lumens resulting in a PETCO2 higher than the PaCO2 (FEDDE 1986). This is contrasting with results encountered with the mammalian alveolar system where the PETCO2 is usually lower then the PaCO2 by 2 to 5 mm Hg due to the presence of alveolar dead space or V/Q mismatch. Measured levels of PETCO2 ranging from 39 and 53 mm Hg were an average 1.5 mm Hg higher then their paired PaCO2 (mean SD, 1.5 5.2 mmHg). Within this range, the difference between PETCO2 and PaCO2 varied from -7 to 13 mm of Hg. All but one of the PETCO2 levels measured above 53 mm Hg underestimated the concomitantly measured PaCO2 level (mean SD, -8,8 6.5 mm Hg). These results suggest that the accuracy of the capnograph used in this study decreases with high values of CO2. However, the error reported for the NPB-75 Nellcor capnograph for values between 39 to 99 mm of Hg is 5%, which is much lower then the discrepancy observed. Consequently, analytic errors do not account, at least entirely, for the differences observed between PETCO2 and PaCO2 during hypercapnia. Another potential explanation for this nding might be the voluminous dead-space of the avian respiratory system. The low respiratory rates needed to obtain elevated PETCO2 (one

PaCO2 (mmHg)

214

breath every 1 or 2 minutes) were probably associated with a transient stagnation of gas exiting the pulmonary parenchyma. Therefore, it is possible that passive mixture occurred between CO2-rich post-parabronchic gas and gas present in the air sacs, resulting in a dilution of the CO2 concentrations and, as a result, an artefactually low PETCO2 at the following expiration. None of the other parameters tested (isourane concentration, arterial blood pressure, weight, respiratory rates) were correlated with the difference between PETCO2 and PaCO2. Interestingly, arterial partial pressure of O2 and O2 saturation of haemoglobin remained within normal limits throughout the procedures. This shows that these parameters are not sensitive indicators of the ventilatory status in anaesthetized birds receiving 100% inspired oxygen. This study shows that, despite some individual variations, the monitoring of PETCO2 with a side-stream Microstream capnometer provides a sufciently accurate estimation of PaCO2 in isourane-anaesthetized birds over 400g receiving manual positive ventilation with a Bain system. Based on the results obtained in the current study, we proposed that anaesthetized birds should be ventilated in order to maintain levels of PETCO2 between 30 and 45 mm Hg.

4 CITATION INDEX
ABOU-MADI N. Avian anesthesia. Vet Clin N Am: Exot Anim Pract 2001; 4: 147 - 167. 2. CASATI A et al. Accuracy of end-tidal carbon dioxide monitoring using the NBP-75 microstream capnometer. A study in intubated ventilated and spontaneously breathing nonintubated patients. European J Anesthesiology 2000; 17: 622 - 626. 3. EDLING TM et al. Capnographic monitoring of anesthetized African grey parrots receiving intermittent positive pressure. J Am Vet Med Assoc 2001; 219: 114 - 1718. 4. FEDDE MR. Respiration. In: STURKIE PD. Avian Physiology. New York: Springer-Verlag, 1986. 5. FORBES NA. Birds. In : SEYMOUR C and GLEED R. Manual of small animal anaesthesia and analgesia. Shurdinton: BSAVA, 1999: 283 - 293. 6. KILEY JP et al. Respiration and cardiovascular responses to exercise in the duck. J Appl Physiol 1979; 47: 827 - 833. 7. NICHOLSON A et al. Neuromuscular and cardiovascular effects of atracurium in isourane-anesthetized chickens. Am J Vet Res 1992; 53: 2337 - 2342. 8. OFLAHERTY D. Capnography Principles and practice series. BMJ Publishing Group, London, 1994. 9. PETTIFER GR et al. The comparative cardiopulmonary effects of spontaneous and controlled ventilation by using the Hallowell EMC anesthesia workstation in Hispaniolan Amazon parrots (Amazona ventralis). J Avian Med Surg 2002; 16: 268 - 276. 10. SELLER TJ. Bird respiration vol.1. Boca Raton: CRC Press 2000. 1.

215

AUTHOR ADDRESS
M. Desmarchelier, DMV, IPSAV Service de mdecine zoologique, Facult de mdecine vtrinaire de Saint-Hyacinthe, Universit de Montral C.P. 5000, St-Hyacinthe, QC J2S 7C6, Canada Email: marion.desmarchelier@wanadoo.fr

216

Clinic for Avian1, Reptile and Fish Medicine, University of Veterinary Medicine, Vienna, Austria; Department of Internal Medicine VI2, Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Germany

PHARMACOKINETICS AND PHARMACODYNAMICS OF THE NEW ANTIFUNGAL AGENT VORICONAZOLE IN BIRDS

A. Scope1, J. Burhenne2, W. E. Haefeli2, M. Hess1

KEYWORDS
Aspergillosis Voriconazole - Antifungal agent Pharmacokinetics - Birds

ABSTRACT
Voriconazole (Vfend, Pzer) is a novel, highly potent triazole antifungal agent with high in vitro activity against a wide variety of fungal pathogens. The present study was performed to gain information on pharmacokinetics and safety of voriconazole in birds, evaluated in chickens as a model. The mean Cmax after IV application of 10mg/ kg voriconazole was ~7.5g/ml, after PO application <1 g/ml. Enteral absorption of the drug was low with a calculated bioavailability of <20% after PO application, the half-life was about 2 hours in chicken. There were no remarkable differences of plasma concentrations of voriconazole between single applications and long-term treatment indicating that there was no accumulation of the drug. During treatment with 10mg/kg PO for 30 days no clinical signs of side effects or disease have been observed. No relevant changes of clinical chemistries were seen, that would indicate organ damage.

1. INTRODUCTION
Aspergillosis is a frequent cause of respiratory disease in birds. It is an important health problem in Psittacines, raptors and waterfowl maintained in captivity, as well as in some zoo-birds. The most common etiologic agent is Aspergillus fumigatus, followed by A. avus and A. niger. Different antimycotics have been used for treatment of birds (OROSZ and FRAZIER 1995, FISCHER and HATT 2003). Because of its broad spectrum and low incidence of toxicity, itraconazole is currently considered as the rst choice for treatment of aspergillosis in birds (OROSZ and FRAZIER 1995, LUMEIJ at al. 1995). There are some concerns that this drug may cause severe side effects like

217

CNS symptoms, vomiting, and even death in African grey parrots (VANDERMAST et al. 1990, OROSZ and FRAZIER 1995, QUESENBERRY et al. 1991). In this species it is recommended to switch to other drugs like ketoconazole or amphotericin B, but with the drawbacks of other possible side effects or lower efcacy. Voriconazole (Vfend, Pzer) is a novel, highly potent triazole antifungal agent that is a derivative of uconazole with high in-vitro activity against a wide variety of fungal pathogens, including Aspergillus, Candida, and Cryptococcus spp. (ABRAHAM et al. 1999, ESPINEL-INGROFF et al. 2001). In humans this drug has been efcacious in treating invasive aspergillosis and is well tolerated (DENNING et al. 2002, MUIJSERS et al. 2002). This new antifungal appears therefore a promising potential alternative for the treatment of aspergillosis in birds, especially in African grey parrots. One case of a cockatiel with suspected aspergillosis that has been treated with voriconazole has been reported (LANGHOFER 2004), but no standardized studies on pharmacokinetics have been published yet. The present study was performed to gain rst information on pharmacokinetics and safety of voriconazole in birds using the chicken as a model.

2. MATERIALS AND METHODS

2. 1. Analytical Methods An analytical method for the determination of voriconazole in plasma was developed utilizing high pressure liquid chromatography (HPLC) and solid phase extraction (SPE) technology. Plasma samples were spiked with internal standard and borate buffer was added. These samples were loaded onto SPE columns and eluted with organic solvent. The extracts were dried, reconstituted and analyzed by HPLC. Detection was performed either by UV detection or mass selective detection (LC/MS). Limits of quantication were 0.20g/ml (HPLC/UV) and 0.05g/ml (LC/MS). The linear calibration range was 0.05 (0.20)g/ml up to 10.0g/ml. Correlation coefcients were always r2>0.99. The analytical methods were validated according to FDA validation guidelines and fullled all quality assurance for accuracy, precision, robustness, and recovery. 2. 2. Performance of the study parts The study was conducted under animal testing permit No. GZ 68.205/192-BrGT/2003 and consisted of three parts. Part 1: For the evaluation of single dose pharmacokinetics and dose dependency single doses of voriconazole administered PO to four groups of chicken. Each group consisted of three chickens to receive 5.0, 7.5, 10, or 15 mg/kg voriconazole PO. Plasma concentrations were evaluated after 0, 0.5, 1, 3, 6, and 12 hours. All other study parts were performed with the 10 mg/kg dose which clinical evidence suggests is effective in aspergillosis treatment.

218

Part 2: For the assessment of the bioavailability of voriconazole in chicken two groups of two chickens were assembled. In a randomized cross-order design, each chicken got 10 mg/kg voriconazole PO or IV separated by a washout phase of 1 week. Plasma concentrations were determined in blood samples taken after 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, and 3.0 hours and were used to calculate bioavailability, maximum plasma concentrations (Cmax), time to reach Cmax (tmax), and the half-life (t1/2) of voriconazole. Part 3: The pharmacokinetics and safety of long-term application of voriconazole was studied in a group of 9 birds receiving 10 mg/kg voriconazole PO for up to 30 days. Plasma concentrations were determined after 10, 20, and 30 days 1 hour and 3 hours after dosing to monitor whether drug accumulation occurs. From the same samples, plasma biochemistries (AST, CA, ChE, CK, GLDH, GLU, LDH, TP, URIC) were also determined for safety assessment.

3. RESULTS
Part 1 Table 1 shows the single dose pharmacokinetic of part 1. Due to the very fast elimination (t1/2~2 h) in chicken only low Cmax values were reached and the AUC values showed no signicant increase with the dose. Table 1: Mean values of pharmacokinetic parameters in chickens. Dose [mg/kg] 5.0 - 15 Part 2 Pharmacokinetics after IV applications are shown in Table 2, oral bioavailability was less than 20%. Table 2: Intravenous pharmacokinetics of 10 mg/kg voriconazole in chickens. Parameter IV Part 3: Plasma values at treatment days 10, 20, and 30 at 1 hour and 3 hours after application were always lower than 3g/ml and showed no meaningful increase with the application duration. No clinically relevant changes of clinical chemistries occurred during the observation period. AUC0-3h [(g/ml)*h] ~11 VD [ml/kg] <1700 Clt [ml/(h*kg)] <600 t1/2 [h] 1.7 0.7 tmax [h] 1.3 0.7 Cmax [g/mL] 0.4 0.1 AUC [(g/mL)*h] 1.25 0.2

219

4. DISCUSSION
The minimum inhibitory concentration (MIC) of voriconazole has been evaluated in vitro (ABRAHAM et al. 1999; ESPINEL-INGROFF et al. 2001) and depends on the infectious agent. The manufacturer recommends 0.05-2g/ml voriconazole for Aspergillus spp. for humans (Vfend information sheet, Pzer, NY, USA). Only for a short period of time this limit has been exceeded after IV application of 10 mg/kg voriconazole and after PO application the mean Cmax was even <0.5g/ml suggesting that antifungal concentrations throughout the dosing interval may be insufcient for some fungi. This is mainly caused by the short half-life of the compound in chickens and the poor oral bioavailability. This is in obvious contrast to the very good absorption of voriconazole in humans (bioavailability up to 96%), as is the fact that during longterm administration no accumulation occurred. The poor bioavailability together with the rather small VD (limited tissue distribution) might indicate that effective therapies would require multiple dosing to reach sufcient plasma concentrations. Because only relatively low plasma concentrations were achieved a nal assessment of the safety of the compound is not possible. The fact that even during treatment with 10mg/kg PO for 30 days no evidence for adverse effects or organ damage emerged may at least suggest that it does not have substantial toxicity. Preliminary results of voriconazole in Psittacine species indicate that there might be big species-specic differences in pharmacokinetics.

5. CITATION INDEX
ABRAHAM OC, MANAVATHU EK, CUTRIGHT JL and CHANDRASEKAR PH. In vitro susceptibilities of Aspergillus species to voriconazole, itraconazole and amphotericin B. Mycology 1999; 33: 7 - 11. 2. DENNING DW, RIBAUD P, MILPIED N, et al. Efcacy and safety of voriconazole in the treatment of active aspergillosis. Clin Infect Dis 2002; 34: 563 - 571. 3. ESPINEL-INGROF A, BOYLE K and SHEEHAN DJ. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: Review of the literature. Mycopathologia 2001; 150: 101-15. 4. FISCHER I and HATT J-M. Antifungal therapy in birds: A review and critical discussion of new trends. 5th ECAMS Scientic Meeting, Tenerife 2003: 21 - 22. 5. LANGHOFER B. Emerging antifungals and the use of voriconazole with amphotericin to treat Aspergillus. Proc Assoc Avian Vet, New Orleans 2004: 21 - 24. 6. LUMEIJ JT, GORGEVSKA D and WOESTENBORGHS R. Plasma and tissue concentrations of itraconazole in racing pigeons (Columba livia domestica). J Avian Med Surg 1995; 9: 32-5. 7. MUIJSERS RB, GOA KL and SCOTT LJ. Voriconazole: in the treatment of invasive aspergillosis. Drugs 2002; 62: 2655 - 2664. 8. OROSZ SE and FRAZIER DL. Antifungal agents: A review of their pharmacology and therapeutic indications. J Avian Med Surg 1995; 9: 8 - 18. 9. QUESENBERRY K, BAUCK L, SPEER B and FLAMMER K. Roundtable discussion: Clinical therapy. J Assoc Avian Vet 1991; 5: 186 - 191. 10. VANDERMAST H, DORRESTEIN GM and WESTERHOF J. A fatal treatment of sinusitis in an African grey parrot. J Assoc Avian Vet 1990; 4: 189. 1.

220

AUTHORS ADRESS
Alexandra Scope, DVM Clinic for Avian, Reptile and Fish Medicine, University of Veterinary Medicine, Veterinrplatz 1, 1210 Vienna, Austria Email: alexandra.scope@vu-wien.ac.at

221

Clinique vtrinaire Sigean, France

CLINICAL ASSESMENT ON THE USE OF FLUCONAZOLE PER OS IN 24 AFRICAN GREY PARROTS (PSITTACUS ERITHACUS) : ACCEPTANCE, SIDE EFFECTS AND EFFICIENCY.
J.M. Pericard DVM

KEYWORDS
Birds - African grey parrot - Fluconazole - Psittacus erithacus - Electrocineresis Aspergillosis

ABSTRACT
Itraconazole is the more commonly used antifungal agent in birds, but in African grey parrots this drug is reported to be toxic. An alternative treatment is uconazole. A retrospective review of 24 African grey parrots treated with uconazole PO allowed us to assess the efciency and tolerance of the protocol used. The 24 birds examined consisted of 19 Psittacus erithacus and 5 Psittacus erithacus timneh. Ages varied from 3 months to 42 years. The birds presented with a range of clinical symptoms including upper and lower respiratory disease, dermatitis, gastro-enteritis and loss of condition. Aspergillosis serology was performed by electrocineresis on 23/24 cases. A positive result was obtained in 19/23 cases, negative in 4/23. Thirteen parrots were treated with uconazole PO as the sole antifungal agent and 10 with a combination of uconazole PO and amphotericin by nebulisation. The uconazole (Triucan TM, suspension at 10mg/ml) was administered at 15mg/kg q12h. The duration of treatment varied from 5 days to 75 days, but more often from 21 to 30 days. The effects were reported by the owners, further observations were made by the author during consultations. Treatment was well accepted in 23/24 cases and refused in only 1/24 cases. No negative clinical effects were recorded in 20/23 birds. Of the remaining, one case (during prolonged treatment) reported decreased appetite, vomiting and yellow or dark faeces. Two birds with severe aspergillosis died 22 and 30 days respectively after the end of treatment. One of them reported tiredness during treatment. These deaths reect probably more a lack of treatment and the severity of the disease than a toxicity of the drug. No difference in negative clinical effects was seen between uconazole alone or associated with amphotericin. Positive clinical results were noticed in all birds: 18/23 recovered, the 5/23 remaining cases improved, but did not fully recover because of chronic lesions on 2/23, old age on 1/23, severe disease and death later on 2/23.

222

1 INTRODUCTION Fungal diseases commonly occur in birds. Aspergillosis of the respiratory tract is common but systemic infections such as fungal dermatitis and fungal disease of the gastro-intestinal tract are also commonly seen. Among psittacine birds, a higher incidence of aspergillosis is seen in the African grey parrot (Psittacus erithacus). Many factors are known to favour aspergillosis, but in that species immunosuppression due to a poor nutrition and husbandry may play the most important role. During the last few years, the treatments of fungal diseases have been improved with the advent of new drugs and better knowledge of the bioavailability in certain avian species. Among these drugs, itraconazole seems to be the preferred choice against Aspergillus. Unfortunately, African greys do not tolerate this drug very well and may show signs of toxicity at standard therapeutic doses (OROSZ et al 1996, POWERS personal communication). In addition, some drugs or galenic forms are not readily available to veterinarians in France. This is the case of injectable antifungal drugs, including the injectable forms of itraconazole and amphotericin. Due to this, the assessment on the use of uconazole PO in the African grey parrot is important.

2 MATERIAL AND METHODS 2. 1 Cases review The review was carried out by selecting cases of our database, based on 3 criteria: a) species: African grey parrot (either nominal, or timneh), b) treatment: including uconazole, c) date: from 1st January 2003. The history of the selected cases was then studied for the chosen data: age, weight, subspecies, symptoms, duration of treatment with uconazole, doses per day, number of administration per day, combination of amphotericin by nebulisation, negative clinical effects, positive clinical effects, serology of aspergillosis by electrocineresis, and duration between beginning of treatment and last contact. The effects were observed by the owners and during consultations. The 24 birds examined consisted of 19 Psittacus erithacus and 5 Psittacus erithacus timneh. Ages varied from 3 months to 42 years. The birds presented with a range of clinical symptoms including upper and lower respiratory disease, dermatitis, gastro-enteritis and loss of condition. Every owner, if not seen at the clinic recently, was contacted by phone to complete the history if needed. 2. 2 Treatments Thirteen parrots were treated with uconazole PO as the only antifungal agent, 10 with a combination of uconazole PO and amphotericin by nebulisation (suspension at 0.5mg/ml). The dose used was 15mg/kg q12h (Triucan TM, suspension at 10mg/ ml). The duration varied from 5 to 75 days, but more often 21 to 30 days.

223

2. 3 Serology of aspergillosis Aspergillosis serology was performed by electrocineresis (Laboratory Ceri, Paris) on 23/24 cases. A positive result was obtained in 19/23 cases, negative in 4/23. The bird not tested was a chick with crop stula under short preventive treatment. This serology is used as a diagnostic tool, and to monitor the recovery by repeated samples.

3 RESULTS
The cases are listed in Table 1. 3. 1 Acceptance Among these 24 African Grey, only one refused to take the drug. This bird was not handled by their owners and did not try again! For the rest, the owners reported that the administration was easy. One owner preferred to give it mixed with jam instead of liquid. 3.2 Clinical effects No clinical negative effects were noticed in 20 on 23 birds treated with uconazole. Two birds died later, but these were birds with severe disease. The rst case (No 3788) had a recurrence infection with severe chronic air sacculitis. Nutrition was very poor. It received uconazole during 36 days, then nothing during 10 days and then again 10 days of uconazole. Other treatments include nebulisation with amphotericin at 0.5mg/ml, injection of vitamin A, pelleted food and fresh fruits, and later doxycycline. The owner noticed tiredness, but also general improvement. The bird died 22 days after the end of uconazole. The owner did not wish a necropsy. The second case (No 4312) was also affected by aspergillosis with weight loss, diarrhoea, polyuria and loss of balance. It received uconazole during more than 21 days, probiotics, a pelleted food and improved. The bird died suddenly 2 months after the beginning of uconazole (and about 1 month after the end of uconazole). The owner did not call us and no necropsy was performed. The only case (No 2450) in which side-effects were noticed was a very old bird (40 years) with weight loss and less active. The bird has a feather picking syndrome for years, a poor nutrition, arthrosis, modied vocalisations for 3 years. At serology the bird was positive for aspergillosis and chlamydophilosis. The blood chemistry was within reference values (uric acid 39mg/l, glucose 2.35g/l, calcium 101mg/l, total protein 38g/l, ASAT 129 UI, CPK 648 UI). The treatment with uconazole as the sole antifungal agent was prolonged because the owner noticed a signicant improvement, and because it was considered that a single 21 days treatment was not sufcient. The bird received uconazole during a total of 75 days on 115 days. Periods of 21 days were separated by 7 to 13 days without treatment. Other treatments included doxycycline PO in water, probiotics, juvenile formula and pelleted

224

food. During the second period of 21 days of uconazole, the owner noticed yellow stools and decrease appetite. No doxycycline was given at that time. The treatment with uconazole was discontinued when the bird started vomiting and passing dark stools, 2 days after the beginning of a new period. The blood sample for chemistry taken 2 days after showed decreased total proteins (uric acid 57mg/l, calcium 91mg/l, total protein 25g/l, ASAT 135 UI, LDH 257 UI, bile acids 56.1mol/l). The symptoms disappeared when the treatment with uconazole stop and were not present at the time of the consultation 2 days after (no vomiting, normal stools). The clinical signs did not reappear after a short treatment with clomipramide and lespedeza extracts. There were no statistical differences between the side-effects during a treatment with uconazole alone or associated with amphotericin (P=0.2). 3. 3 Positive clinical effects 3. 3. 1 Clinical recovery Recovery was seen by disappearance of the symptoms in 18 of the 23 birds treated with uconazole. Eleven were treated with uconazole alone, 7 with a combination of uconazole and amphotericin in more severe cases. The negative results of the electrocineresis for aspergillosis and the duration between the treatment and the last contact were also good indicators of recovery (see table 1). 3. 3. 2 Clinical improvement The other 5 birds of the 23 treated improved. Even for the 2 who died later, improvement was noticed: able to speak again, more active, better appetite (No 3788), weight gain, reduction of polyuria (No 4312). The one who had side-effects also improved considerably, limited by its old age: behaviour more active, better vocalisation, able to y and later able to y without dyspnoea, mconnaissable (No 2450). The 2 others were considered only improved because the opacity of air sacs remains; one of them is still under treatment.

4 DISCUSSION
4. 1. Doses and frequency of uconazole treatment The doses and frequency we chose for most of these treatments were 15mg/kg q12h (RUPIGER unpublished, in OLSEN and OROSZ, 2000), a lot higher than 20mg/kg q48h or 10mg/kg q24h as proposed by FLAMMER (1996). Our results show a good clinical efcacy and few side effects at this dose. However, this cannot stand in for the lack of studies on the bioavailability of uconazole in the African grey. 4. 2. Duration of treatment The duration of treatment is chosen according to the disease and results. Many authors think that for aspergillosis the duration must be at least several weeks and

225

sometimes several months (review in ABUNDIS-SANTAMARIA 2002, ANDRE 2004). We share that opinion and often made treatments of about 1 month. For longer treatments, we made interruption of about 10 days between duration of 3 to 5 weeks. The idea is to minimise the possible toxicity by accumulation (see under). 4. 3. Side-effects and toxicity Fluconazole has negligible hepatic metabolism, while itraconazole has extensive hepatic metabolism. Fluconazole preferentially accumulates in the kidneys and is excreted in the urine. It is not linked to proteins contrary to itraconazole. It is water soluble and well absorbed PO, and has a neutral pH, well tolerated by mucosa (review in OROSZ 2003). Then the potential toxicity is very different from itraconazole. But it is not easy to separate the effects of the treatments and of the disease itself! Some African greys are reported partially to totally anorexic during uconazole treatment (OROSZ 2003). In the present cases, only one of 23 had a transient lower appetite, and another one was noticed to have a better appetite! The effects observed on case No 2450 (lower appetite and yellow stools, then vomiting, dark stools, and hypoproteinemia) might be linked to renal insufciency, but we could not conrm this. Symptoms disappeared immediately when the treatment stop and before consultation. Urine analysis was impossible in that bird since it had a large amount of mixed stools due to a dilated cloaca. Because amphotericin also has a renal metabolism (OROSZ 2003), cumulative toxicity could be possible when uconazole is used at the same time. In this case review, no statistical difference was seen on side effects and deaths. 4. 4. Efciency The global results of the treatments (18/23 recovered, 21/23 survived) is very good. It seems as good as treatments with itraconazole Per Os in experimental aspergillosis: 41/60 quails survived in the itraconazole treatment group, 0/60 in the untreated group (GUMUSSOY et al. 2004). The apparent better results of the uconazole alone compared to uconazole and amphotericin, is probably due to a selective bias: more severe cases were treated with the association of antifungal agents, less severe ones with uconazole alone.

5 ACKNOWLEDGEMENTS
I thank Dr Florence Buronfosse, Centre de Pharmacovigilance Veterinaire de Lyon, Ecole Veterinaire de Lyon, for her help and advice with this study and Dr Glen Cousquer for kindly reviewing the manuscript.

226

6 CITATION INDEX
1. ABUNDIS-SANTAMARIA E. Aspergillosis in birds of prey. AGUILAR R. (ed): Mexico: Cervantes Olivares, 2002: 31. www.aspergillus.man.uk/secure/veterinary/ Aspergillosis.pdf. 2. ANDRE J.P. Oiseaux de cages et de volires. De la maladie la bonne sant. Published by the author, La Teste 33 France, 2004, 490. 3. FLAMMER K. Fluconazole in psittacines birds, Proc Assoc Avian Vet, Tampa 1996: 203 - 204 4. GUMUSSOY KS, UYANIK F, ATASEVER A, CAM Y. Experimental Aspergillus fumigatus infection in quails and results of treatment with itraconazole. J Vet Med B Infect Dis Vet Public Health. 2004; 51(1): 34 - 38. 5. OROSZ S.E. Antifungal drug therapy in avian species. Vet Clin Exot Anim 2003, 6: 337 - 350. 6. OROSZ S.E., FRAZIER D.L., SCHROEDER E.C., et al. Pharmacokinetic properties of itraconazole in blue-fronted Amazon parrots (Amazona aestiva aestiva) J Avian Med Surg 1996; 10: 168 - 173. 7. RUPIGER D.J. Unpublished data in OLSEN G.H. and OROSZ S.E.: Manual of Avian Medicine. St Louis: Mosby, 2000.

AUTHORS ADRESS
J.M. Pricard, DVM, CES de pathologie aviaire, diplme dpidmiologie Clinique vtrinaire, 24, rue du Cers, 1113 Sigean, France Email: jm.pericard.vetoiseaux@wanadoo.fr

227

Table 1. Results of the clinical assessment on the use of uconazole PO in 24 African Grey parrots (Psittacus erithacus)

No Ampho

Age (years) 23 30 31 30 15 31.2 2 No None Recovery 35.2 2 Yes None Recovery 29.7 2 Yes None Recovery 23.7 2 Yes None Recovery 27.3 2 No None Gain of weight Recovery 1 arc then 0 1 arc then 0 1 arc then 0 2 arcs then 0 1 arc

Weight (g)

Symptoms

Duration uconazole days Dosis mg/kg per day

Nb of admin. per day

Negative clinical effects

Positive clinical effects

Serology electrocineresis aspergillosis

Last contact after treatment (month) 8 24 4 15 19

4972

4?

512

Regurgitation

4233

422

4246

5.5

538

4451

398

4484

12

256

Dyspnea, loss of voice, nasal granuloma, gout Air sac opacity Loss of voice, prostration, air sacculitis Rhino-sinusitis, low appetite 36, stop 10 10 30 2 Yes 33 21? 21 29.3 2 No 30 2 No 29.7 2 Yes None None None Tiredness Died 22 days after end of uconazole Improvement: voice, can speak, but chronic lesions. Recovery Recovery Recovery

228
>60 31.2 2 Yes None >30 21 25 30 2 2 Yes No None None

3788

18,5?

356

Recurence of aspergillosis with severe chronic air sacculitis, voice loss, prostration, thinness

0 arc then 2 then 1

4429

18

471

Nasal granuloma, sinusitis

1 arc then 0 1 arc then 1 then 0 1 arc

19 16 14

4639

0.7

410

Thinness

4754

5?

477

Vomiting, loss of voice, prostration

4361

? Adult

448

Severe dyspnea,air sacculitis, rhinosinusitis, +gout, bullet & ancient fracture, chlamydophilosis

Improvement but chronic lesions

1 arc then 0

19

1 arc 1 arc then 0

22 16

4636

0.4

480

Thinness, control after purchase

Improvement but chronic lesions Gain of weight Recovery

4312 21,stop 13 21, stop 7 30 2 No yellow stools, decrease appetite 30 2 No 2 arcs then 3

? Adult >21 32.2 2 No 2 arcs 2

372

Loss of weight, diarrhea, polyuria, trouble of equilibrium

2450

40

427

Loss of weight, less active

None but sudden death 2 months later None

Improvement: gain of weight, reduction of polyuria Improvement

21,stop 10

30

No

Improvement: behaviour, ight Improvement: ight without dyspnea, muscle development, voice... then 0

10 15 8 8 15 5 21 21 30 not given 30 35 37, stop 7, 15 30 30 30 2 2 2 30.3 2 10.1 1 No No Yes Yes No No None None None 11.9 1 Yes 23.8 1 Yes 26 2 No None None None None None Recovery Improvement then Recovery Recovery Recovery Improvement Recovery 27 2 No None 29 2 No None 36 2 No None Recovery Recovery Recovery Recovery 30 2 Yes None Recovery

10, stop 10 2, stop 30 2 No

Vomiting, dark stools

4 0 arc 0 arc no test 0 arc 0 arc 1 arc 25 1 arc then 1 1 arc then 0 1 arc then 0 1 arc then 0 2 arcs then 1 0 then 1 then 1 then 0 25 9 5 1.5 4 13 9 0.4 24 19

4787

375

4964

275g Timnhe

229

4694

0.25

488g

Dyspnea during effort, loss of voice Prostation, anorexia, automutilation, nervous troubles Crop stula

2565

42

368g

Diarrhea, prostration, anorexia, polydypsia,

4313

3.5

305g Timnhe

Dermatitis, loss of voice

4198

12,5?

421

Rhinitis, keratitis, conjonctivitis, thinness

4165

? Adult

496

Almost dead (abandonment), rhinitis, taeniasis

560

5000

1.75

423

5293

1.6

232g Timnhe

5457

14

259g Timnhe

710

35

390

Prostration, sinusitis, low appetite Vomiting, prostration, dark green stools, asthma Dyspnea, anorexia, air sacculitis Loss of weight, less active

College of Veterinary Medicine, University of Georgia, United States of America

PHARMACOKINETICS AND USE OF MELOXICAM IN PSITTACINE BIRDS

G. H. Wilson, DVM, Dipl ABVP (avian), S. Hernandez-Divers, BVetMed,DZoo Med, MRCVS, DACZM, S. C. Budsberg, DVM, DACVS, K. S. Latimer, DVM, PhD, DACVP, K. Grant, LVT, and M. Pethel

KEYWORDS
Meloxicam - Pharmacokinetics - Ring necked parakeets - Psittacula krameri

ABSTRACT
This study evaluated the pharmacokinetics of meloxicam given to ring-necked parakeets (Psittacula krameri) orally and intravenously at a dose of 0.5mg/kg. The elimination half-life was 16 hours after oral administration and 4 hours after intravenous administration. Peak serum concentrations were 1.38ug/ml and 3.86ug/ ml after oral and intravenous administration respectively. The results suggest that meloxicam should be administered at 0.5mg/kg twice a day if given orally or six times a day if given intravenously.

1 INTRODUCTION
Reduction of pain and inammation is an important part of medical and surgical management of avian patients. Meloxicam (Metacam, Merial, Duluth, GA USA), which has been used in veterinary medicine for many years in Europe, is a NSAID that has recently become available in the U.S. At this time, it has only been licensed for use in dogs, for which the reported dosage is 0.1 to 0.2mg/kg PO q 24h. Meloxicam has been shown to have nearly 100% bioavailability when administered orally with food in dogs. Frequently, drug dosages for birds are extrapolated from mammalian dosages, but this method has been fraught with potential complications in the past. While NSAIDs have been used and studied in many mammalian species, there is little data on their use in birds. (BUDSBERG et al. 2002, POULSEN et al. 1999, BUSCH et al. 1998, TURCK et al. 1996, LEES et al. 1991) Furthermore, the studies that have been performed in birds are predominately in species not commonly kept as companion animals, such as poultry, quail, ostriches, and ducks. (BAERT et al. 2002,

230

BAERT et al. 2002, BAERT et al. 2003, GRAHAM et al. 2001) Also, the previous studies in chickens, ducks, ostriches, turkeys, and pigeons, have not looked at oral bioavailability. When meloxicam was given to these birds in the injectable form, there were signicant differences in elimination rates of the drug between different species. The pharmacokinetics of meloxicam have not been established in any parrot species, although there are anecdotal reports of its usage in pet birds in Europe at a dose of 0.3 to 1.0mg/kg orally q 12h. Therefore, the purpose of this study was to establish the pharmacokinetics of meloxicam given orally and intravenously and to evaluate bioavailability in one species of psittacine birds. 2 MATERIAL AND METHODS Twenty adult ring-necked parakeets (Psittacula krameri) of both sexes, weighing between 110 and 140 grams each, were used. A dose of 0.5 mg/kg was given intravenously (IV) to each of the 20 birds. Blood samples were taken prior to administration of the drug (time 0) and from each bird again at two of several randomized time samples over a 24 hour period. After a two week washout period the subjects then received the same dose (0.5 mg/kg) administered orally (PO). Blood samples were again taken from each bird at several times over a 24 hour period. No more than 10% of the estimated total blood volume for any individual was removed within a 24 hour period. The collections times for each bird were randomized to minimize the effect of iatrogenic anaemia on drug dosage concentrations and health of the animal. Plasma concentrations of the drug were determined by highperformance liquid chromatography methods.(VELPANDIAN et al. 2000) The UGA Institutional Animal Care and Use Committee approved the protocol. 3 RESULTS There were no apparent clinical side effects noted in the birds in this study at a dose of 0.5mg/kg given orally or IV. Results showed that the half-life of meloxicam in ring-necked parakeets was of longer duration than in any of the previously studied non-psittacine avian species for both the oral and IV routes. Bioavailability was 100% for intravenous administration and was also high for oral administration. Based on these results a dose of 0.5mg/kg twice a day orally or every 4 hours intravenously is recommended in psittacine birds. 4 DISCUSSION Interestingly, based on the results of this study and previous studies in other species of bird, it appears that the half-life is inversely proportional to the weight; so the larger the bird, the shorter the half-life. (BAERT and DEBACKER 2003) In humans and rats and presumably in birds, meloxicam is eliminated partly in urine and partly in faeces. Pharmacodynamic studies are needed to further understand the disposition of meloxicam in psittacine birds.

231

5 CITATION INDEX
1. BAERT K and DEBACKER P. Disposition of sodium salicylate, unixin and meloxicam after intravenous administration in broiler chickens. J Vet Pharmacol Therap 2002, 25; 449 - 453. 2. BAERT K, NACKAERTS J and DE BACKER P. Disposition of sodium salicylate, unixin, and meloxicam after intravenous administration in ostriches (Struthio camelus). J Avian Med Surg 2002; 16(2): 123 129. 3. BAERT K and DE BACKER P. Comparative pharmacokinetics of three non-steroidal anti-inammatory drugs in ve bird species. Comp Biochem and Physi 2003; 134(1): 25 - 33. 4. BUDSBERG SC, CROSS AR, QUANDT JE, et al. Evaluation of intravenous administration of meloxicam for perioperative pain management following stie joint surgery in dogs. Am J Vet Res 2002, 63(11): 1557 - 63. 5. BUSCH U, SCHMID J, HEINZEL G, et al. Pharmacokinetics of meloxicam in animals and the relevance to humans. Drug Metab Dispos 1998; 26(6): 576 - 584. 6. GRAHAM JE, TELL LA, KOLLIAS-BAKER C and CRAIGMILL AL. Pharmacokinetics of ketoprofen in adult Japanese quail. Proc Assoc Avian Vet, Orlando 2001: 19 - 21. 7. LEES P, SEDGWICK AD, HIGGINS AJ, et al. Pharmacokinetics and pharmacodynamics of meloxicam in the horse. Br Vet J 1991; 147(2): 97 - 108. 8. POULSEN NB and HORSTERMANN D. Pharmacodynamic and pharmacokinetic aspects of the non-inammatory non-steroidal agent meloxicam in dogs. Dtsch Tierarztl Wochenshcr 1999; 106(3): 94 100. 9. TURCK D, ROTH W and BUSCH U. A review of the clinical pharmacokinetics of meloxicam. Br J Rheumatol 1996; 35(1): 13 - 6. 10. VELPANDIAN T, JAISWAL J, BHARDWAJ RK and GUPTA SK. Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples. J Chromatogr B Biomed Sci Appl 2000; 738(2): 431 - 436.

AUTHORS ADRESS
G. Heather Wilson, DVM, Dipl. ABVP-avian Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 31522, United States of America Email: hwilson@vet.uga.edu

232

Clinic for Birds and Reptiles University of Leipzig, Germany

THE USE OF ENALAPRIL IN BIRDS : INDICATIONS, CLINICAL EXPERIENCES, PHARMACOKINETICS AND POSSIBLE SIDE EFFECTS
M. Pees, DrMedVet, K. Kuhring, M.E. Krautwald-Junghanns, ProfDrMedVet, DiplECAMS

KEYWORDS
Birds Cardiac Disease ACE-inhibitor - Enalapril

ABSTRACT
Cardiac disease in birds occurs more frequently than suspected previously. With the ongoing technical progress and increasing experience, it is possible to assess cardiac function and to diagnose alterations intra vitam, today. First experiences indicate the high potential value, but also possible side effects of the ACE-inhibitor enalapril in birds.

1 INTRODUCTION
Recent studies show the incidence of cardiac disease as a common nding in post mortem examinations (KRAUTWALD-JUNGHANNS et al. 2004). In contrast, intra vitam diagnosis is comparably rare due to the non-specic clinical signs and accompanying diseases overlying the cardiovascular symptoms. Nevertheless, several studies about the examination of cardiac function have been done over the last years (KRAUTWALD-JUNGHANNS et al. 1995, BOSKOVIC et al. 1999, CARRANI et al. 2003, PEES et al. 2004). Radiology, electrocardiography (ECG) and especially echocardiography have been found to be of diagnostic value. With the ability to diagnose cardiac alterations ante mortem, the possibility of an efcient therapy becomes more important. Unfortunately, concerning this point, the state of scientic knowledge is far below that of mammal medicine. Only few studies and some case reports about cardiac therapy in birds exist. The only group of cardiac drugs for which pharmacokinetic data is available for birds are heart glycosides (HAMLIN and STALNAKER 1987, WILSON et al. 1989). Heart glycosides (digoxin, digitoxin) have a positive inotropic effect on the contraction of cardiac muscle, improve

233

its relaxation and decrease the heart rate. Unfortunately, their therapeutic margin is very small, and long-time application seems to be critical in birds due to difculties in controlling effect and plasma levels. Sudden death has been reported after some days of application (PEES et al. 2001). In our experience, their main indication is stabilizing birds in emergency situations. ACE-inhibitors (captopril, enalapril) are frequently used for therapy of cardiac problems in human and mammal medicine. Their mechanism is based on the inhibition of the conversion from angiotensin I to angiotension II. This enzyme is responsible for the contraction of the vessels and the retention of sodium and water in the kidneys. Therefore ACE-inhibitors lead to an improvement of renal function, an increase of diuresis and a decrease of blood pressure resulting in a decreased pre- and afterload of the heart. Since the half-life period and effectivity of enalapril is higher compared to captopril (SPONER 2002), this drug is preferable for use in birds. In mammals, the therapeutic margin of enalapril is not as critical as the margin of cardiac glycosides (SPONER 2002). The aim of this study was to summarize clinical experiences with this drug in birds, and to give rst guidelines for the application and the control of the therapeutic success.

2 MATERIAL AND METHODS

2.1. Clinical experiences In our clinic, enalapril is used for the therapy of cardiovascular disease since 4 years. During this period, the effects of the administration on the clinical picture, the survival time and side-effects that are potentially related to the drug administration have been noted. In birds that have died, necropsy was performed to evaluate the cause of cardiovascular disease and accompanying diseases. 2.2. Pharmakokinetics and dosis-dependent side effects To examine the pharmacokinetics and the dosis-dependent effects of enalapril, 39 adult pigeons were included in the study. Before administration of enalapril, they underwent a thorough clinical, parasitological and microbiological examination. Only healthy birds without signs of any disease were used for the study. The birds were housed in single caged and fed with a commercial seed diet and water ad libitum. For the application, enalapril tablets (Enacard Tablets, Intervet, Germany) were dissolved in sterile water and applied with a crop cannula following body mass. For the examination of the pharmacokinetics, enalapril was administered orally to 15 pigeons. Dosage was 5 mg/kg body mass. Blood was taken on dened time intervals (0h, 0.5h, 1.0h, 2.0h, 4.0h, 6.0h, 8.0h, 12.0h, 16.0h, and 24.0h) to check for drug concentration. For the examination of dosis-dependent side-effects, 24 pigeons were divided into groups with each 8 animals. Besides clinical examination, radiography was performed and the heart was examined by means of electrocardiography and echocardiography. Blood was taken from the birds and examined (WBC, blood chemistry) after an acclimatisation period of 7 days. Enalapril was given over a period of 21 days with regular control examinations (body weight, clinical examination, blood

234

examination, echocardiography, and electrocardiography). One group was treated without application of enalapril (placebo), one group was treated with 5 mg/kg orally once daily, and the third group was given 10 mg/kg orally once daily.

3 RESULTS AND DISCUSSION

3.1. Clinical experiences The main indications for the use of enalapril in birds were alterations of the right ventricle (hypertrophy, dilatation, insufciency of the muscular atrioventricular valve) and following congestion into the large circulatory cycle (resulting in liver congestion, hydropericardium and ascites). Only in one bird, a left ventricular dysfunction was diagnosed and treated with enalapril. Ideopathic hydropericardium was diagnosed several times and also treated. Diagnosis was made in all cases by means of ultrasound (after preceding hints for cardiovascular disease from the clinical examination and/or radiology). The clinical use of enalapril over the last 4 years indicates a broad safety margin of this drug in birds, too. As in mammals, the drug was administered orally, not as tablets but as solution of the tablets in water. With this solution, dosing and application was easier than with tablets. Concerning the application, a regular observation was that the acceptance of this drug was extraordinary well. It seems that the taste of the solution is favoured by the patient since the acceptance is better than the acceptance of pure water or any other drug used in the clinic. There is nothing known if this depends on the drug itself or on accompanying ingredients of the tablets. In consequence of this good acceptance most owners could apply the drug regularly and without problems. Concerning the effect of enalapril on the cardiovascular system and the general health status, an improvement of the general body condition (activity, appetite, coordination) could be seen in the majority of the treated patients. This improvement was always connected with an improvement of the status of the cardiovascular system. Correspondingly, in birds that not reacted to the enalapril administration, there was also no change in the cardiovascular function. The improvement of cardiovascular function could be seen in a decreased congestion, visible in a reduction of pericardial effusion, liver congestion and free uid in the thoraco-coelomic cavity. The effect on the cardiac function could be proven with a long-term therapy in an amazon bird suffering from right-sided heart failure. In this birds the size of the ventricles, the wall thickness of the interventricular septum and the contractility of the ventricles was measured by means of echocardiography (values see table 1). An improvement of the cardiac function (increase of the fractional shortening (FS)of the right ventricle from 26.8% (day 1) to 36.6% (day 140)), and also the nal decompensation (day 825) could be seen (decrease of contractility of both ventricles). In birds with high-grade pericardial effusion and/or ascites, the use of enalapril was combined with the surgical removal of uid from the thoraco-abdominal/pericardial cavity by means of ultrasound-guided abdominocentesis/ pericardiocentesis

235

(STRAUB et al. 2003). In these cases, the reduction of uid from the pericardial cavity seemed to have an inuence on the cardiac function (decreased pressure on the heart). The remaining uid could be resorbed after administration of enalapril (in some cases in combination with furosemide 0.1 to 2 mg/kg/day). In birds with high-grade pathological alterations of the heart, the therapy with enalapril (and additional therapeutic measures) could improve the life quality and the survival time of the birds (up to 2 years). In birds with idiopathic signs of congestion or only low-grade alterations cardiac function seemed to be normal after the signs of congestion had vanished. Admittedly it has to be said that the diagnosis of lowgrade heart alterations is still difcult and based on individual experience rather than objective measurements. The only side effect that has been observed clinically during long-term therapy in dosages of 5 mg/kg/day orally was an increase of the PCV, that was possibly caused by an increased diuresis. Since this dehydration was severe, the long term application of enalapril should be monitored carefully with regular blood examinations. A dosis reduction after improvement of the general health status is advisable. On the other hand, clinical experiences show that with dosages of 1 mg/kg/day, also the effectivity on the cardiovascular systems seems to be reduced. Practical cases will be presented at the conference. 3.2. Pharmacokinetics and dosis-dependent side effects Results of the examinations concerning the pharmacokinetics and dosis-dependent side-effects will be presented at the conference (ongoing research).

4 CITATION INDEX
1. BOSKOVIC M, KRAUTWALD-JUNGHANNS ME, FAILING K, et al. Mglichkeiten und Grenzen echokardiographischer Untersuchungen bei Tag- und Nachtgreifvgeln (Accipitriformes, Falconiformes, Strigiformes). Tierarztl Prax 1995; 27: 334 - 341. CARRANI F, GELLI D, SALVATORI M, et al. A preliminary echocardiographic initial approach to diastolic and systolic function in medium and large parrots. Proc Eur Assoc Avian Vet, Tenerife 2003, 145-149. HAMLIN RL, STALNAKER PS. Basis for use of digoxin in small birds. J Vet Pharmacol Therap 1987; 10: 354 - 356. KRAUTWALD-JUNGHANNS ME, SCHULZ M, HAGNER D, et al. Transcoelomic two- dimensional echocardiography in the avian patient. J Avian Med Surg 1995; 9: 19 - 31. KRAUTWALD-JUNGHANNS ME, BRAUN S, PEES M, et al. Research on the Anatomy and Pathology of the Psittacine Heart. J Avian Med Surg 2004; 18(1): 2 - 11 PEES M, STRAUB J and KRAUTWALD-JUNGHANNS ME. Insufciency of the muscular atrioventricular valve in the heart of a blue fronted amazon (Amazona aestiva aestiva). Vet Rec 2001; 148(17): 540 - 543.

2. 3. 4. 5. 6.

236

7.

PEES M, STRAUB J and KRAUTWALD-JUNGHANNS ME. Echocardiographic examinations of 60 African grey parrots and 30 other psittacine birds. Vet Rec 2004; 155: 73 - 76 8. SPONER G. Pharmakologie des Herz-Kreislauf-Systems. In: Frey HH, Lscher W (eds): Lehrbuch der Pharmakologie und Toxikologie fr die Veterinrmedizin. Stuttgart: Enke 2002: 147 - 169. 9. STRAUB J, PEES M, ENDERS F and KRAUTWALD-JUNGHANNS ME. Pericardiocentesis and the use of enalapril in a schers lovebird. Vet Rec 2003; 152: 24 - 26. 10. WILSON RC, ZENOBLE RD, HORTON CR, et al. Single dose digoxin pharmacokinetics in quaker conure. J Zoo Wildl Med 1989; 20: 432 - 434.

AUTHORS ADDRESS
M. Pees, DrMedVet, ResECAMS Clinic for Birds and Reptiles, University of Leipzig, An den Tierkliniken 17, 04103 Leipzig, Germany Email: pees@vmf.uni-leipzig.de

237

Clinic for Birds and Reptiles, University of Leipzig, Germany

PHARMACOKINETICS, BIOAVAILABILITY AND COMPATIBILITY OF DOXYCYCLINE IN BIRDS AFTER ORAL ADMINISTRATION

E. M. Weilacher, M. E. Krautwald-Junghanns, Prof DrMedVet, Dipl ECAMS, F. R. Ungemach, Prof DrMedVet

KEYWORDS
Birds - Pigeons - Chlamydophilosis - Antibiotic therapy - Doxycycline - Oral administration

ABSTRACT
Chlamydophilosis in racing pigeons, as in any pet bird species, is still common. Investigations by several authors show, that doxycycline is the drug of choice to treat the clinical signs and to handle the elimination of the pathogens. There have been several studies about the possible administration of doxycycline (PRUS 1992, POWERS 1997).

1 INTRODUCTION
For decades doxycycline is a frequently used antibiotic in veterinary medicine. In recent years this drug regained importance again because of its effectiveness against a broad antibacterial spectrum with good compatibility and low costs. In avian medicine doxycycline is known especially for its effectiveness in treatment of Chlamydophila infections. Therefore the aim of this study was to develop an oral therapeutic regime and to determine an appropriate dosage of doxycycline with optimal effectiveness and lower side effects for treating chlamydophilosis in pet birds using the racing pigeon as a model.

2 MATERIAL AND METHODS

For all studies, adult, healthy racing pigeons (Columbia livia domestica) of both sexes were used. They were housed in individual cages with a wired oor at room temperature and an articial daylength of 16 hours. The birds had free access to

238

water and a standard breeding grain-mix for racing pigeons. No grit-mineral mixture was given during the experiments. Blood samples were taken from the ulnar vein at various times before and after administration of doxycycline. The blood and organ concentrations of doxycycline were determined with HPLCanalysis with UV-detection after reconditioning the samples extracted by solid phase extraction. The study was divided into four sections: The aim of section 1 was to assess whether a sufcient doxycycline absorption in the gastrointestinal tract after oral administration had been achieved and to examine pharmacokinetic parameters. For this purpose a single dose of doxycycline of 60mg/ kg was administered to 15 pigeons via crop cannula. Blood was taken at dened intervals after the application (0, 0.5, 1, 2, 4, 6, 8, 12, 16, 24, 36, 40, 48, 72 h) and examined for doxycycline concentration. On the basis of these results in the following section 2 36 racing pigeons were examined over 14 days. The main question was whether a reduced dose of 30mg/kg/d maintains constantly high and effective plasma levels for treating Chlamydophilosis. Considering the half-life of doxycycline the drug was distributed in two equal doses twice a day and administered with a crop cannula into the crop. Doxycycline blood concentrations were determined of expected peak and trough values on day 2, 7 and 13. After 14 days of doxycycline administration levels in lungs and liver of 12 pigeons were examined 2 and 5 hours after the last doxycycline application. This was done to check for effective doxycycline concentrations in the organs needed for therapy of Chlamydophila infections. Section 3 was conducted to simulate the conditions in the eld. Twenty racing pigeons received doxycycline medicated drinking water at a concentration of 750mg/l ad libitum for 10 days. The voluntary intake of doxycycline medicated drinking water and the resulting doxycycline plasma levels were tested. At day 1, 2, 4, 6, 8, 10 blood samples were collected two times a day (8 am and 4 pm) and the doxycycline concentrations determined. In the last section (4) the doxycycline tolerance was examined over 20 days after oral administration. Sixty pigeons were divided into groups of 12 animals each. One group was treated with a placebo (0mg/kg/d), one group was treated with the therapeutic dose of doxycycline of 30mg/kg/d, one group with 45mg/kg/d, one group with 60mg/kg/d and the fth group with 150mg/kg/d. The doxycycline solution was administered divided into two equal dosages with a crop cannula. Before and during the study the pigeons were checked for their health status (clinical, microbiological and parasitological). Blood samples were taken and examined (haematology, blood chemistry, doxycycline concentration), food and water consumption were recorded. After 20 days a pathological examination was performed. Half of the pigeons were necropsied the day after the last application, half of the animals one week later.

239

3 RESULTS
The evaluation of section 1 (checking for pharmacokinetic parameters) resulted in the following average values: cmax = 8.1g/ml; tmax = 6.0 h; t1/2 = 11.3 h; VZ/f = 5.3 l/kg. Doxycycline was well absorbed in the gastrointestinal tract of pigeons after oral administration. Table 1. Pharmacokinetic parameters after a single application of 60mg/kg doxycycline Parameter Cmax Tmax Z t1/2 AUC0- MRT0- CL/f VZ/f Unit [g/ml] [h] [h-1] [h] [gh/ml] [h] [ml/minkg] [l/kg] Section 1: single application of 60mg/kg BW 8.1 6.0 0.0615 11.3 184.1 18.8 5.4 5.3

In section 2 (oral doxycycline administration) all doxycycline blood levels were above 1 g/ml, necessary for treating chlamydophilosis. The determined values uctuated between 1.5 and 2.7g/ml. Doxycycline plasma concentrations could be measured up to 73 h after the last application, this resulted in a calculated elimination half-life of 12.5 h. Table 2. Pharmacokinetic parameters after application of 30mg/kg (2x 15mg/kg) doxycycline for 10 days Parameter Cmax Tmax Z t1/2 AUC0- MRT0- CL/f VZ/f Unit [g/ml] [h] [h ]
-1

Section 2: twice a day application of 15mg/kg BW 2.4 (peak in the steady state) 1.8 (trough in the steady state) steady state 0.0554 12.5 37.7 18.4 6.6 7.2

[h] [gh/ml] [h] [ml/minkg] [l/kg]

240

The doxycycline levels in liver and lungs, after 14 days of doxycycline administration reached 14.3 and 9.7g/g 2 hours past the last doxycycline application, respectively 4.4 and 2.8g/g 5 hours past the last doxycycline application. At any time the concentrations were above 1 g/g. The organ/plasma-level of the doxycycline concentrations were about 8 for the liver and about 2 for the lungs and also conrmed an accumulation of the high lipophilic doxycycline in the organs of racing pigeons. Section 3 showed a good acceptance and voluntary intake of doxycycline medicated drinking water and constant doxycycline plasma levels for 10 days. In accordance with an average intake of medicated drinking water of 18.0ml/kg/d, the mean daily doxycycline dose ingested was 27mg/kg. In the blood samples taken two times a day (8 am, 3 pm) average doxycycline concentrations of 1.62 up to 2.20g/ml were detected. Some birds failed to maintain doxycycline concentrations above 1g/ml for the entire period, but the majority of birds reached or exceeded these levels during treatment. In section 4 no signicant differences between the placebo group, the group receiving the therapeutic dose, the 1.5fold, and the 2fold therapeutic dose were observed by the clinical examination, for food and water intake, blood chemistry, haematology and at necropsy. However, the results of the group with the 5fold therapeutic dose differed signicantly from the other groups. The pigeons of this group showed regurgitation after administration of the drug. They had polyuria and yellowish uric acid. The water intake increased from an average of 20 ml to an average of 50 ml/pigeon. A loss of bodyweight was registered. AST, GGT, bile acids and cholinesterase were signicantly increased during the application period. Pathological gross ndings were diffuse yellow-brown or yellow-red coloured livers. Microscopically an extramedullar haematopoesis (2 pigeons), acute hepatitis (1 pigeon) and fatty liver cell degeneration (1 pigeon) was seen.

4 DISCUSSION
Due to practical considerations and despite known possible disadvantages the application of antibiotics via drinking water is preferred. This offers a possibility to avoid production of medicated feeding stuff that is often not very well accepted or injections that may produce muscle necrosis. In pet bird medicine this is for example especially important in racing pigeons to keep the birds in good ight condition. The results of our studies show that doxycycline is well absorbed in the gastrointestinal tract of pigeons after oral administration. Constant doxycycline blood levels were achieved exceeding 1g/ml needed to effectively treat chlamydophilosis. PADILLA (2003) reported comparable data in fruit doves, FLAMMER (2000) in psittacines. Furthermore, high doxycycline levels observed not only in the plasma, but also in the target and elimination organs for Chlamydophila (lungs and liver). The organ/plasmalevel indicated an accumulation of the highly lipophilic doxycycline in these organs. Under eld conditions with a voluntary intake of drinking water a good acceptance of doxycycline medicated drinking water was found. At a concentration of 750mg/l of

241

doxycycline in the drinking water again plasma levels above 1 g/ml were reached. In section 4 no side effects of doxycycline for the therapeutic dose and with an application period of 20 days could be found. This is in accordance with other studies (PADILLA 2003). In contrast to studies in psittacines however (FLAMMER 2000) we did not see any side effects in racing pigeons for the 1.5 fold and 2 fold therapeutic dose. On the other hand the pigeons which received the 5 fold therapeutic dose showed side effects like regurgitation and loss of body weight, liver damage with following polydipsia, polyuria, yellowish colouring of the uric acid and increased liver enzymes. It can be assumed that the tolerance of doxycycline for racing pigeons after oral administration for 20 days is excellent up to the twofold therapeutic dose. Higher doses should be avoided, because they could induce liver damage and therefore impair the well being of the animals. This fact has to be considered especially in diseased birds with a higher water intake.

5 CITATION INDEX
1. 2. 3. 4. FLAMMER K: Preliminary notes on treatment of chlamydiosis with doxycycline medicated water. Proc Assoc Avian Vet, Portland 2000: 3 5. PADILLA LR, MILLER RE and FLAMMER K. Doxycycline in drinking water for treatment of Chlamydophila psittaci in fruit doves. Proc Am Assoc Zoo Vet, Minneapolis 2003: 267 - 268. PRUS SE, CLUBB SL and FLAMMER K. Doxycycline plasma concentrations in macaws fed a medicate corn diet. Avian Dis 1992; 480 - 483. POWERS L and FLAMMER K. Dosing methods for administration of doxycycline in cockatiels. Proc Assoc Avian Vet, Reno 1997: 57 58.

AUTHORS ADRESS
E M Weilacher, DVM Clinic for Birds and Reptiles University of Leipzig An den Tierkliniken 17 04103 Leipzig, Germany Email: weilacher@vmf.uni-leipzig.de

242

Clinic for Birds and Reptiles University of Leipzig, Germany

ORAL FURAZOLIDONE AND CHLORAMPHENICOL FOR TREATMENT OF GASTROINTESTINAL BACTERIAL INFECTIONS

M.E. Krautwald-Junghanns, ProfDrMedVet, DiplECAMS, V. Schmidt Res ECAMS and N. Reitz

KEYWORDS
Furazolidone Chloramphenicol - Oral application, E. coli Salmonella - racing pigeon

ABSTRACT
In two similar studies, the clinical efcacy of furazolidone for treatment of gastrointestinal E. coli infections (study 1) rsp. the clinical efcacy of chloramphenicol for treatment of a S. typhimurium infections (study 2) was investigated by examination of 36 rsp, 24 adult pigeons. The pigeons originated from conventional breeders and were housed in different groups (control -, capsule and powder group). After infection with an E. coli strain that proved to be pathogenic for pigeons rsp, a S. typhimurium var. Copenhagen strain, the animals developed clinical signs of disease. After this time, the treatment with different modes of oral application was started. This phase was followed by an adspectory phase and/or by a pathological examination in study 2. The negative identication of the marked bacteria was determined as main parameter for the clinical efcacy of the treatment. Both furazolidone preparations proved to be effective in treating gastrointestinal E. coli infections in racing pigeons in a dosage of 12.5 mg /pigeon, however best results were obtained by application via capsule for 5 days. In contrast to study 1, a clinical effectiveness could not be postulated for the treatment with chloramphenicol against a S. typhimurium infection.

1 INTRODUCTION
Changes in drug regulations and requirements often cause an omission of different effective drugs from the market. Particularly minor species are concerned by that, animals, whose number is too small to animate pharmaceutical enterprises to investigate the costs of the admission. In Germany, furazolidone and chloramphenicol are allowed only for veterinary purposes and here only for racing pigeons. By legal

243

conversion they may be used also for other pet and zoo birds. The permission is limited to the treatment of bacterial gastro-intestinal tract infections caused by E. coli for furazolidone and those caused by Salmonella typhimurium for chloramphenicol. These bacteria rank among the most frequent causes of bacterial diseases of the avian intestinal tract. However when furazolidone and chloramphenicol are considered, they have to be used under critical consideration among others of their side effects. Another important point for the mode of application of these drugs is their half-life and whether sufcient blood and tissue levels may be achieved for example by the common oral application. In this context it was the aim of our study, to examine the clinical effectiveness of furazolidone and chloramphenicol as chemotherapeutic agents in gastro-intestinal tract diseases caused by E. coli rsp., S. typhimurium var. Copenhagen using different oral modes of application. Drugs used Furazolidone: In 1995 the use of nitrofuranes in food-supplying animals was already forbidden in all member states of the EU (KROKER et al. 2002). Nitrofuranes have a small therapeutic margin; in poultry the double therapeutic dose can lead to cardiomyopathy (KROKER et al., 1996). Waterfowl may react particularly sensitive to furazolidone and should therefore not be treated with this antibiotic drug (GYLSTORFF and GRIMM, 1998). Besides that the nitrofuranes possess a spermicidal effect, so their application is contraindicated in breeding animals. Due to the bad systemic compatibility after parenteral application furazolidone has to be administered locally or orally (KROKER et al. 1996). The advantage of the bacteriostatic furazolidone is its broad antibacterial spectrum and the insignicant formation of resistance. Furazolidone is called a cavity therapeutic agent, since its therapeutic effect is only in the intestine and it is hardly absorbed from there. It was predominantly used therefore in former times in poultry for the treatment of infectious enteritis. Main indication is the E. coli infection of the gastro-intestinal tract, but many other sensitive strains like Klebsiella, Campylobacter, Salmonella and Shigella may be covered as well (KROKER et al. 1996). The resistance situation of nitrofuranes is generally good. For the pigeon however there are only little current publications. Chloramphenicol: Already in 1990 the use of chloramphenicol was forbidden in foodsupplying animals in all member states of the EU. This was due to the potential side effects chloramphenicol may cause in men like aplastic anaemia, contact dermatitis. Other side effects that have been described may be regurgitation in pigeons after oral application and bone marrow depression in embryos (so chloramphenicol should not be used in breeding hens). The veterinary importance of chloramphenicol is based on its broad antibacterial spectrum and a slow development of resistance, which is mainly plasmid bound. Chloramphenicol may penetrate the cell wall, so it is effective against intracellular bacteria. Due to its lipophilic effect it may be used to treat bacterial central nervous diseases. The effectivity spectrum of the bacteriostatic chloramphenicol covers many sensitive strains of most gram positive and gram negative bacteria. Mycobacterium and Pseudomonas possess a natural resistance (LSCHER et al. 1999). In racing pigeons this antibiotic is mainly used to treat samonellosis caused by Salmonella enterica subsp. enterica Serovar Typhimurium

244

var. Copenhagen (called S. typhimurium in the text). After oral administration the absorption is almost complete. The apparent distribution volume is very high. The elimination is predominantly renal (LSCHER et al. 1999). However the half-life of chloramphenicol in pigeons reaches only 0.26 h (CLARK et al. 1992).

2 METHODS
The studies were performed according to valid regulations as well as according to the principles of the Good Clinical Practice (GCP). The controlled clinical studies were performed as parallels group study. The pigeons originated from conventional breeders and were housed in different groups a 12 birds (control - capsule and powder group). After infection with an E. coli strain that proved to be pathogenic for pigeons rsp. a S. typhimurium var. Copenhagen strain, the animals developed clinical signs of disease. After this time started the treatment with different mode of application (capsule, drinking water). This phase was followed by an adspectory phase and in case of the Salmonella infection by a pathological investigation. The negative identication of the marked bacteria was determined as main parameter for the clinical efcacy of the treatment.

2. 1 STUDY 1 - FURAZOLIDONE
Dosage and application Two different furazolidone preparations were applied (powder and capsule form), which are used in practice for oral administration over the drinking water or instilled into the crop. The furazolidone powder contained 500 mg furazolidone per 7.5 g packing unit according to manufacturer data in powder form; the furazolidone in capsule form contained 12.5 mg furazolidone per capsule. The theoretical therapy dose was 25 mg/kg BW furazolidone, so that each pigeon got (average weight 500g) daily 12.5 mg furazolidone over 5 days. Powder: Since the drinking water admission is dependent on numerous factors in pigeons, the animals got a more exact dosage into the crop 2 x daily every 12 hours - once in the morning and once in the evening. Like that an application volume of 20 ml/ kg BW of the accordingly medicated drinking water was instilled in each pigeon by crop cannula. Directly before the application of the furazolidone a solution of the powder with warm water was prepared in each case. Capsule: The daily dosage was one capsule/ pigeon; the application of the capsules took place once daily in the morning one hour before feeding. In order to ensure a better admission of the capsules, these were dipped directly before the installation into parafn oil. Expiration of study 1 The pigeons of the two therapy-groups (capsule, powder) were infected with a marked, inoculated, pathogenic E. coli strain (infection dose 1 x of 109). Twenty four hours after the inoculation the marked E. coli strain could be re-isolated from the cloaca of all pigeons. Then, started the therapy for 5 days as described. After completion of the therapeutic part on day 5 and 6 swabs were taken from choana, crop and cloaca and examined for the presence of the inoculated E. coli.

245

Results of study 1 At day 5 and 6 in none of the treated pigeons the marked E could be isolated in contrast to the birds of the control group. In the adspection and clinical investigation already 24 hours after beginning of treatment, a clear improvement of almost all investigation criteria could be seen in the capsule group. In the powder group this improvement was seen approx. 48 hours after beginning of therapy. At the last day of drug application, 3 pigeons in the capsule group showed regurgitation. Due to the orange colour of the regurgitated material it was most likely that this contained the dissolved capsules. Other side effects could not be found. Only after completion of the therapy phase a normalization of the faeces in the two treated groups could be seen. However a positive tendency of this clinical criterium (towards physiological faeces) was already seen in the capsule and powder group from day 5 on.

2. 2. STUDY 2 - CHLORAMPHENICOL
Dosage and application The chloramphenicol powder used was certied for oral administration in the drinking water and contained (according to manufacturer data) per 6.5g packing unit in powder form in powder form 850 mg of the active substance cloramphenicol. The dosage was determined on the basis of the following formula: 650 mg chloramphenicol /kg BW/day x BW (kg) of the animal treated mean daily drinking water admission (l)/ animal =... mg chloramphenicol per l water The middle daily drinking water admission was determined with 40ml/kg BW. Directly before the application a solution with a concentration of 16.25mg chloramphenicol/ ml was made with warm water in each case. Since the drinking water admission is dependent of temperature, air humidity and other factors, the animals were treated in contrast to study 1 and due to the bad results of various pre-studies with lower application intervals - ve times daily (every 3 hours) with an application volume of 20ml/kg medicated drinking water by crop cannula. Expiration of study 2 The pigeons were infected orally with a chloramphenicol sensitive, pathogenic S. typhimurium var. Copenhagen strain. The infection dose was 1x109. 3 to 7 days p. infect. clear signs of clinical disease could be found and all animals shed the inoculated S. typhimurium var. Copenhagen strain, so that the therapeutic part of the study began. The application intervals of the drug had to be shortened to 3 hours (see above) and the application duration was extended to 10 days. The re-isolation was performed due to described methods after euthanasia of the animals from the individual organs.

246

Results of study 2 6 days after the inoculation the inoculated S. typhimurium strain could be re-isolated from all pigeons from the faeces. In the daily adspection and the clinical investigations, an improvement could be documented in the phase of acclimatisation (infected but not treated), at the beginning and at the end of the therapy in the following points, behaviour, body attitude and plumage in the treated groups. The clinical improvement of the treated pigeons however could not be brought in connection with an efcient destruction of the pathogen. Both in the treated as well as (as expected) in the untreated group pathological-anatomical ndings in the liver could be found, which hint at an infection with S. typhimurium. Interestingly most of these alterations were seen in the pathological-anatomical investigation immediately after the end of treatment (day 10). The inoculated S. typhimurium strain could be re-isolated in all pigeons of the treated group. After the therapy with chloramphenicol, on day 10 and 16 organ samples were taken, all 12 animals of the treated group were S. typhimurium positive. Signicant differences in the isolation from the single organs could not be determined. As expected, the 12 animals of the untreated group were S. typhimurium positive as well; all reisolated S. typhimurium strains were chloramphenicol sensitive.

3 DISCUSSION
Both furazolidone preparations proved to be effective in treating gastrointestinal E. coli infections in racing pigeons in a dosage of 12.5mg/ pigeon, however best results were obtained by application via capsule for 5 days. For the treatment with chloramphenicol it has to be noticed in summary that the main criterion for the evaluation of the clinical effectiveness of chloramphenicol in treating S. typhimurium infection was not fullled. Due to the short half-life of chloramphenicol in racing pigeons of 0.26 hours and the peculiarities of Salmonella infections this result is explainable. Concerning the oral modes of application in these studies and in the eld: Since the housing conditions in the single cages in an air-conditioned room do not correspond to the actual housing conditions in the eld (with free ight etc.), a strongly reduced drinking water admission under these condition was expected due to various previous experiences. In order to ensure the admission of an effective dose in the two studies an application with crop cannula was necessary instead of the application via drinking water. In contrast to the latter, an exact dosage per animal could be achieved with the crop installation like by capsule application. Under practice conditions without doubt substantially larger uctuations in the actually intake of drinking water have to be expected: Thus among other things hot weather conditions, kind of food, taste of the drug and actual diseases may play an important role in this context. Generally, for an antibiotic treatment of larger bird collections the antibiotic is usually applied within the drinking water despite the well-known disadvantages just due to practicability reasons. Due to the inaccurate dosage with this kind of application however actually only preparations should be used, which have large therapeutic margin. The latter does not apply to furazolidone and chloramphenicol; however incompatibilities as described for water fowl (GYLSTORFF and GRIMM, 1998), were so far not observed in the pigeon. Nevertheless when these drugs are administered via drinking water the actual seasonal drinking water intake of the individual bird group should be considered in each case.

247

Without doubt the mode of application by capsule or tablet is - besides the unusual application by crop cannula - the most exact possibility of the oral administration of a drug in birds. The disadvantages of the application by capsule are that it is time consuming and impracticable for the treatment of a larger collection (KAMPHAUSEN 2003). As a further disadvantage it is described that the pigeons may vomit the capsules/ tablets, without this is noticed by the owner. In our own study, regurgitation could be seen after capsule application in 3 pigeons. A clear improvement of the clinical symptoms occurred quite early (as opposed to the powder group) in the capsule group - already 24 hours after beginning of therapy. Possibly the noticed temporal difference lies in the high concentration of the active substance in the capsule. Another point that has to be considered is, that even under natural conditions with free access to the medicated drinking water, a sufcient effective blood level may not be kept constant, since particularly in the night the pigeons do not drink and thus a sub-therapeutic effect may develop.

4 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. CLARK CH, THOMAS JE, MILTON JT. et al. Plasma concentrations of chloramphenicol in birds. Am J Vet Res 1982; 43: 1249 1253. GYLSTORFF I AND GRIMM F. Vogelkrankheiten. 2. Auage, Stuttgart: Ulmer Verlag 1998: 148. KAMPHAUSEN L. Antibiotikatherapie bei Brieftauben. Tagungsberichte der 2. Leipzig: Leipziger Samstagsakademie 2003. KROKER, R., LSCHER W., SIMUNEK J. et al. Nitrofurane. In: FREY, H.H. and W.LSCHER (eds.): Lehrbuch der Pharmakologie und Toxikologie fr die Veterinrmedizin. Stuttgart: Enke Verlag 1996: 499 - 501. KROKER, R. Nitrofurane. In: LSCHER, W., UNGEMACH F., KROKER R. (eds.): Pharmakotherapie bei Nutz- und Haustieren. Berlin: Blackwell Verlag 1999: 243 - 244 KROKER, R., SCHERKL R., UNGEMACH F. Nitrofurane. In: FREY, H.-H., LSCHER W. (eds.): Lehrbuch der Pharmakologie und Toxikologie fr die Veterinrmedizin. Stuttgart: Enke Verlag, 2002: 387 - 388 LSCHER W., UNGEMACH F., KROKER R. Pharmakotherapie bei Hausund Nutztieren, Berlin: Parey Buchverlag 1999: 230 - 231

AUTHORS ADDRESS
M. E. KRAUTWALD-JUNGHANNS, ProfDrMedVet, DiplECAMS Clinic for Birds and Reptiles, University of Leipzig, An den Tierkliniken 17, 04103 Leipzig, Germany Email: krautwald@vmf.uni-leipzig.de, pees@vmf.uni-leipzig.de

248

Klinik fr Vgel, Ludwig-Maximilian-Universitt Mnchen, Germany

EVALUATION OF VARIOUS TISSUES FOR DIAGNOSIS OF PSITTACINE BEAK AND FEATHER DISEASE (PBFD)
C. H. Grund, B. Khler, R. T. Korbel

KEYWORDS
PBFD - PCR - Acute outbreak - Diagnostic - African grey parrots

ABSTRACT
Psittacine beak and feather disease (PBFD) is an important viral disease of psittacine birds, caused by psittacine beak and feather disease virus (PBFDV). To detect diseased birds or virus carriers, PCR is the diagnostic method of choice. In order to evaluate the reliability of PCR results obtained by testing skin-feather samples, we screened skin-feather samples and primary lymphoid tissues (spleen, bursa of Fabricius) in parallel, as well as liver. Samples were derived from a ock of young African grey parrots (Psittacus erithacus), which suffered an outbreak of acute PBFD. Of a total of 34 birds investigated, all 29 were tested PBFDV-positive by PCR with a positive PBFD-signal predominantly in skin-feather-samples (n=26). Whereas in 21 birds viral DNA was detected in more than one sample (skin 18/21, liver 21/21, spleen 9/21, bursa 9/19), in eight birds only the skin-feather sample was PBFDVpositive. Nonetheless, in birds tested PBFDV-negative in the skin, viral DNA could be detected in liver (3/3), spleen (1/3) and bursa (2/3). The results indicate that in African grey parrots, despite the observed immune-depression in young birds of this species, the liver is the primary target organ. Considering the high risk of contaminated PCRsamples from the skin and/or feathers by dust, biopsy specimen from the liver seems to be an alternative for individual diagnostic of PBFDV in African grey parrots.

1 INTRODUCTION
Beak and feather disease (PBFD) is a severe disease in most species of the order Psittaciformes (RITCHIE 1995). In addition, infection with the aetiological agent, the psittacine beak and feather disease virus (PBFDV) has been reported in some other bird species (RAHAUS and WOLFF 2003, EISENBERG et al. 2003). PBFDV is, like the porcine circovirus, a member of the genus Circovirus, of the family Circoviridae (RITCHIE et al. 1989). By homologies of the viral genomes, pigeon circovirus, goose circovirus and canary circovirus should also be grouped in this genus (MANKERTZ

249

et al. 2000, TODD et al. 2001). Depending on the species and age of birds, clinical outcome of infection may vary. Characteristic signs common in chronic cases, are a slow progressive loss of feathers in conjunction with beak elongation and necrosis of beak and palate. In young birds however, the course of the disease is dominated by an acute onset of a severe immunosuppression, which may result in lethal secondary infections and sometimes accompanied by an irreversible loss of feathers. In particular, in African Grey parrots this acute course of PBFDV infection has been recognised in young birds that died with no obvious symptoms (SHOEMAKER et al. 2000). Diagnosis of PBFDV infection relies most often on detection of viral DNA by polymerase chain reaction (PCR). However, in order to obtain reliable results by this fast and sensitive method, selection of the sample is crucial. So far, independent of the species tested, blood and feathers are used routinely for diagnostic PBFDVPCR. Considering, that virus load might vary considerably in different species depending on the course of infection we investigated tissue distribution in 34 African grey parrots that died during an outbreak of acute PBFDV infection by PCR. Testing skin-feather samples, primary lymphoid organs (spleen and bursa of Fabricius) as well as liver samples in parallel, reliable diagnostic samples for PBFDV-PCR in African grey parrots should be identied.

2 MATERIAL AND METHODS

2.1. Animals and experimental design From a ock of African grey parrots (Psittacus erithacus) that suffered an acute outbreak of PBFD, 34 birds were sent in for pathological examination. The birds were delivered together in big plastic bags in frozen condition. Samples taken for PCR-test included the spleen, liver, bursa of Fabricius and skin with feathers. All samples were stored at -70C until further processing. 2.2. DNA extraction DNA extraction was done by a commercial DNA-isolation kit (DNA easy tissue kit, Quiagen, Hilden, Germany) according to the manufacturers instructions. Skin with feather samples was cut with fresh razor blades, the other organs with two injection needles, paying attention to change instruments between each sample. 2.3. Polymerase chain reaction Isolated DNA was tested for the presence of circoviral DNA by PCR amplifying a part of the genomic region within ORF 1 applying the protocol (primer no. 2 and 4) described by YPELAAR et al. (1999) with some modications. In brief, we used 25l reaction mixture containing 25pmol of each primer, 0,2mM dNTP`s, 1mM MgCl2 and 0,2 IU of taq-Polymerase (peqlab, Erlangen, Germany). The thermocycler conditions were: 15 min 95C (activation of the polymerase and initial denaturation) followed by 32 cycles of 30s 95C, 20s 57C, 30s 72C and a nal extension at 72C for 5 min. The 717bp long PCR products were separated in a 2% agarose gel and stained with ethidium bromide.

250

3 RESULTS
For this study, a total of 34 African grey parrots from a suspected outbreak of acute PBFD were investigated for gross anatomic changes as well as for viral infection by PCR. Overall, the birds` body condition was good except for seven birds with a light to moderately reduced bodyweight. At external inspection birds had complete plumage with normal formed feathers and the horn of beak and claws was intact. In eight birds a discreet red discolouration of the contour feathers in the dorsal and ventral caudal abdominal region was visible. Predominant alterations of internal organs included hepatomegaly (n=15) and splenomegaly (n=12), as well as a hydropericardium (n=11). Testing for PBFDV by PCR a total of 29 birds were tested PBFDV-positive. Viral DNA was predominantly detected in samples of the skin and feathers (26/32) and liver (21/33). In contrast a positive PBFDV-signal in bursa and spleen was obtained only randomly (spleen: 9/31, bursa: 9/27). Within the group of PBFDV-positive birds (n=29), 21 had a positive PCR-signal in two or more tissues. Investigating the birds where PBFDV was detected in the skin-feather-samples (n=26) only three quarter of the liver-samples (n=18) and one third of the lymphoid organs (spleen n=8, bursa n=7) were tested PBFDV-positive (Fig. 1A). However, in 3 birds tested PBFDVnegative in the skin, viral DNA could be detected in liver (3/3), spleen (1/3) and bursa (2/3) (Fig 1B).

Fig. 1: PCR-results of birds from an acute PBFD-outbreak.

251

From 34 African grey parrots, 29 birds were tested positive for PBFDV by PCR. Shown are the PCR-results of these 29 birds divided in groups with A PBFDVpositive skin/feather sample (n=26), B PBFDV-negative skin/feather sample (n=3), C PBFDV-positive liver sample (n=21), D PBFDV-negative liver sample (n=7), E PBFDV-positive spleen sample (n=9) and F PBFDV-positive bursa sample (n=9). Columns in blue represent the number of samples tested and in red, PBFDV-positive samples, with the respective percentage given above. Comparing the PCR-results of the internal organs, it became evident, that within the group tested positive in the liver (Fig. 1C, n=21) almost 1/3 of the lymphoid organs (spleen n=9, bursa n=9) were tested PBFDV-positive as well as 18 skin-feather samples. In contrast, in the group of PBFD-positive birds with negative PCR-results of the liver-samples (Fig 1D; n=7), the lymphoid tissues (bursa and spleen) gave also negative results (Fig 1D). Similar results were observed when PBFDV-DNA was detected in the spleen (Fig 1E; n=9) or bursa of Fabricius (Fig 1F; n=9). In both groups viral DNA was detected also in the liver, but not always in the skin-feather samples.

4 DISCUSSION
In the present study of 34 African grey parrots from a ock that suffered an outbreak of acute PBFD, 29 birds were tested PBFDV-positive by PCR. In addition to skin-feather samples (n=26) viral DNA was detected regularly in the liver (n=21). This frequent detection of PBFDV in the liver is in agreement with observations by Shoemaker et al. (SHOEMAKER et al. 2000). In a study on 14 young African grey parrots, suffering acute PBFD, he was able to demonstrate moderate to severe coagulative necrosis in the liver. In addition, he reported, that viral inclusion bodies were detected regularly in the bursa (7/7). In our study we were able to demonstrate viral DNA in the bursa as well as in the spleen, but not as often as in the liver (spleen 9/21, bursa 9/19). In particular, the comparison of the PCR results from spleen and liver proves that the birds did not die during viremia and thus the positive PCR-signal indicates replication within the liver. In addition, the high frequency of PBFDV in the liver emphasise the relevance of the liver for pathogenesis of acute PBFD in young African grey parrots. Considering that acute PBFD is seldom associated with feather manifestations (SHOEMAKER et al. 2000), the regular detection of PBFDV-DNA in skin-feather samples is somewhat surprising. However, it is known, that feathers harvest virus, even without alterations. For example, in a longitudinal study of PBFDV infected budgerigars (Melopsittacus undulatus) viral DNA could be detected in feathers of birds without clinical signs for as long as 38 weeks (HESS 2004). Nonetheless, in particular a positive PCR-signal only in a skin-feather sample, as observed in 7 birds in this study has to evaluated critically. Considering that PBFDV can spread by feather dust, PCR-samples from the skin and/or feathers have a high risk of contamination. Concerning our results in African grey parrots, biopsy specimen from the liver for PCR seems to be an alternative for the diagnosis of PBFDV in individual birds in this species.

252

5 CITATION INDEX
1. EISENBERG SWF. Detection of circovirus with a polymerase chain reaction in the ostrich (Struthio camelus) on a farm in the Netherlands. Vet Microbiol 2003; 95: 27 - 38. 2. HESS M. Comparative sensitivity of polymerase chain reaction diagnosis of psittacine beak and feather disease on feather samples, cloacal swabs and blood from budgerigars (Melopsittacus undulatus, Shaw 1805). Avian Pathol 2004; 33: 477 - 481. 3. MANKERTZ A. Cloning and sequencing of columbic circovirus (CoCV), a new circovirus from pigeons. Arch Virol 2000; 145: 2469 - 2479. 4. NIAGRO FD. Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses. Arch Virol 1998; 143 (9): 1723- 1744. 5. RAHAUS M. Psittacine beak and feather disease: a rst survey of the distribution of beak and feather disease virus inside the population of captive psittacine birds in Germany. J Vet Med B 2003; 50: 368 - 371. 6. RITCHIE BW. Circoviridae. In B.W. RITCHIE (Ed.), Avian Viruses: Function and Control. Lake Worth: Wingers Publishing 1995: 223 - 252. 7. RITCHIE BW. Characterization of a new virus from cockatoos with psittacine beak and feather disease. Virology 1989; 171(1): 83 - 88. 8. SCHOEMAKER NJ. Severe leukopenia and liver necrosis in young African grey parrots (Psittacus erithacus erithacus) infected with psittacine circovirus. Avian Dis 2000; 44(2): 470 - 478. 9. TODD D. Genome sequence determination and analyses of novel circoviruses from goose and pigeon. J Virol 2001; 286; 354 - 362. 10. YPELAAR IA. Universal polymerase chain reaction for the detection of psittacine beak and feather disease virus. Vet Microbiol 1999; 68: 141 148.

AUTHORS ADRESS
C. H. Grund, Klinik fr Vgel, Ludwig Maximilian Universitt, Sonnenstrasse 18, 85764 Oberschleiheim, Email: Christian.Grund@lrz.uni-muenchen.de

253

Koret School of Veterinary Medicine1, Hebrew University, Jerusalem, Kimron Veterinary Institute2, Bet Dagan, Israel

RELIABILITY OF CROP BIOPSY AS A DIAGNOSTIC TOOL FOR PROVENTRICULAR DILATATION DISEASE IN PSITTACINE BIRDS
U. Bendheim1, DVM, Dip ECAMS, A. Lublin 2 DVM, PhD, N. Edery2, DVM

KEYWORDS
Proventricular Dilatation Disease (PDD) - Crop biopsy

ABSTRACT
A pair of blue and yellow macaws (Ara ararauna) was introduced into a breeding farm in Israel and produced chicks that presented with proventricular dilatation disease (PDD) signs at the age of 10-14 wks. Forty two grey parrots (Psittacus erithacus) and 8 blue and yellow Macaws were hatched in this aviary in that period, about 27 of whom died and demonstrated lesions typical of PDD. Birds intended for breeding were euthanased. 19 of the euthanased birds were necropsied for comparison of crop vs. proventricular and brain lesions to test whether crop biopsy may be a reliable means for the clinical diagnosis of PDD. Square samples of 1 cm2 including blood vessels and probably nerves were taken from these tissues, and were processed for histological examination. Histology demonstrated multifocal lymphohistiocytic inltration with gliosis in brain and inltration in ganglia of the muscularis externa of the proventriculus in 10 birds out of 19, while only 4 birds had similar microscopic changes in the crop. The rest of the birds did not present any histologic changes. We conclude that the crop should not be recommended for clinical PDD diagnosis due to the high prevalence of false-negative cases. Also brain and proventriculus may present negative histological results even in birds with PDD.

1 INTRODUCTION
Proventricular dilatation disease (PDD) was rst described in macaws as a malabsorption/maldigestion disorder. Common clinical signs are weight loss, regurgitation and indigestion, but central nervous signs may also be observed, as central and peripheral nervous tissues are involved. Clinically PDD is not easy to conrm in the live bird, because x-ray imaging does not give a clear diagnosis (GREGORY 1995) and because of the lack of serology. Nor does a histopathological

254

evaluation of suspected cases give a clear diagnosis. GERLACH (1991) found microscopic lesions in only 62% of birds with typical PDD clinical signs. However, 33% of birds with lymphoplasmacytic proliferation in the proventriculus did not have proventricular dilatation (GREGORY 1995). In a group of 15 psittacine birds with proventricular dilatation, typical lesions were found in the proventriculus of all birds, but only in 67% in the crop (GREGORY 1995). Sporadic cases of PDD have been observed in adult psittacine birds (various species) in Israel in the past, but have not been published. In a severe outbreak in a psittacine breeding farm with high mortality which was diagnosed as PDD the owners, following the veterinarians advice, agreed to euthanase the clinically affected birds intended for breeding, in order to prevent further spreading of the disease.

2 CASE HISTORY
A severe outbreak of PDD occurred in year 2004 in chicks of a psittacine breeding farm in Israel, about one year after the introduction of a blue and yellow macaw (Ara ararauna) breeding pair to the farm. Most of the chicks became sick at 10-14 wks, one chick at 30 wks. Distribution of morbidity and mortality of the various species is presented in Table 1.

Table 1. Distribution of morbidity and mortality of various species is a breeding

farm in which PDD occurred. Species Grey parrot Blue and yellow macaw Citron-crested cockatoo Galah Eclectus N 42 8 6 8 5 Healthy 3* 0 4 7 3 17 Sick birds euthanased 17 1 0 0 0 18 Died 22 7 2 1 2 34 %Affected (morbidity & mortality) 93 100 33 12.5 40 75

Total 69 * Recovered after treatment.

In most cases the disease lasted for 2-10 days before death. The clinical signs were: stasis of the crop; refusal to eat pelleted food; towards weaning time the chicks refused to eat spontaneously and even refused to accept the usual amount of forcefeeding; growth retardation and in most cases also regurgitation. Three of the chicks also had central nervous signs (torticollis), and one chick had blood in the faeces and in the regurgitation content. One bird revealed severe respiratory distress. In post mortem, all birds presented dilatation of the proventriculus, with histological conrmation of PDD in 12 out of 19 cases (63%). All sick chicks except 3 were euthanased. The three remaining chicks were treated for 1 month with 10 mg per day of celecoxib (CELCOX 100), which belongs to a new family of non-steroidal antiinammatory drugs (NSAIDs) (DAHLHAUSEN and ALDRED 2004), and were left for follow up.

255

Several species remained free of any clinical signs. These included: Indian ringneck parakeet (n=20), Jardines parrot (n=6), Pionus spp (n=6), black-headed caique (n=5), sun conure (n=3), and lory (n=2). The macaws that had been suspected as the origin of this outbreak of PDD were transferred to a non-breeding aviary. All premises in which the affected birds were kept were disinfected by formaldehyde evaporation.

3 PATHOLOGY
Nineteen of the dead birds were necropsied (14 grey parrots, 3 blue and yellow macaws and 2 citron-crested cockatoos). Dilatation of the proventriculus was observed in all necropsied birds. The diagnosis of PDD was conrmed in 12 of the birds following a histopathological examination of the brain, proventriculus or crop (at least in one of these organs). From these birds, segments of 1 cm2 were cut from crop and proventriculus and were xed in a 10% formaldehyde solution, together with a brain sample, and were stained with hematoxylin and eosin. The typical microscopic lesions were multifocal lymphohistiocytic inltration with gliosis in brain and lymphohistiocytic inltration in ganglia of the muscularis externa of the proventriculus and/or the crop. The histological changes in the examined birds are summarized in Table 2. Table 2. Summary of histological changes in 19 birds with clinical PDD. Signs of clinical PDD 19 PDD in gross pathology 19 Histological changes in brain 10 Histological changes in proventriculus 10 Histological changes in crop 4

Differential diagnosis: To eliminate other diseases that may be involved in nervous signs, tests for Newcastle disease, avian inuenza, West Nile disease (serology), bacterial and mycotic agents in brain, and Chlamydophila psittaci (serology and immunouorescence) were carried; the rest results were all negative. In addition, the crops were examined for fungi, and in 3 of them, Candida albicans was isolated.

CITATION INDEX
1. 2. 3. GERLACH H. Macaw wasting disease. A 4 year study on clinical case history, epizootiology, analysis of species, diagnosis and virological results. Proc Euro Assoc Avian Vet, Vienna 1991; 273 - 81. GREGORY CR. Proventricular dilatation disease. In: RITCHIE BW (ed): Avian Viruses Function And Control. Lake Worth: Wingers Pub 1995; 444 - 445. DAHLHAUSEN B and ALDRED S. (2004); Resolution of clinical proventricular dilatation disease by cyclo-oxigenase 2 inhibition.t

AUTHORS ADDRESS

U. Bendheim, DVM, DIP ECAMS P.O.Box 196, Shavey Zion 25227, Israel Email: dr-uri@012.net.il

256

Veterinary practitioner1, Parque Zoologico de Barcelona2, Barcelona, Maragall ExoticsVeterinary Centre3, Barcelona, Departamento de Medicina y Cirugia3, Facultad de Veterinaria, Universidad Autonoma de Barcelona, Barcelona, Spain

MYENTERIC GANGLIONEURITIS IN FOUR NON-PSITTACINE BIRDS

D. Perpin1, LV, MS, H. Fernndez-Bellon2, LV, MS, C. Lpez3, LV, A. Ramis, LV, PhD, Dipl ECVP

KEYWORDS
Birds - Passeriformes - Piciformes - Myenteric ganglioneuritis - Proventricular dilatation disease

ABSTRACT
A clinical syndrome similar to proventricular dilatation disease of psittacines, and characterized histologically by lymphoplasmacitic inltrates of the myenteric plexus of the gastrointestinal tract, is described in 4 species of non-psittacine birds (3 of the order Passeriformes and 1 of the order Piciformes).

1 INTRODUCTION
Proventricular dilatation disease (PDD) is a condition of psittacine birds characterised histologically by an inammatory response with accumulation of lymphocytes and plasma cells in the nervous system, especially the nerves that supply the muscles in the proventriculus and other digestive organs including crop, ventriculus and small intestine (RITCHIE et al. 1998). The most common clinical signs of PDD include depression, weight loss, regurgitation, passage of indigested seeds on the faeces and/or central nervous system signs (RITCHIE et al. 1998). The disease has also been called neuropathic gastric dilatation, myenteric ganglioneuritis, and splachnic neuropathy (SCHMIDT et al. 2003). Although this syndrome is typical of psittacine birds, suggestive lesions compatible with PDD have also been reported in toucans, honey-creepers, canaries, weaver nches, red-tailed hawks, Canada geese and rosette spoonbills (RITCHIE et al. 1998, DAOUST et al. 1991, SCHMIDT et al. 2003). This paper describes 4 non-related cases of a PDD-like syndrome in 3 species of Passeriformes (a canary Serinus canaria, a chafnch Fringilla coelebs, and an Amazon umbrella bird Cephalopterus ornatus) and 1 species of Piciformes (a bearded barbet Lybius dubius).

257

2 CLINICAL CASES
Two of the birds, the bearded barbet and the Amazon umbrella bird, were housed at the Barcelona Zoo, and the canary and the chafnch were kept as pets and were presented to the Maragall Exotics Veterinary Centre in Barcelona, Spain. The bearded barbet was found dead in its enclosure, without previous signs, and the other animals showed varied clinical signs before their death, including polyphagia, weight loss, weakness, and/or ataxia. Gross necropsy ndings were variable and included emaciation, hepatic and renal atrophy or enlargement, gallbladder dilatation, and intestinal and ventricular dilatation. Histologically, all 4 cases presented multifocal lymphoplasmacytic myenteric ganglioneuritis. The intensity of the lesion, as well as the degree of involvement of different GI sections, varied for each case. Cardiac lymphoplasmacytic ganglioneuritis was also observed in all cases.

3 DISCUSSION
Proventricular dilatation disease (PDD) was rst described in the late 1970s. Initially, the disease seemed to be limited to macaws. However, since then PDD has been reported in more than 50 species of Psittaciformes (GREGORY et al. 1994, GREGORY et al. 2000). Although suggestive lesions of PDD in some non-psittacine species have also been orally communicated (GREGORY et al. 2000, SCHMIDT et al. 2003), there is only one published article that reports a condition similar to PDD in 2 Canada geese (DAOUST et al. 1991). In the present paper we describe 4 non-related cases that clinically, macroscopically, and microscopically are similar to psittacine PDD, since they combine gastrointestinal signs and lesions, and the characteristic myenteric ganglioneuritis. Three of the species affected (canary, chafnch and Amazonian umbrella bird) were Passeriformes and the fourth (bearded barbet) was a member of the family Piciformes. The description of myenteric ganglioneuritis (or PDD-like syndrome) in multiple families of birds may suggest that its cause is not restricted to a particular host (GREGORY et al. 2000). Although myenteric ganglioneuritis can show a range of clinical signs, this condition must be suspected in birds with wasting disease or crop problems, especially when the results of crop cytology are unrewarding. Although specic treatment was not attempted in the present cases, cyclooxigenase-2 inhibitors have been used successfully in psittacine birds with PDD (DALHAUSEN et al. 2002) and their use should be also considered in non-psittacine birds.

4 CITATION INDEX
1. 2. DALHAUSEN B, ALDRED S and COLAIZZI, E. Resolution of clinical proventricular dilatation disease by cyclooxigenase 2 inhibition. Proc Assoc Avian Vet, Monterey 2002: 9 - 12. DAOUST P-Y, JULIAN RJ, YASON CV and ARTSOB H. Proventricular impaction associated with nonsuppurative encephalomyelitis and ganglioneuritis in two Canada geese. J Wildl Dis 1991; 27(3): 513 - 517.

258

3. 4. 5. 6.

GREGORY CR, LATIMER KS, NIAGRO FD, et al. A review of proventricular dilatation syndrome. J Assoc Avian Vet 1994; 8(2): 69 - 75. GREGORY CR, RITCHIE BW, LATIMER KS, et al. Progress in understanding proventricular dilatation disease. Proc Assoc Avian Vet, Portland 2000: 269 - 275. RITCHIE BW, GREGORY CR, LATIMER KS, et al. Progress in preventing PDD, Polyomavirus, and PBFD virus. Proc Assoc Avian Vet, St Paul 1998: 25 - 40. SCHMIDT RE, REAVILL DR and PHALEN DN. Gastrointestinal system and pancreas. In: Pathology of pet and aviary birds 2003, Iowa: Iowa State Press: 41 - 65.

AUTHORS ADDRESS
David Perpin C/ Balmes, 17, 08918 Badalona (Barcelona), Spain Email: dperpinan@yahoo.es

259

Service de Parasitologie-Mycologie1, UMR BIPAR, Ecole Nationale Vtrinaire dAlfort, Maisons-Alfort; Centre de Sauvegarde2; Ecole Nationale Vtrinaire de Nantes3; Service de Pasitologie, Facult de Mdicine de Crteil, France

EVALUATION OF THE SEROLOGICAL TEST PLATELIA ASPERGILLUS FOR THE DIAGNOSIS OF ASPERGILLOSIS
G. Le Loch1, M. Deville1, E. Risi2, S. Bretagne3, J. Guillot1

KEYWORDS
Serology - Serological - Test - Platelia - Aspergillus fumigatus - Aspergillosis

ABSTRACT
Aspergillosis remains a major cause of mortality in birds, especially in psittacine, raptors and aquatic birds in captivity. Avian aspergillosis usually presents fairly nonspecically with inappetance, depression, dyspnoea, cyanosis or sudden death without any clinical signs. The objective of the present study was to evaluate the accuracy of the serological test Platelia Aspergillus for diagnosis and follow up of cases of aspergillosis in different avian species. This test is based on the detection of fungal antigen (galactomannan) by sandwich ELISA. We conducted a prospective study during the years 2003 and 2004. A total number of 141 serum samples from 33 different species were collected from privately-owned psittacines or wild birds at two wildlife refuges in France: Nantes and Alfort. For all these birds aspergillosis was suspected on the basis of clinical signs and/or radiographical examination. Using the Platelia Aspergillus kit, 38 avian sera were considered positive for antigenemia analysis. In 14 out of these sera, the presence of precipitating antibodies to Aspergillus spp. was detected by counter-immunoelectrophoresis. This study clearly indicated that the detection of Aspergillus antigens should not be considered as a valuable test for the diagnosis of aspergillosis in birds.

1 INTRODUCTION
Aspergillosis is a common and life threatening disease in wild and domestic birds, especially in birds of prey, psittacine and waterfowl. The condition is caused by saprophyte fungi of the genus Aspergillus, with Aspergillus fumigatus being the predominant species involved. Usually the disease does not occur when the bird is healthy and exposed to a low concentration of fungal conidia. On the other hand, under certain circumstances (e.g. increase of fungal exposure, weakened immunity)

260

Aspergillus can develop in the body and lead to non-specic clinical signs. Sometimes, the bird dies before any clinical signs appear (BAUCK 1994, GRACZYK et al. 1998, JONES and OROSZ 2000, KAWAZU et al. 2004, LATGE et al. 1994, MENNINKKERSTEN et al. 2004, OGLESBEE 1997, OROSZ 2000). The diagnosis of avian aspergillosis remains a challenge for veterinarians. Clinical signs are diverse and often late in becoming evident. History and clinical examination of the bird, combined with radiography, endoscopy and a complete blood count, provide clues to the presence of lesions due to Aspergillus. The detection of Aspergillus is achieved by cytology, histology and culture, but these tests require the anaesthesia of the bird, and the necessary handling can threaten the life of a bird under respiratory distress (KAWADU et al. 2004). The treatment of aspergillosis is difcult and often not efcient because the diagnosis is made too late. It has recently been shown that the use of serological testing could resolve this problem. Serological testing for the anti-Aspergillus antibodies assay is used and usually allows an efcient and early diagnosis. An indirect ELISA has been developed specically for birds in raptor centers. This ELISA is not available in France, where veterinarians use electrosyneresis to detect precipitant antibodies. Moreover, studies for the detection of Aspergillus DNA have been made and are promising (PHALEN 2001, REDIG 1993). In human medicine, aspergillosis is a major disease in haematology and oncology care units. Diagnosis must be made early to allow efcient treatment. Galactomannan (gure 1), a polysaccharide composing the cell wall of Aspergillus fumigatus, is the antigen whose detection by ELISA (Platelia Aspergillus) allows diagnosis of invasive aspergillosis in humans. A comparative study in humans has showed that the sensitivity of ELISA Platelia Aspergillus is higher than the sensitivity of a PCR test (test GeniQ-Asper) (100% and 55%, respectively) and that specicity is the same (93%) (LATGE et al. 1994, MENNINK-KERSTEN et al. 2004). We recently conducted a feasibility study on birds to assess the efciency of Platelia Aspergillus for the diagnosis of aspergillosis.

2 MATERIALS AND METHODS

One hundred and twenty-three sera collected from 116 birds were sent to the Parasitology department at the veterinary college of Alfort (ENVA) by veterinarians or wildlife care centers. The sera were collected either because birds showed clinical signs of aspergillosis, or because the results of complementary tests suggested the presence of aspergillosis. Some serum samples were also collected because the birds situation rendered it vulnerable to aspergillosis: e.g. young birds recently purchased, wild birds held in wildlife centers, birds exposed to excessive stress, etc. The serum was separated by centrifugation at 1000g for ve minutes. Stored in 1.5ml Eppendorf tubes, the sera were conserved at 4C until the moment they underwent electrosyneresis (a few days). After electrosyneresis, the sera were frozen at -20C

261

up to the moment of undergoing the ELISA test. Each serum sample was subject of a serological test to detect anti-Aspergillus antibodies (electrosyneresis), as well as a test to detect galactomannan (Platelia Aspergillus test).

Fig 1: structure of galactomannan (MENNINK-KERSTEN et al. 2004) For electrosyneresis, 30l of each antigen and 15l of sera to be tested were placed in the presence of an electric eld on cellulose acetate strips, on which they migrated towards the anode and cathode, respectively. The positive control serum came from a dog in which aspergillosis was conrmed. Contact between antigens and antibodies leads to the formation of one or several precipitation arcs, visible after staining. Two types of antigens were used in the electrosyneresis: metabolic and somatic Aspergillus fumigatus antigens (Bio-Rad). The number and intensity of the precipitation arcs varied as a function of the concentration of precipitant antibodies. A serum sample is considered to be positive when it forms at least one clear precipitation arc with at least one of the two types of antigens. The Platelia Aspergillus test is an immuno-enzymatic assay directly on a solid phase, called an ELISA sandwich, which allows the semi-quantitative detection of galactomannan circulating in the serum. This test has a positive threshold xed at 1ng/ ml of serum tested. An IgM antibody was used. This is a monoclonal antibody, EBA-2, which recognizes the (BAUCK 1994, GRACZYK et al. 1998, JONES and OROSZ 2000, KAWAZU et al. 2004, LATGE et al., 1994) galactomannan galactofurans. It has a double use because it is xed at the bottom of the microtitre wells, and, when marked with peroxidase, it is used as a conjugate for the detection of antigens. The ELISA test was conducted using the Platelia Aspergillus Bio-Rad kit instructions (GRACZYK et al. 1998). These instructions advise using 300l of sera, but considering the small volumes of the sera we collected, we used only 100l. The rst step in the reaction consists of treating the sera in order to free the circulating immuno-

262

complexes and to precipitate the proteins that can interfere during the immunoenzymatic reaction. To achieve this, 100l of each serum is vigorously mixed with 33l of an EDTA acid solution (ethylene dinitrolo tetra acetic acid), heated at 100C for three minutes, and then centrifuged for 10 min at 10,000g. Four control sera supplied with the kit were prepared in the same manner. They consist of one negative control serum, two threshold sera (1 ng/ml of galactomannan) and one positive control serum (10ng/ml of galactomannan). Once the sera were prepared, the microtitre wells (in which the EBA-2 antibodies are xed) were lled with 50l of conjugate (EBA-2 antibodies coupled with peroxidase), then with 50l of supernatant for each serum treated. The support plate was then incubated at 37C for 90 minutes. In the presence of antigens, complexes of antibodyantigen-antibody + peroxidase form. Following incubation, the plates were washed ve times with a TRIS-NaCl buffer solution. The peroxidase enzymatic activity was revealed by the addition of 200l of dimethylsulfoxide (DMSO) in each well. The reaction was ended after 30 minutes by the adding of 100l of a sulfuric acid stopping solution to the wells. The readings were taken by a spectrophotometer at a wavelength of 620nm. For each well, an optical density (OD) was determined. On determining a threshold value (TV) corresponding to the average OD of the wells containing the threshold serum (R4), an index (I) was calculated for each serum tested as follows: I = OD sample / TV The aim of this calculation is to limit the variations in the inter-trial and inter-laboratory OD. The test is validated if the following criteria are respected: 0.3 TV 0.8 I (R5) > 2.0 I (R3) < 0.5 The results can then be interpreted as follows: I < 1.0: serum negative for the presence of galactomannan 1.0 I < 1.5: serum intermediate for the presence of galactomannan I 1.5: serum positive for the presence of galactomannan

3 RESULTS AND DISCUSSION

Among the 116 birds from which serum samples was collected, 38 were sampled by simple screening i.e. the birds showed no clinical signs. Thirty species were represented, with the majority being Psittacidae (17 species), 6 raptor species, and 7 species belonging to various taxonomic families. Of those birds for which an age could be determined, there were 39 birds aged one year or less, and 38 birds older than one year.

263

Of the 123 serum samples tested, 35 gave a positive Platelia Aspergillus result (gure 2), and for at least one of these birds (a gannet - Sula bassana), aspergillosis was conrmed by culture and isolation of Aspergillus fumigatus from a pulmonary granuloma. The average index of the 35 positive serum samples was 3.39 (approximately 11.2ng/ml of galactomannan) and 18 of these sera had an index above 3.5 (15.2ng/ml of galactomannan). As a consequence, when galactomannan is present in a bird serum, it is present in high concentrations. We can suppose that the immune system, insofar as it remains competent, allows the bird to maintain a relatively low level of antigens. But as soon as it is surpassed, immuno-suppression becomes very important and the circulating level of galactomannan rises sharply. Fifty-six per cent of the birds whose index was higher than 3.5 were in poor general condition. Conversely, only 18 per cent of the birds whose index was between 1.5 and 3.5 were in poor general condition.
55 50 45 40

Number of sera

35 30 25 20 15 10 5 0 I < 0,5 1,5 > I 1 2,5 > I 2 3,5 > I 3 4,5 > I 4 5,5 > I 5 6,5 > I 6 I6,5

1>I 0,5

2>I 1,5

3>I 2,5

4>I 3,5

5>I 4,5

Index

Figure 2 : distribution of sera according to their index According to clinical signs and electrosyneresis results the birds were categorized into three groups: Group A (aspergillosis considered as highly probable) is made up of birds giving a positive electrosyneresis result and showing symptoms compatible with aspergillosis; Group B (presumptive aspergillosis) comprised birds showing only one of the two characteristics shown by group A; and Group C (aspergillosis considered as unlikely) comprised birds showing a negative electrosyneresis result and no clinical signs. Considering birds of group A as infected and birds of group C as healthy, we were able to calculate the sensitivity and specicity of the Platelia Aspergillus test: 30% and 86%, respectively. Given the low sensitivity of the test in

264

6>I 5,5

the present study, the usefulness of this test for diagnosing aspergillosis in birds, when used alone, appears to be limited. The Platelia Aspergillus test is capable of detecting galactomannan at a concentration of 1ng/ml. It thus seems that the circulating concentrations of galactomannan in birds infected by Aspergillus are frequently lower than 1ng/ml. This could be explained partly by the low vascularisation of certain infected organs such as the air sacs. On the other hand, only a few false positive results occurred with the Platelia Aspergillus test and the specicity (86%) and the positive predictive value (75%) of the test were high. In humans, false positive results have been explained by positive reactions of ELISA with the lipoteic acid in Bidobacterium. Furthermore, moulds of the genera Penicillium, Alternaria, and Paecilomyces all contain galactomannan, which can give a positive result on the ELISA test. Some of these fungi are occasionally found in birds. The Platelia Aspergillus test is thus relevant for the detection of circulating galactomannan, but not for the diagnosis of avian aspergillosis. Recent study demonstrated that a low antigenemia level may be common in healthy birds (JONES and OROSZ 2000, OGLESBEE 1997). The Platelia test could be useful in the detection of avian aspergillosis before the onset of clinical signs, since eight birds out of the 38 tested by random screening (i.e. without any signs of the infection) showed a positive result for the Platelia Aspergillus test. It would be interesting to know if these birds develop aspergillosis later on, as well as the time between galactomannan detection and the appearance of the rst clinical signs. Finally, we observed that birds showing signs of invasive aspergillosis yielded a positive result for the Platelia Aspergillus test more frequently (45%) than those showing only signs of respiratory aspergillosis (23%). We may thus presume that the Platelia Aspergillus test is better advised for the diagnosis of invasive aspergillosis than for localized forms of the infection. An investigation with conrmed cases of invasive aspergillosis would be necessary to conrm the usefulness and the performance of the Platelia Aspergillus test in these specic cases.

4 CITATION INDEX
1. 2. 3. 4. BAUCK L. Mycoses. In: RITCHIE BW, HARRISON GJ and HARRISON LR (eds): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing 1994, 997 - 1006. GRACZYK TK, CRANFIELD MR and KLEIN PN. Value of antigen and antibody detection, and blood evaluation parameters in diagnosis of avian invasive aspergillosis. Mycopathologia, 1998, 140; 121 - 127. JONES MP and OROSZ SE. The diagnostic of aspergillosis in birds. Sem Avian Exot Pet Med 2000, 9(2); 52 - 58. KAWAZU M, KANDA Y, NANNYA Y, et al. Prospective comparison of the diagnostic potential of real-time PCR, double-sandwich enzyme-linked immunosorbent assay for galactomannan, and a (13)--D-Glucan test in weekly screening for invasive aspergillosis in patients with hematological disorders. J Clin Micro 2004, 42; 2733 - 2741.

265

5.

LATGE JP, KOBAYASHI H, DEBEAUPUIS JP, et al. Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus. Infection and Immunity 1994, 62; 5424 - 5433. 6. MENNINK-KERSTEN M, KLONT R, WARRIS A, et al. Bidobacterium lipoteichoic acid and false ELISA reactivity in Aspergillus antigen detection. The Lancet 2004, 363; 325 - 327. 7. OGLESBEE BL. Mycotic diseases. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM, et al. (eds): Avian medicine and surgery. Philadelphia: WB Saunders 1997, 323 - 331. 8. OROSZ SE. Overview of aspergillosis: pathogenesis and treatment options. Sem Avian Exot Pet Med 2000, 9(2); 59 - 65. 9. PHALEN DN. The use of serologic assays in avian medicine. Sem Avian Exot Pet Med 2001, 10(2); 77 - 89. 10. REDIG PT. Avian aspergillosis. In: FOWLER ME (ed): Zoo and Wild Animal Medicine Current Therapy, 3th ed. Philadelphia: WB Saunders 1993, 178 - 181.

AUTHORS ADDRESS
G Le Loch Service de Parasitologie-Mycologie, UMR BIPAR, Ecole Nationale Vtrinaire dAlfort, Maisons-Alfort, France Email: guillaume_ll@hotmail.com

266

Great Western Referrals1, Swindon, Strathmore Veterinary Clinic2, Andover, TDDS3, Exeter, United Kingdom

ASSESSMENT OF DISC DIFFUSION TESTS TO EVALUATE THE EFFECTS OF DIFFERENT NEBULISATION AGENTS AGAINST BACTERIA ISOLATED FROM THE AVIAN TRACHEA
D. Monks1 BVSc (Hons) MACVSc (Avian Health) CertZooMed MRCVS, P. Zsivanovits1 DMedVet MRCVS, J. Chitty2 BVetMed CertZooMed MRCVS, C. Fisk3 BSc (Hons), N. Forbes1 BVetMed CBiol MIBiol DipECAMS FRCVS

KEYWORDS
Disc diffusion test - Nebulisation - Trachea - Bacterial ora

ABSTRACT
Nebulisation therapy is widely used in avian medicine for the treatment of respiratory tract infections. Although there are published dose rates of commonly nebulised agents, the efcacy of those dilutions has not been tested against common avian tracheal isolates. Disc diffusion testing is a technique with which to assess the antibacterial efcacy of varying agents on isolated organisms. This study aimed to establish a method of in-house disc diffusion testing for specic concentrations of nebulising agents and to evaluate the antibacterial efcacy, at recommended dilutions, of three commonly used antibacterial nebulising agents. These agents were enrooxacin, gentamicin and F10, a disinfectant containing quaternary ammonium and biguanide compounds, tetrasodium ethylene diamine tetraacetic acid (EDTA) and non-toxic ampholytic surfactants. Tracheal swabs were taken from fty-three birds, incorporating healthy and unhealthy raptors and psittacine birds. Culture and sensitivity tests were performed, using discs containing 0.5mg enrooxacin, 0.25mg gentamicin and F10 at dilutions of 1:250 (0.4%), 1:125 (0.8%), 1:62.5 (1.6%). Thirtyseven bacterial isolates were cultured from the raptors, of which 22 (59.5%) were Gram positive while 15 (40.5%) were Gram negative. Thirty-ve isolates were cultured from the psittaciformes, of which 14 (40%) were Gram positive and 21 (60%) were Gram negative. The most common isolates for both groups of birds were coliforms (both lactose and non-lactose fermenting), Enterococcus and Staphylococcus species. Pseudomonas species were recovered from ve birds. Only Enterococci and a single Staphylococcus showed resistance to either enrooxacin or gentamicin with the methodology used in this study. The F10 results were highly variable and the methodology did not appear transferable to this agent.

267

1 INTRODUCTION
Nebulisation therapy is widely used in avian medicine for the treatment of respiratory tract infections. It offers a method of topical administration of antimicrobial agents, which may achieve better concentrations than parenteral antimicrobials in organs with poor perfusion, such as air sacs. Potentially nephrotoxic agents such as gentamicin can be used safely due to limited drug absorption across respiratory membranes. Nebulisation minimises patient stress by reducing patient handling, increases hydration of respiratory epithelium and may increase dissolution of necrotic debris. Although there are published dose rates of commonly nebulised antimicrobial agents, the efcacy of those dilutions has not been tested against common tracheal isolates. Enrooxacin is a bactericidal, broad spectrum uoroquinolone antibiotic that inhibits bacterial DNA-gyrase. Activity is concentration dependent and the spectrum of action includes a wide range of Gram negative bacteria, some Gram positive aerobes and organisms such as Mycoplasma and Chlamydophila (KROKER 1997). Gentamicin, an aminoglycoside antibiotic, acts by interference with bacterial protein synthesis and is bactericidal. Aminoglycosides are generally active against Gram negative aerobes including Enterobacteriaceae and Pseudomonas (KROKER 1997). The active constituents of F101 Super Concentrate Disinfectant (F10) include quaternary ammonium and biguanide compounds, tetrasodium ethylene diamine tetraacetic acid (EDTA) and non-toxic ampholytic surfactants, with the exact formulary remaining a commercial secret. F10 is said to be virucidal, bactericidal, fungicidal and sporicidal (VERWOERD 2001). Biguanide compounds and quaternary ammonium compounds act via interference with cytoplasmic membrane function, causing, amongst other actions, coagulation of cytoplasmic constituents. Additionally, quaternary ammonium compounds cause increased permeability of the outer member in Gram negative bacteria (MAILLARD 2002). EDTA disrupts cell wall integrity by chelating metal ions (FOSTER and DEBOER 1998). Aerosol therapy with F10 has been successfully used in cases of lower respiratory tract infections at a dilution of 1:250 and is well tolerated by patients (CHITTY 2002). The aim of this study was to demonstrate the tracheal bacterial ora in healthy and unhealthy raptors and psittacine birds, to trial disc diffusion testing as a practical means of assessing the efcacy of commonly used nebulising agents (enrooxacin, gentamicin and F10) in an in-house setting and to evaluate the efcacy of these agents at recommended concentrations.

2 MATERIALS AND METHODS

Blank discs were purchased from Tekia2 and sterilised in an autoclave. Various concentrations of enrooxacin, gentamicin and F10 were prepared using sterile water as diluent. Food dye was added to facilitate identication of each disc type. The solutions were then placed onto the discs in a sterile fashion (0.05ml of solution per disc) and the discs were left to dry before being sent to the laboratoryc. A preliminary test was conducted in which the discs were prepared as above, but lacking food dye and using commercial concentrations of both enrooxacin (5ug) and gentamicin

268

(10ug). Disc diffusion testing was performed simultaneously with the experimental and commercial discs and the zones of inhibition were comparable. New discs with added food dye were also tested, with no appreciable difference in effect. Discs were then prepared using solutions of enrooxacin (10mg/mL), gentamicin (5mg/ml) and F10 (1:250) as above and sent to the TDDS laboratoryc. This gave disc concentrations of 0.5mg, 0.25mg and 0.4% for enrooxacin, gentamicin and F101, respectively. Some samples were subsequently tested with concentrations of F10 (1: 125, 0.8%; 1:62.5, 1.6%). Additionally, a lter paper method of F10 testing was used. This involved placing a strip of lter paper over the inoculated agar plate, and wetting it with F10 at identical concentrations to those used in the disc testing. Bacterial growth around and immediately under and over the paper strip was assessed after incubation. Tracheal samples were collected on an opportunistic basis at two veterinary centresa,b. These were taken after mask induction with oxygen isourane, using commercial culturettes and then sent in transport media to the TDDS laboratoryc. All solid and liquid media used in the laboratory was supplied by Oxoid Limited3. Swabs were initially plated onto two 5% columbia whole debrinated columbia horse blood agar plates, one CLED (cystine lactose electrolyte decient) agar plate, one Staphylococcus/ Streptococcus selective agar plate and a section of Sabourauds agar. Blood agar was used to estimate total bacterial population, CLED agar was used to identify lactosefermenting isolates and Sabourauds agar was used for the isolation of yeast. One of the horse blood agar plates was then incubated under anaerobic conditions and the other four plates were incubated in air, at 370C for a maximum of 48 hours. The plates were then examined and colony types were recorded. Further basic identication tests such as Gram stain, oxidase and coagulase were carried out to conrm genus and species. Sensitivity testing was performed by selecting several colonies of a particular species and emulsifying them in 5ml quarter strength ringer solution. A sterile swab was used to inoculate dried isosensitest plates3 with the ringer solution, and impregnated discs or lter paper were applied to the surface using sterile forceps. Sterile water was used on the lter strips as control. Plates were incubated overnight at 370C, and then examined for growth. Zones of inhibition around the discs were measured and organisms designated sensitive or resistant according to laboratory protocol. Initially, organisms were only designated as sensitive or resistant, whereas later in the study, the zones of inhibition diameters were reported in millimetres.

3 RESULTS
Fifty-three birds were sampled, of which ve samples did not culture. Of the remaining 48 samples, 19 (39.6%) originated from raptors and 29 (60.4%) from psittacine birds. Table 1 shows the distribution of organisms recovered from both raptors and psittaciformes. The most common isolates are shown in Table 2. The coliform group included lactose-fermenting coliform species (not identied further) and Klebsiella species. Of the 19 raptors, four (21.1%) were reported as healthy. Of the 29 psittaciformes, ten (34.5%) were reported as healthy. The four isolates from healthy raptors were

269

Enterococcus species (one moderate and two heavy growth), Klebsiella species (one heavy growth), Staphylococcus species (moderate growth) and a coliform (heavy growth). The six isolates from healthy psittaciformes were Candida (three scanty growths), coliforms (one scanty growth, three moderate growth) and a Streptococcus species (one very scanty growth). Nine birds in total had signs of respiratory disease (psittaciforme 17.2%; 5/29: raptors 21.1%; 4/19). Enterococcus species were isolated from six psittaciformes and twelve raptors. Of those birds, three psittaciformes (50%) and three raptors (25%) showed signs of respiratory disease. Coliforms were isolated from in 16 psittaciformes and seven raptors, of which three (18.8%) and two birds (28.6%), respectively, showed signs of respiratory disease. Staphylococcus species were isolated from six psittaciformes and ten raptors. Of those birds, two psittaciformes (33.3%) and two raptors (20%) showed signs of respiratory disease. Table 3 shows the distribution of resistance against enrooxacin and gentamicin amongst the isolates obtained. Some isolates were resistant only to enrooxacin or gentamicin while some were resistant to both. Enterococcus species accounted for the majority of the resistant isolates, with 14 of the 18 (77.8%) Enterococcus isolates showing resistance to at least one antibiotic. One psittacine Staphylococcus isolate was resistant to both enrooxacin and gentamicin. There was signicant variability in the F10 results, between different isolates, different disc concentrations and discs versus lter strips. No distinct trends were noted for any variable. There are no guidelines for the determination of appropriate zones of resistance and susceptibility for F10 using disc diffusion test methods. A total of eight yeast were isolated; two from raptors and six from psittacine birds. As expected, all seven isolates were resistant to enrooxacin and gentamicin. Again, the zones of inhibition for F10 were variable and unreliable for all seven isolates.

4 DISCUSSION
Although the total distribution of Gram positive and Gram negative bacteria was equal (36/72, 50% each), there were differences in distribution of raptors and psittaciformes. Psittaciforme birds had Gram negative bacteria comprising over half (60%, 21/35) of the total isolates, whereas 40.5% (15/37) of the bacteria isolated from raptors were Gram negative. The majority of birds in our study were unwell, which may have biased the results. The expected tracheal ora of healthy raptors and psittaciformes is not well-documented. In agreement with previous studies on unhealthy birds, our study found coliforms, Enterococcus, Pseudomonas and Staphylococcus species as common tracheal isolates (MARTEL et al. 2004). Importantly, though, these bacteria were also isolated from birds that did not show evidence of respiratory disease. In fact, coliforms were isolated from four healthy psittaciformes and two healthy raptors while Enterococcus species were isolated from three healthy raptors. Enterococcus species are generally considered to be of low pathogenicity and can form part of the normal ora of some mucosal surfaces. Enterococci commonly

270

exhibit antibiotic resistance, and sensitivity testing is considered clinically important (SELBITZ 1992). Interestingly, Enterococcus species accounted for all but one of the resistant isolates in our trial. Only 3/18 (16.7%) Enterococci were sensitive to both enrooxacin and gentamicin. Another Gram positive coccus, Staphylococcus was the other resistant isolate. Given this pattern, the presence of Gram positive cocci on tracheal cytology may be a useful marker of potential resistance. It is important to note that the presence of these bacteria was not pathognomonic for clinical signs of respiratory disease in our study. Enrooxacin and gentamicin discs were produced at concentrations used for nebulisation. By that, the actual concentrations of the enrooxacin and gentamicin discs were 100 and 25 times more concentrated than commercial discs, respectively. This contrasts to principles of zone inhibition testing, which generally use the lowest concentration that is still effective. Furthermore, the zones of inhibition were measured from the edge of the disc to the edge of the zone in the current study, while generally zones of inhibition are measured across the diameter of the inhibition zone and the disc. The presence of resistance is interesting, given the much larger concentration of drugs in the in-house discs (0.5mg enrooxacin and 0.25mg gentamicin) compared to the commercial discs (5ug enrooxacin, 10ug gentamicin). None of the Gram negative isolates in our study, including Pseudomonas, showed resistance to enrooxacin or gentamicin. The F10 disc diffusion and lter paper methods of testing generated highly variable results. The biocidal label claims of F10 against bacteria have been approved and validated by testing agencies in a number of countries (http://www. healthandhygiene.co.za/product_details.asp?ContentId=68&FormDocumentsLi st_Page=3#DocumentsList) Testing usually involves determination the minimal inhibition concentration (MIC) of an agent, addition of the known concentration into a broth of known antimicrobial concentration, then inoculation of agar plates with the broth after a set incubation time. That is, the organism is suspended in aqueous solution with the biocide. To the authors knowledge, there is no published information about impregnation of quaternary ammonium or biguanide compounds into discs for antibiotic sensitivity testing. Studies investigating the use of EDTA-impregnated discs reported conicting results with poor appearance and reproducibility in several strains of bacteria (ARAKAWA et al. 1999; YONG et al. 2002). However, unpublished data using agar diffusion techniques (with the bacteria incorporated into the agar itself) as well as wet discs (using the disc diffusion method) has given less variable results (J. Temperley, Elize Lloyd, personal communication). The differences in variability may relate to the use of different disc methodology and the use of agar diffusion versus disc diffusion techniques. An appropriate zone of inhibition for a particular biocide requires correlation of the zone of inhibition with the MIC of that biocide, at known bacterial loads according to standard procedures. However, the aim of this study was to assess the disc diffusion method as commonly used in diverse hospitals and laboratories as an in-house test, using whatever bacteria isolated from the trachea, without known bacteria loads, at the concentration that the tested agents are used for nebulisation. Dry, ready-to-use F10 impregnated discs gave inconsistent results when used in disc diffusion tests. Different disc methodologies and the correlation of MIC with zones of inhibition for F10 are areas worthy of further research.

271

5 ACKNOWLEDGEMENTS
We would like to acknowledge the co-operation we have received from Health and Hygiene (Pty) Ltd and the Microbiology Department of the South African Bureau of Standards.

6 CITATION INDEX
1. 2. 3. 4. ARAKAWA Y, SHIBATA N, SHIBAYAMA K, et al. Convenient test for screening metallo-B-lactamase-producing Gram-negative bacteria by using thiol compounds. J Clinical Microbiol 2000; 38(1): 40 - 43. CHITTY J. A novel disinfectant in psittacine respiratory disease. Proc Assoc Avian Vet, Monterey, California, USA 2002; 25 - 27. FOSTER AP and DEBOER DJ. The role of Pseudomonas in canine ear disease. Comp Cont Ed 1998; 20(8): 909 - 918. KROKER R. Pharmak zur Behandlung und Verhutung bakterieller Infektionen In: LOSCHER W, UNGEMACH FR and KROKER R (eds): Pharmakotherapie bei Haus-und Nutztieren. Parey Buchverlag Berlin, Germany, 1997: 211 246. MARTEL A, RAUE R, VERMINNEN K, et al. Microbial pathogens associated with respiratory disorders in psittacine birds. Proc Assoc Avian Vet, New Orleans 2004: 369 - 371. MAILLARD JY. Bacterial target sites for biocide action. J Applied Microbiol Symp Suppl 2002; 92: 16S - 27S SELBITZ HJ. Lehrbuch der veterinarmedizinischen Bakteriologie. Gustav Fischer Stuttgard, Germany 1992; 171. VERWOERD D. Aerosol use of a novel disinfectant as part of an integrated approach to preventing and treating aspergillosis in falcons in the UAE. Falco 2001; 17: 17 - 18. YONG D, LEE K, YUM JH, et al. Imipenem-EDTA disk method for differentiation of metallo-B-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clinical Microbiol 2000; 240; 10: 3798 - 3801.

5. 6. 7. 8. 9.

AUTHORS ADDRESS
Deborah Monks BVSc (Hons) MACVSc (Avian Health) CertZooMed MRCVS Great Western Referrals, County Park Estate, Shrivenham Road, Swindon, SN1 2NR, United Kingdom Email: d.monks@gwreferrals.co.uk

272

Table 1. Summary of isolated organisms. Yeast Raptors Psittaciformes Total 2 6 8 Gram positive (% of total) 22 (59.5) 14 (40) 36 (50) Bacteria Gram negative (% of total) 15 (40.5) 21 (60) 36 (50) Total isolates 37 35 72

Table 2. Common isolates by taxonomic group. Number % gram isolates + isolates Raptors: Coliforms 7 NA N=22 gram positive Pseudomonas 5 NA (Gram +) N=15 gram negative Enterococcus 12 54.5 (Gram ) Staphylococcus 10 45.5 Psittaciformes: Coliforms 16 NA N=14 gram positive Pseudomonas 1 NA (Gram +) N=21 gram negative Enterococcus 6 42.9 (Gram ) Staphylococcus 6 42.9

% of gram isolates 46.7 33.3 NA NA 76.2 4.8 NA NA

% of total isolates 18.9 13.5 32.4 27.0 45.7 2.9 17.1 17.1

Table 3. Sensitivity spectrum for enrooxacin and gentamicin. Enrooxacin Gentamicin Sensitive Resistant Sensitive Resistant Raptors: Coliforms 7 0 7 0 Pseudomonas 5 0 5 0 Enterococcus 6 6 5 6 Staphylococcus 10 0 9 0 Total Gram 16 6 16 6 Positive Total Gram Negative 15 0 15 0 Psittaciformes: Coliforms 16 0 16 0 Pseudomonas 1 0 1 0 Enterococcus 5 1 3 3 Staphylococcus 5 1* 5 1* Total Gram 12 2 10 4 Positive Total Gram 21 0 21 0 Negative

Total 7 5 11 9 22 5 16 1 6 6 14 21

273

Kimron Veterinary Institute, Bet Dagan, Israel

DIAGNOSES OF ROTAVIRUS IN PET BIRDS VS POULTRY

A. Lublin, DVM, PhD, S. Mechani, DVM, V. Bumbarov, DVM

KEYWORDS
Rotavirus Malabsorption syndrome VP6 - Immunohistochemistry

ABSTRACT
Rotavirus is one of the causes of enteritis in birds. Infection by rotavirus includes growth retardation, lack of uniformity in ock, low food conversion, diarrhoea, anaemia, abnormal feathering and sometimes bone lesions. Immunosuppression and outbreaks of other intestinal pathogens such as Clostridium sp., is common in rotavirus infection. In broilers we found association between rotavirus infection and malabsorption syndrome, while association with reovirus infection, as had been suggested by some researchers, could not be demonstrated. Eight of 18 ocks (mostly broilers) with signs of stunting, undigested food and dilatation of the proventriculus were positive to the major inner capsid protein VP6 of group A-rotavirus by ELISA and by immunohistochemistry, and in few of the cases, also by immuno-electron microscopy-negative staining. Rotavirus have been found especially in intestinal mucosal cells, but also in the inner connective tissue of the mucosa, i.e. lamina propria, and inside intestinal contents. In pet birds, rotavirus is one of the causes to gut dilatation and impaired food absorption. The virus was diagnosed by ELISA only in 3 of 18 suspected birds (two grey parrots and one ring-necked parakeet), but also in two birds without clinical suspicion (grey parrot, cockatoo). It seems that rotavirus is more abundant in stunted poultry than in pet birds with similar signs.

1 INTRODUCTION
Rotavirus is a double-stranded RNA virus of the Reoviridae family with a three-layer capsid 70-80 nm in diameter lacking an external envelope. The virus is one of the causes of enteritis with diarrhoea in birds, as in mammals, but there is no consensus about cross infection between the two groups. Rotavirus invades the intestinal mucosal cells, especially at the edges of the intestinal villi. Viral replication causes lysis of the host cells and impairing of absorption. The main replication site is in the enterocytes of the small intestines, but also in the colon and cecum. Clinical signs of rotavirus infection include growth retardation, lack of uniformity in a ock,

274

low food conversion, diarrhoea, anaemia, abnormal feathering and sometimes bone lesions. Immunosuppression and outbreaks of other intestinal pathogens such as Clostridium sp. or coccidia, are common in rotavirus infection. The virus is excreted in high numbers in faeces and can be transmitted directly and indirectly. The most important portal of entry is by ingestion. Most reports are derived from poultry and less information is known about pet birds. 1.1. Rotavirus in poultry In broilers we found an association between rotavirus infection and malabsorption syndrome, that according various researchers, reovirus either rotavirus, is its main aetiology. The main pathological lesions of the syndrome consist of white-coloured or transparent intestinal walls, enlarged gall bladder, sometimes proventriculitis, pancreatic atrophy, degeneration of the bursa of Fabricius and rickets. Serological surveys of reovirus in ocks were negative in an experimental model in which infected intestinal contents from retarded birds were inoculated into specic pathogen free 1day-old chicks and follow-up for antibody titres in affected birds (as most of the inoculated chicks became). In contrast, in 8 out of 18 ocks (mostly broilers) with signs that may indicate rotavirus, a group A-rotavirus antigen major inner capsid protein VP6, could be detected by ELISA and by immunohistochemistry, with signicant correlation between the two diagnostic methods (chi-square coefcient 4.04, P<0.05). Sixteen tested ocks out of 21 received the same result with both methods. In a few of the cases the virus was also diagnosed by electron microscopy using a negative staining technique, and by immuno-electron microscopy with rotavirus antiserum. Diagnosis of the virus by immunouorescence was also tested, but the results were not satisfactory. One of the advantages in using immunohistochemistry is the ability to locate the virus in tissue (as red colour appears at the sites of antigen localization). Rotavirus was diagnosed particularly in intestinal mucosal cells and to a lesser extent in the inner connective tissue of the mucosa i.e. lamina propria. The virus was also observed in intestinal contents, making the excreted faeces a route of spreading the virus. Clinical disease (retarded growth with or without diarrhoea) was reproduced in 12 inoculation experiments with ltered intestinal contents after direct administration into the crop of specic pathogen free 1-day-old chicks. The syndrome could not be reproduced in chicks older than 2 days. Inoculation of turkey poults did not reproduce the disease. Thus, sensitivity to rotavirus infection is limited to broiler chicks younger than 1-2 days. In testing the various segments of the intestines for rotavirus infection, the highest rate of infection was found in cecum, in 42% of the cases, in comparison to 15% of cases in duodenum and 29% in jejunum-ileum. Rotavirus was also detected by ELISA in poultry manure, on average 37% of the manure. Virus prevalence was relatively high in turkey manure (about 40%), but was found also in manure from broilers, breeders, layers, pullets and organic chickens. The high prevalence in manure is probably due to its accumulation in manure and its high resistance in the environment

275

1.2. Rotavirus in pet birds In pet birds rotavirus is one of the causes of gut dilatation and impaired food absorption, widespread especially in lovebirds and pigeons. Watery diarrhoea may occur in infected pigeons. Some of the strains do not cause clinical symptoms in carrier birds. In pet birds rotavirus is less familiar than in domesticated birds. In a survey conducted in our laboratory, rotavirus was diagnosed by ELISA only in 3 of 18 birds suspected according to clinical and pathological signs: two grey parrots (Psittacus erithacus) with pathological presentation of proventricular dilatation disease (PDD) and a ringnecked parakeet (Psittacula krameri) from a ock with respiratory disturbances and mortality, while necropsy ndings in this case included diarrhoea, proventricular dilatation and undigested food as in other cases. Rotavirus was also detected in two birds without clinical suspicion (grey parrot, cockatoo).

2 CONCLUSIONS
1. 2. 3. Rotavirus is more abundant in stunted poultry than in pet birds with similar signs. In poultry rotavirus is usually associated with a clinical manifestation of diarrhoea/stunting and in pet birds this association is less obvious. More information concerning rotavirus in poultry exists compared with pet birds, therefore more research needs to be done in pet birds.

AUTHORS ADDRESS
A. Lublin, DVM, PhD Division of Avian & Fish Diseases, Kimron Veterinary Institute, POB 12,Bet Dagan 50250, Israel Email: lublina@int.gov.il

276

Institute for Virology1, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Munich2, Germany

PHYLOGENETIC ANALYSIS OF THE HEXON LOOP 1 REGION OF ADENOVIRUS ISOLATED FROM PSITTACINE BIRDS INDICATES THE EXISTENCE OF A NEW PSITTACINE ADENOVIRUS
R. Raue1, H. Gerlach2 and H. Mller1

KEYWORDS
Psittacine adenovirus - PsAdV - PCR - Hexon gene - L1 region

ABSTRACT
Adenovirus infections in psittacine birds have been well known and were mainly caused by fowl adenoviruses (FAdV). Liver samples collected from an outbreak of acute disease in Poicephalus spp., showing morphological and histological signs of an adenovirus infection, were investigated in this study. A PCR amplifying the variable loop 1 region of the hexon gene was developed using primers located in two conserved pedestal regions. A PCR product of approximately 590 bp in size was amplied and sequenced. In a phylogenetical analysis the sequence obtained grouped outside of the FAdV reference strains of all 12 serotypes, egg drop syndrome virus and hemorrhagic enteritis virus, indicating that the sequence originated from a new avian adenovirus. In a comparison to the FAdV reference strains, the percentage of identical nucleotides ranged between 60.3 and 67.0, and that of identical amino acids (aa) between 51.3 and 61.0. In this region, 37 unique aa exchanges were observed. Out of these, 27 aa are located in the 4 hypervariable regions of loop 1, which encode the serotype-specic epitopes. Therefore, it is obvious that a new conventional adenovirus from psittacine birds has been identied. According to the classication of the International Committee on Taxonomy of Viruses, the designation psittacine adenovirus (PsAdV) is proposed.

1 INTRODUCTION
The classication of the family Adenoviridae is currently under consideration. Avian adenoviruses characterised by a common group-specic antigen were previously classied as group I and still will represent the genus Aviadenovirus. Prototypes of this genus are the fowl adenoviruses (FAdV) with 12 serotypes. Further group I

277

avian adenoviruses have been isolated from turkey, duck and pigeon, which can be differentiated by cross neutralization tests. The former group II egg drop syndrome virus (EDSV) and group III turkey adenovirus 3 (TAdV-3) recently have been moved to the new genera Siadenovirus and Atadenovirus, respectively. Adenoviruses are non-enveloped particles of 70-90 nm in diameter with a single linear molecule of double-stranded DNA of variable size between approximately 26 and 45 kbp (BENK et al. 2000). The capsid consists of 252 capsomers, 240 hexons and 12 pentons. The bre is bound non-covalently to the penton base. As a particularity, all FAdV strains have two bres at each penton base. Serotype-specic determinants are located on both, the bre and the hexon. The three-dimensional structure of human adenovirus 2 has been identied (ROBERTS et al. 1986). On the outer surface of the hexon 3 loop (L) regions designated as L1, L2 and L4 are located. The L3 region combines the outer surface regions and the conserved pedestal (P) regions P1 and P2 forming the inner surface. As demonstrated by the analysis of L regions of different avian adenoviruses, L1 represents the most variable region (RAUE and HESS 1998). With a length of more than 130 amino acids (aa) it is the largest one, showing only 42.5% homology between FAdV1 and FAdV10. Seven hyper variable regions (HVR) were identied in the L regions of the hexon gene, 4 HVR in L1, 2 in L2 and 1 in L4 (CRAWFORD-MIKSZA and SCHNURR 1996). It has been demonstrated that the type-specic antigenic determinant is encoded by the HVR, particularly of L1 and L2. In this study liver samples showing typical histological signs of an adenovirus infection were collected from Poicephalus spp. with acute disease. A PCR amplifying the L1 region of the hexon gene was developed. The nucleotide (nt) sequence obtained was analysed at the nt and the aa level. In a comparison with various avian adenovirus reference strains, the phylogenetic analysis of the sequenced L1 region showed remarkable differences supporting the proposal for a new psittacine adenovirus (PsAdV).

2 MATERIALS AND METHODS

Two 42 days-old Senegal parrots (Poicephalus senegalus) hatched out in an aviary died within 24 hours after the onset of severe clinical disease demonstrated by rufed feathers and anorexia. Liver samples (M158-04 and M159-04) were collected and investigated by histological examination. Intranuclear eosinophilic inclusion bodies in hepatocytes of these samples indicate the presence of an adenovirus. From the samples, total DNA was isolated using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) as recommended by the supplier. By the alignment of published nt sequences of all 12 FAdV serotypes, two conserved regions were identied for primer annealing and amplication of the L1 region of the hexon gene. The sense primer Hex L1-s (5-atgggagcsacctayttcgacat-3) is located in a region from nt 301 to nt 323 of the hexon gene of FAdV1; the antisense primer Hex L1-as (5-aaattgtccckraanccgatgta-3) corresponds to a region spanning nt 890 to nt 868. The expected fragment size was approximately 590 bp.

278

Isolated DNA was tested for the presence of adenovirus DNA by PCR as follows: 5 units of Taq-DNA-polymerase (Peqlab, Erlangen, Germany), 5 l of 10x reaction buffer (200 mM Tris-HCl (pH 8.55), 160 mM (NH4)2S04 and 20 mM MgCl), 10 mM of each dNTP (Peqlab, Erlangen, Germany) and 100 pmol of primers Hex L1-s and Hex L1-as were mixed with 4 l of template DNA in a total volume of 50 l. The PCR was started with an initial denaturation step of 5 min at 95 C. The temperature prole of the following 40 cycles consisted of 30 s at 94 C for denaturation, 30 s at 56 C for primer annealing and 45 s at 72 C for elongation. The reaction was terminated by a nal elongation step of 5 min at 72 C. The PCR products (5 l aliquots) were separated on a 1.5% agarose gel stained with ethidium bromide and visualised by UV transillumination. The PCR product of liver sample M158-04 was puried (Qiaquick gel purication kit; Qiagen, Hilden, Germany), cloned in to the pCR4-TOPO vector as recommended by the supplier (Invitrogen, Karlsruhe, Germany) and sequenced for phylogenetic analysis. The nt sequence was determined using both M13 and M13 reverse primers and an automated 377 ABI DNA Sequencer together with the Dye Terminator Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, USA). The Nt sequence of the 587 bp PCR products and the deduced aa sequence, were aligned with the published sequences of the reference strains of avian adenoviruses using the Clustal W method of the MegAlign program (DNA Star Inc., Wisconsin, USA).

3 RESULTS
For the specicity testing of the developed PCR, a liver sample (107-04) taken from a 78 days-old Jardins parrot (Poicephalus gulielmi) was used. This bird did not show any clinical or pathological signs of an adenovirus infection, but had died following acute psittacine beak and feather disease. Using the primer combination Hex L1-s and Hex L1-as, the presence of adenoviral DNA was demonstrated in fresh liver samples collected from two Senegal parrots (Poicephalus senegalus) with clinical signs of an adenovirus infection (M158-04; M159-04) whereas sample 107-04 reacted negative. Both samples reacting positive in the PCR showed the same restriction enzyme pattern after cleavage with different restriction enzymes. Therefore, only the nt sequence of the PCR product obtained with sample M158-04 was established and compared with the sequences of other avian adenoviruses. The length of the fragment compared varied between 575 nt (strain TAdV-3) and 602 nt (strains 340 and IBH-2A). The overall percentage of identity among the sequences obtained from the FAdV reference strains and M158-04 ranged between 60.3 to 67.0 %, whereas that of the TAdV-3 sequence and M158-04 was only 14.1 %. With 12.5 %, the sequence of EDSV showed the lowest percentage of identity when compared with that of M158-04.

279

A phylogenetic tree was established (Fig. 1) showing 3 major branches for the proposed genera, Aviadenovirus (FAdV1-12), Atadenovirus (EDSV) and Siadenovirus (TAdV-3). Within the Aviadenovirus branch, 7 minor branches were observed. Six out of the seven branches formed the groups representing the genogroups as published (ZSAK and KISARY 1984). However, within this major branch a sole minor branch was formed by M158-04 indicating that M158-04 represents a new avian adenovirus genotype. The phylogenetic tree of the deduced aa sequences was comparable to the one described for the nt sequences. The length of the aa sequences varied between 191 (strain TAdV-3) and 200 aa (strains 340 and IBH-2A). The overall percentage of identity among the sequences obtained from the FAdV reference strains and M158-04 ranged between 51.3 to 61.0 %, whereas that of the sequences obtained from EDSV and M158-04 was only 34.0 %. With 32.5 %, the sequence of TAdV-3 showed the lowest percentage of identity as compared to sequence of M158-04. The alignment of deduced aa sequences revealed that unique aa exchanges were observed at 30 positions in the M158-04 sequence: twelve of these were located in the rst variable region A1, 5 in A2, 4 in A3, 1 in A4 and 8 in the conserved regions. Furthermore, 7 aa of the M158-04 sequence were identical to those in the EDSV and TAdV-3 sequences, respectively, but not to those observed in the sequences of the FAdV strains; fe out of these were located in the variable regions A1-A4 (3 in A1, 1 in A2 and 1 in A3) and 2 in the conserved regions. The evidence that the M158-04 sequence belongs to the group I avian adenoviruses is supported by the analysis of its g/c content. With a g/c content of 52.99 %, the M158-04 sequence was a g/c-rich sequence and within the range observed in the FAdV sequences (48.81-59.03 %). Both, the sequences of EDSV and TAdV-3 showed lower g/c contents. The charge at pH 7.0 and the isoelectric point of the M158-04 sequence are in a range observed in the case of the FAdV strains. It is evident that the sequence M158-04 represents a new group I avian adenovirus which should be designated as PsAdV.

4 DISCUSSION
This is the rst report of a specic psittacine adenovirus detected by PCR from Senegal parrots with signs of acute disease. Avian adenoviruses have been detected in psittacine birds by electron microscopy many times. However, only limited data exist on the classication of adenoviruses infecting psittacines. So far, the existence of FAdV2, FAdV3, FAdV4 and FAdV8, respectively, was demonstrated in psittacines with or without clinical signs by neutralisation tests. Since it was demonstrated recently that FAdV strains can be typed by their hexon L1 sequences (MEULEMANS et al. 2001), a fast and convenient method for the characterisation of avian adenoviruses became available. Therefore, a PCR protocol was developed amplifying the L1 region of the hexon gene. As this region encodes most of the serotype-specic epitopes, it may be expected from the deduced amino acid sequence to show whether a new serotype exists or not. The sequence obtained here revealed signicant differences on the nt as well as on the deduced aa level (Fig. 1). In accordance with the

280

nomenclature used by the International Committee of Taxonomy of Viruses (ICTV) it is proposed to designate this new avian adenovirus as PsAdV. The identication of new adenoviruses by sequencing is a common technique, i.e. an adenovirus from white sturgeon was recently identied by its hexon sequence and based on that a new adenovirus group was predicted (KOVACS et al. 2003). Here, 37 out of 195 (19.0 %) aa of the PsAdV sequence were not observed in any of the FAdV reference strains; 27 out of them were located in the 4 hypervariable regions of loop 1 carrying most of the serotype-specic epitopes. These observations might indicate that PsAdV is a new avian adenovirus with special (antigenic) characteristics. On the other side, PsAdV shows common characteristics of group I avian adenovirus such as its g/c content, isoelectric point and charge at pH 7.0. Furthermore, sera of birds living in the same aviary as those investigated here and surviving the disease reacted positive with common group I avian adenovirus-specic antigens in an immunodiffusion test (H. M. Hafez, unpublished data). So far, nothing is known about the distribution and signicance of PsAdV and its possible role for a specic disease in psittacines. The PCR described here is a suitable and convenient method to identify PsAdV DNA in liver samples. Furthermore, the sequence obtained allowed the development of specic and sensitive PCR protocols which can be used for epidemiological studies in the future.

5 CITATION INDEX
1. BENK M, HARRACH B and RUSSELL WC. Family Adenoviridae. In: VAN REGENMORTEL MHV, FAUQUET CM, BISHOP DHL, et al. (eds): Virus Taxonomy, Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press 2000, 227 - 238 CRAWFORD-MIKSZA LK. Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specic residues. J Virol 1996; 70: 1836 - 1844 KOVACS GM. Phylogenetic analysis of the hexon and protease genes of a sh adenovirus isolated from white sturgeon (Acipenser transmontanus) supports the proposal for a new adenovirus genus. Virus Res 2003; 98: 27 - 34 MEULEMANS G. Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses. Avian Pathol 2001; 30: 655 - 660 RAUE R. Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus. J Virol Methods 1998; 73: 211 - 217 ROBERTS MM. Three-dimensional structure of the adenovirus major coat protein hexon. Science 1986: 232: 1148 - 1151 ZSAK L. Grouping of fowl adenoviruses based upon the restriction patterns of DNA generated by BamHI and HindIII. Intervirol 1984; 22: 110 - 114

2. 3.

4. 5. 6. 7.

281

AUTHORS ADRESS
Dr. Rdiger Raue Institute for Virology, Faculty of Veterinary Medicine, University Leipzig, An den Tierkliniken 29, D-04103 Leipzig, Germany Email: rraue@vetmed.uni-leipzig.de

Fig. 1. Phylogenetic tree established with the nucleotide sequences of the approximately 590 bp fragment of the hexon gene containing the L1 region (clustal W method). Numbers in parenthesis correspond to the accession numbers. The box indicates sequences belonging to group I avian adenovirus classied as Aviadenovirus. Typing of FAdV into genogroups (A-E) according to ZSAK and KISARY (1984) is also mentioned.

282

Parc de Clres Jean Delacour Musum National dHistoire Naturelle, Ecole Nationale Vtrinaire de Nantes, France

PLASMA ELECTROPHORESIS REFERENCE RANGES IN VARIOUS BIRD SPECIES

D. Ordonneau, Vet student, Y. Roman , DVM, MsC, D. Chaste - Duvernoy, Biol, M.C. Bomsel, PhD.

KEYWORDS
Protein Electrophoresis Reference ranges.

ABSTRACT
Plasma protein electrophoresis is generally recognised to be a very reliable diagnostic tool in birds for many pathologic conditions. Unfortunately, wide variations of electrophoretic patterns have already been described between some bird taxa. In the daily practice, these variations lead to difculties in the interpretation of results and are therefore responsible for the under use of plasma protein electrophoresis in avian medicine. This study focuses on plasma protein electrophoresis of birds belonging to species that are not well documented, insisting on peculiarities of each species. 291 birds belonging to 8 different species (62 bar headed geese (Anser indicus), 42 white storks (Ciconia ciconia), 36 rock doves (Columbia livia domestica), 44 jackdaws (Corvus monedula), 34 black kites (Milvus migrans migrans, Milvus migrans parasitus), 40 peafowls (Pavo cristatus), 13 African grey parrots (Psittacus erithacus), 18 blacksmith lapwings (Vanellus armatus) were blood sampled to establish reference ranges and to compare interspecic differences.

1 INTRODUCTION
Plasma protein electrophoresis is generally recognised to be a very reliable diagnostic tool in birds for many pathologic conditions (WERNER and REAVILL 1999; CRAY and TATUM 1998). Unfortunately, wide variations of electrophoretic patterns have been described between some bird taxa (BLANCO and HOFLE 2003, CRAY and TATUM 1998, ZAIAS et al. 2000). Practitioners should be aware of the species reference ranges to be condent in their results and to interpret variations of the different fractions concentrations. However, at this time, most of the data available in the literature deals with raptors or psittacine birds and there is a lack of information

283

about other bird taxa. Furthermore, the variations between bird taxa make it difcult to dene the different protein fractions in a standardised way. Some data is available about the protein content of some fractions in psittacine birds (albumin; -lipoprotein, -antitrypsin, 2-macroglobulin, haptoglobulin in -globulins; brinogen, transferrin, complement, -lipoprotein in -globulins, immunoglobulins in -globulins) (CRAY et al. 1996), but much of it seems to have been extrapolated from mammals (KANEKO 1989). In daily practice, these variations lead to difculties in the interpretation of the results and are therefore responsible for the under use of plasma protein electrophoresis in bird medicine. The purpose of the study reported here was to dene plasma protein electrophoresis reference ranges on various bird species in order to extend the use of this test to other avian species and to comment on interspecic differences.

2 MATERIALS AND METHODS

This study was conducted on 291 adult, presumed healthy, birds belonging to 8 phylogenetically distant species: 62 bar headed geese (Anser indicus) from the zoological parks of Clres (France) and Branfr (France); 42 white storks (Ciconia ciconia) from the zoological parks of Clres, La Haute Touche (France) and Branfr; 36 free ranging rock doves (Columbia livia domestica) from the Mnagerie du jardin des plantes de Paris (France) (epidemiological study); 44 free ranging jackdaws (Corvus monedula) from the zoological park of Clres (epidemiological study); 34 black kites (Milvus migrans migrans and parasitus) from the falcon house of the Puy du Fou (France); 40 peafowls (Pavo cristatus) from the zoological park of Clres;13 African grey parrots (Psittacus erithacus) from the zoological park of Clres, the Mnagerie du jardin des plantes, and some private owners; 18 blacksmith lapwings (Vanellus armatus) from the zoological park of Clres. In each species, we studied the largest population sample as possible, in order to minimise intraspecic variations. From each bird, 1 to 5 ml of blood was collected from the right jugular or brachial vein, depending on the species. Since haemoglobin is known to migrate in the fraction (WERNER and REAVILL 1999), care was taken to avoid haemolysis by taking blood samples with 20 G needles. Protein electrophoresis was carried out on plasma, since it is commonly admitted that plasma is preferable to serum for protein electrophoresis in birds, because it is less prone to haemolysis and contains brinogen, which is a characteristic acute phase protein (KANEKO 1989, WERNER and REAVILL 1999). Blood was therefore collected in lithium heparin. Heparinised blood was immediately centrifuged and plasma samples were stored in cryotubes at -20C for a variable duration before being send to the laboratory. Samples were then allowed to thaw and were rehomogenised 1 hour before analysis. Total protein concentration was determined by the biuret method with the Roche integra system. Agarose gel electrophoresis was performed with Hygragel protein 15/30 kits (Sebia, France). Plasma aliquots were loaded onto the gel and allowed to migrate for 8 min at 296 V (6mA) in a Sebia Hydrasis system. Once dried and coloured with amidoblack in the same system, gels were scanned by a high resolution Epson expression 1680pro at scanner. The electrophoresis curves and dosages of the different fractions were

284

made with Phoresis Sebias software. Systat 7.0 software (SPSS INC 1997) was used for all analyses. Considering the small size of the population studied, we used basic statistic and Kruskal and Wallis one way analysis of variance.

3 RESULTS
Without any information about a standardized method of dening the different protein fractions, the fractions were dened by comparison with other bird species, considering the fact that brinogen seems to be the only protein migrating in a consistent way, whatever the species may be (ORDONNEAU unpublished data), and that albumin fraction was the rst high peak in the electrophoretic patterns. Electrophoretic patterns appeared to be divided in 5 fractions (.Albumin, 1-globulin, 2-globulin, -globulin, -globulin) (Fig. 1-8). Prealbumin fraction values were very low and were included to albumin fraction values. Results are documented in Table 1. The bar headed gooses curve was very simple with one peak for each fraction. The white storks curve was similar, with a higher 1-fraction. The rock doves curve was characterised by an unapparent 1-fraction, a very important peak in 2-fraction, and 2 peaks in the -fraction. In the jackdaws curve, we observed an unapparent 1-fraction and 2 peaks in -fraction. In the black kites curve, there was one peak for each fraction and a tray between 2-fraction and -fraction. The peafowls curve was characterised by 2 peaks in the 2-fraction. The African grey parrots curve had the same characteristics as the rock doves with a smaller peak in 2 and -fractions. Finally, the blacksmith lapwing showed one peak for each fraction except for 1-fraction, which was unapparent. Interspecic differences were highly signicant for all parameters: total protein concentration (Kruskal and Wallis test: df = 6, Z = 41,724, p < 0,001), albumin / globulin ratio (Kruskal and Wallis test: df = 6, Z = 193,141, p 0,001) and all protein fractions (Kruskal and Wallis test: df = 6, p < 0,001). No intraspecic differences were found whatever the species may be (Kruskal and Wallis test).

4 DISCUSSION
This study highlights that there is no intraspecic variability for the parameters studied. Electrophoretic patterns and values are completely species specic (Fig. 1-8). However, in a dened taxon, electrophoretic patterns of different species generally share many resemblances, showing that there is a great homogeneity within taxonomic groups (Phasianidae, Anatidae, Columbidae) (ORDONNEAU unpublished data). Without any standards for dening protein fractions, it is generally accepted that the most relevant parameters for the interpretation of avian proteinograms are total protein concentration and albumin / globulin ratio (FUDGE 2000). In this study, these parameters appeared to be species specic. This stresses the importance of dening reference ranges in main taxonomic groups.

285

In the rock doves curve, 2-fraction concentration was higher than albumin fraction concentration. This result is quite surprising and was not documented in other publications about this species (LUMEIJ and BRUIJNE 1987). This difference could be related to the use of different kinds of gels (cellulose acetate instead of agarose). Agarose gels are more discriminating and allow a better separation of protein fractions (LE CARRER 1998). Further studies will focus on the identication of this important protein fraction in Columbidae. One must choose a standardised technique to allow comparison between further studies. In this study, the use of the Sebia system enabled us to use gels that were the same, and there was no variation related to the operators handling. The practitioner should always pay attention to the technique used in his usual laboratory to be able to compare his own results with published reference ranges (CRAY and TATUM 1998). In this study, the black kites and African grey parrots data appeared to be similar to information about raptors (BLANCO and HOFLE 2003) and African grey parrot (CRAY and TATUM 1998). However, different ways of dening protein fractions could have led to different results. In further studies, authors should always illustrate the way they dened each protein fraction. Without this information, publication of reference values is not useful for the bird practitioner. Due to important taxonomic differences, it appears very difcult to dene a standardized way of dening protein fractions in birds. Some data was published about the composition of protein fractions, but it often appears to have been extrapolated from mammal serum protein electrophoresis (KANEKO 1989). Further studies will focus on identication of proteins that make up each fraction, by 2D electrophoresis and mass spectroscopy. Identication of these proteins should allow better denition of protein fractions in order to standardise plasma protein electrophoresis in various bird taxa. At this time, without this information, the practitioner must be very familiar with the electrophoretic patterns of bird species he deals with. Without this knowledge, he cannot be condent with the interpretation of results.

5 CITATION INDEX
1. 2. 3. 4. 5. BLANCO JM and HOFLE U. Plasma protein electrophoresis as diagnostic an prognostic tool in Raptors. Proc Euro Assoc Avian Vet, Tenerife 2003: 256 - 261. CRAY C, BOSSART GD and HARRIS D. Plasma protein electrophoresis : an update. Proc Assoc Avian Vet, Tampa 1996: 97 - 100. CRAY C and TATUM LM. Applications of protein electrophoresis in avian diagnostics. J Avian Med Surg 1998; 12(1): 4 - 10. KANEKO JJ. Serum proteins and the dysproteinemias. In: KANEKO JJ. (ed): Clinical Biochemistry of Domestic Animals. 4th ed. San Diego: Academic Press Inc, 1989; 142 - 165. LE CARRER D. Electrophorse et Immunoxation des protines sriques. Laboratoires SEBIA, 1998.

286

6. 7. 8. 9.

LUMEIJ JT and BRUIJNE JJ. Blood chemistry reference values in racing pigeons (Columbia livia domestica). Avian Pathol 1987; 14: 401 - 408. ROSENTHAL K.L., Avian protein disorders. In: FUDGE AM. (ed): Laboratory Medicine : Avian and Exotic Pets. Philadelphia: WB Saunders 2000; 171 - 173. WERNER LL and REAVILL DR. The diagnostic utility of serum protein electrophoresis. Vet Clin North Amer: Exot Anim Prac 1999; 2: 651 - 662. ZAIAS J, FOX WP, CRAY C and ALTMAN NH. Hematologic, plasma protein, and biochemical proles of brown pelicans (Pelecanus occidentalis). Am J Vet Res 2000; 61(7): 771 - 774.

6 AKNOWLEDGMENTS
We thank Sebia France for their generous gift of Hydragel protein 15/30 kits, the falcon house of the Puy du Fou, and specially J.L. Ligeois, from the Mnagerie du jardin des plantes, N. Chai, from the zoological park of la Haute Touche and F. Huyghe from the zoological park of Branfr.

AUTHORS ADRESS
D. Ordonneau, Vet student N11 Rsidence Plein Sud, Av dAunis, 17570 La Palmyre, France Email: ordonneau.do@voila.fr

287

Table 1 : Plasma protein electrophoresis mean values and SD in g/L and percentages of healthy birds of 8 different species A/G* mean 1,34 57,02 3,45 39,98 4,94 29,32 5,28 39,64 3,22 43,87 2,46 56,1 4,07 53,04 4,07 0,76 0,14 15,51 42,89 1,15 0,2 19,99 2,48 4,59 2,66 4,55 2,46 1,28 0,13 24,88 2,5 2,04 4,6 0,63 1,68 1,94 5,39 4,1 7,21 0,79 0,14 17,65 2,48 2,91 1,34 3,23 0,38 0,78 0,14 0,47 0,45 1,03 5,2 2,76 0,54 0,67 0,15 14,17 2,24 0,99 2,76 8,19 22,87 10,34 25,91 7,52 16,94 9,07 23,81 9,32 26,01 5,23 2,42 0,51 36,21 0,42 0,11 10,65 1,57 0,88 0,18 13,3 1,75 2,52 1,59 3,68 2,3 6,15 0,67 1,27 1,94 3,31 1,96 5,21 2,98 21,02 3,98 15,11 2,31 0,67 0,08 16,06 1,7 8,45 1,7 6,09 1,12 4,34 10,77 9,7 25,92 10,58 29,08 6,16 15,23 5,51 12,4 5,71 15,14 6,39 17,54 3,82 5,86 1,33 18,29 2,64 13,86 0,2 25,39 2,66 2,39 0,55 7,59 1,79 5,77 1,64 2,9 0,95 2,01 2,75 5,22 2,73 4,76 1,6 3,27 0,67 1,12 0,99 2,32 2,02 4,24 mean mean mean mean mean 2,01 4,95 5,3 13,14 3,36 6,29 2,08 5,7 3,13 7,8 4,41 9,93 2,34 6,23 2,91 8,05 Albumin Alpha 1 Alpha 2 Beta Gamma 0,71 1,87 1,1 2,57 1,08 2,56 0,84 1,82 0,86 2 1,02 2,09 0,44 1,25 0,59 1,16

Total protein 5,11

mean

Anser indicus

g/L

41,16

n=62

Ciconia ciconia

g/L

40,21

n=42

Columbia livia domestica

g/L

36,83

n=36

288

Corvus monedula

g/L

36

n=44

Milvus migrans

g/L

40,18

n=34

Pavo cristatus

g/L

44,38

n=40

Psittacus erithacus

g/L

37,17

n=13

Vanellus armatus

g/L

36,11

n=18

A/G* = albumin / globulin (1+2++) ratio

A/G* = albumin / globulin (1+2++) ratio

289

Parc de Clres Jean Delacour, Musum National dHistoire Naturelle, France

EARLY DETECTION OF AN ACUTE INFLAMMATORY CONDITION BY PLASMA PROTEIN ELECTROPHORESIS AND HAEMATOLOGY IN PEAFOWLS
Y. ROMAN, DVM, MSc D. ORDONNEAU, D. CHASTE - DUVERNOY, Biol, M. SAINT JALME, PhD, M.C. BOMSEL, PhD, Y. HINGRAT, MSc.

KEYWORDS
Protein - Electrophoresis - Haematology - Inammation - Peafowl.

ABSTRACT
This study was conducted at Clres Zoological Park (France) where we followed the inammatory kinetic after a supercial pectoralis tendonectomy on ten adult peafowl (Pavo cristatus). This surgery was considered to be the origin of an iatrogenic inammatory condition. We assessed the inammatory kinetic by performing plasma protein electrophoresis and haematology on blood samples drawn at intervals of 3, 6, 11, 16, 28 and 52 hours after surgery. The inammatory condition led to visible changes as early as 3 hours after surgery for haematology and 16 hours after surgery for protein electrophoresis. Changes on the electrophoretic patterns occurred mainly in the fraction - where brinogen is known to migrate - and in the fraction. Plasma protein electrophoresis and haematology thus both seem to be reliable laboratory examinations allowing early detection of acute inammation conditions.

1. INTRODUCTION
Serum protein electrophoresis has been extensively used for 3 decades in human diagnostics, but its use in avian medicine is far more recent. Haematology has been used in avian medicine for a far longer time. These examinations are both commonly recognized to be reliable diagnostic tools for many pathologic conditions in birds (WERNER 1999, CAMPBELL 1995). Like haematology, protein electrophoresis is quite a non specic laboratory examination which is thought to react rapidly. In acute phases of diseases, when serological tests fail to detect the agent of a disease, signicant changes in the electrophoretogram may already be recorded (CRAY and

290

TATUM 1998, BLANCO and HOFLE 2003). However, we lack information about the delay between the beginning of an inammatory process and the changes in the haemogram or in the proteinogram. This kind of missing information often makes it difcult to be condent about the interpretation of the results and is therefore responsible for its underused in avian medicine. The purpose of the study reported here was to determine the period of time between the beginning of an inammatory condition and its detection by haematology and plasma protein electrophoresis. This study also aims at describing the kinetics of a normal inammatory condition resulting from soft tissue surgery and to compare the efciency of haematology versus plasma protein electrophoresis to diagnose such an acute inammatory condition.

2. MATERIAL AND METHODS

This study was conducted at the Clres zoological park (France) on ten healthy adult peafowl (Pavo cristatus) which were prevented to y by supercial pectoralis tendonectomy (OLSEN, 1994) for legal and safety reasons. This surgery, performed under sterile conditions was considered to be the origin of an iatrogenic inammatory condition. We followed the inammatory kinetic by performing plasma protein electrophoresis and haematology on blood samples drawn at the right jugular vein at intervals of 3, 6, 11, 16, 28 and 52 hours after surgery. Since haemoglobin is known to migrate in the fraction, care was taken to avoid haemolysis by taking blood samples with 20 G needles. For the needs of this study, no anti-inammatory drugs were used postoperatively. All surgeries went well and all the birds were released back into the park after the last blood sample was taken. For plasma protein electrophoresis, we collected 2 ml of blood on lithium heparin. Protein electrophoresis were carried out on plasma, since its commonly accepted that plasma is preferable to serum, for protein electrophoresis on birds, because it is less prone to haemolysis and contain brinogen which is a characteristic acute phase protein (CRAY and TATUM 1998, HOCHLEITHNER, 1994). Heparinised blood was immediately centrifuged and plasmas were stored in cryotubes for one week at -20C (-4F) to guarantee all reagents would be the same for the ulterior analysis. Samples were then thawed and re-homogenised 1 hour before analysis. Total protein concentration was determined by the biuret method with the Roche integra system. Agarose gel electrophoresis was performed with Hydragel protein 15/30 kits (Sebia, France). Plasma aliquots were loaded onto the gel and allowed to migrate for 8 min at 296 V (6mA) in a Sebia Hydrasis system. Once dried and coloured with amidoblack in the same system, gels were scanned by a high resolution Epson expression 1680 pro at scanner. The electrophoretic curves and dosages of the different fractions were made with Phoresis Sebias software. Albumin concentration includes prealbumin fraction. Globulins were divided into 5 fractions: 1, 2, 3, and , as explained later in the discussion. For haematology, we collected 1 ml of blood on EDTA. Blood smears were made within 20 min after blood sampling to avoid artefacts caused by prolonged exposure to EDTA. Smears were made with the microscope slide and coverslip technique, and

291

dried immediately with a hair drier. Smears were coloured with the Wright and Giemsa coloration, and used to perform differential counts. Counts were made by counting 100 WBC which were distributed into 6 cell classes: heterophil granulocytes, eosinophil granulocytes, basophil granulocytes, lymphocytes, monocytes and immature WBC. TWBCs were performed within 5 hours after blood sampling, under phase contrast microscopy, using the indirect method described in avian haematology and cytology (CAMPBELL 1995), but replacing the Neubauer haemocytometer by a Mallassez haemocytometer, and the eosinophil Unopette 5877 system (not available in France) by a 1/200 dilution of the blood with eosinophil dilution liquid containing B phloxine (LMR5004, Labo Moderne, France). Systat 7.0 software (SPSS Inc 1997) was used for all analyses. Considering the small size of the population studied, we used Friedmans two way analysis of variance and Wilcoxons signed rank test.

3. RESULTS
3. 1 Protein electrophoresis Peafowl electrophoretic patterns appeared to be divided into 7 fractions as illustrated in Fig. 1. From our interpretation of these fractions albumin showed the biggest peak. Prealbumin values were very low and were included to albumin values. The and globulin were identied on the electrophoretic curves, since looking similar to and peaks in other species, but we found 3 fractions in the region. Total protein and protein fraction values all signicantly varied with the time after surgery as illustrated in Fig. 2 (Friedmans test: p<0.001). They all dramatically decreased between T0 and T3 (Wilcoxons test: p<0.05), without any signicant difference in the fraction rates and in albumin / globulin ratio between these two times. The fraction values then signicantly increased from T11 to T52 (Wilcoxons test: p<0.013). The fraction values signicantly decreased between T6 and T11 and signicantly increased between T16 and T28. The fraction appeared to be divided into 3 sub-fractions (1, 2, 3) which varied independently, as represented on Fig. 3. All of them decreased between T0 and T3. The 2 fraction concentration then signicantly decreased until T11 (Wilcoxons test: p<0.041) and then appeared to be stable, whereas 1 and 3 fraction concentrations respectively increased after T16 (Wilcoxons test: p<0.07) and T11 (Wilcoxons test: p<0.07). Albumin and globulin concentration slowly decreased from T3 to T16. After that, albumin concentration signicantly increased between T16 and T28. The albumin / globulin ratio did not appear to signicantly vary between T0 and T6. It signicantly decreased from T6 to T28 (Wilcoxons test: p<0.022). Difference with T0 became signicant after T16 (Wilcoxons test: Z=-2.981, p=0.003).

292

3. 2 Haematology TWBCs, heterophil granulocyte and lymphocyte counts signicantly varied with the time after surgery (Friedmans test: p<0.008) (Fig.4). Eosinophil granulocyte, basophil granulocyte, monocyte and immature cell counts did not signicantly vary with the time after surgery. Heterophil granulocyte counts dramatically increased between T0 and T3 (Wilcoxons test: Z=2.803, p=0.005). None of the variations recorded later appeared to be signicant. Lymphocyte counts appeared to signicantly decrease between T0 and T6 (Wilcoxons test: p<0,037). They signicantly increased between T6 and T11 (Wilcoxons test: Z=2.293, p=0.022) and decreased again between T11 and T16 (Wilcoxons test: Z=-2.191, p=0.028). Signicant increases were recorded after T16 (Wilcoxons test: p<0.028).

4. DISCUSSION
This study highlights the problems encountered when dealing with electrophoretic patterns of bird Taxa other than raptors or psittacine birds, which are well described in the literature. It stresses the importance of explaining in each study how the author dened protein fraction. Unfortunately, much data available in the literature was extrapolated from mammal serum protein electrophoresis. Further studies should be made to determine a standardised method of dening protein fractions on bird electrophoretic patterns. Without these precautions, the publication of reference values is completely useless for the bird practitioner. In this study, we found that total protein and protein fraction concentrations all signicantly decreased within 3 hours after surgery. Since there were no signicant differences between the different fraction rates at T0, T3 and T6 and between albumin / globulin ratios at T0, T3 and T6, we can state that this protein leakage does not specically affect one kind of protein, like it should in an active inammatory condition (exudate). This protein loss may be a passive phenomenon, like massive blood loss or transudate occurring per and immediately postoperatively. Another hypothesis could be a response to isourane anaesthesia (DRESSEN et al. 1999). After the decrease occurring in the rst 3 hours, and fraction values increased until the end of the study at T52. The and fractions contain acute phase proteins which rise during the rst hours after an acute inammatory condition like surgery. The main acute phase protein in the peak is generally accepted to be brinogen. These results agree with what is usually described in the literature about acute inammatory condition (CRAY and TATUM 1998). However sub-fractions appeared to react in different ways. In peafowl, only 1 and 3 fractions seem to contain acute phase proteins and may therefore be of diagnostic interest in an acute inammatory condition. Without the massive protein leakage occurring within the rst 6 hours, and the inevitable

293

Figure 1 : example of a peafowl electrophoretic pattern at T0

Figure 2 : variation of protein fractions concentrations (g/dl) with the time after surgery (h) Asterisks represent signicant differences between two successive measures (Wilcoxons test: p<0.05).

294

Figure 3: variation of fractions concentrations (g/dl) with the time after surgery (h) Asterisks represent signicant differences between two successive measures (Wilcoxons test: p<0.05).

Figure 4: variation of WBC counts (cell/l) with the time after surgery (h) Asterisks represent signicant differences between two successive counts (Wilcoxons test: p<0.05).

295

Dilution artefact related to successive blood samples (DRESSEN et al. 1999), the rise of acute phase protein concentrations should have probably been far more precocious and massive than demonstrated in this study. The slow decrease of fraction values is normal because this fraction is commonly known to contain immunoglobulin in birds (CRAY and TATUM 1998) and is therefore supposed only to increase in chronic inammatory conditions (>10 days). In this study, the decrease could be related to the haemodilution due to frequent blood samples. Without standards for dening each protein fraction, the most relevant criteria allowing the assessment of the birds health status seems to be the albumin / globulin ratio, as other authors reported in previous publications (LUMEIJ 1997). Albumin / Globulin ratio decreases during an inammatory condition, due to increased globulin fractions and decreased albumin fraction (mainly in chronic inammatory conditions). This study highlights the fact that A/G ratio values must be interpreted in the light of total protein concentration, since A/ G ratio was stable within the rst 11 hours even though total protein was decreased. In avian medicine, protein electrophoresis was rst recognised for the detection of chronic subclinical inammatory conditions such as aspergillosis, tuberculosis and psittacosis as well as egg peritonitis (MARGOLIN 1995). Further studies concluded that plasma protein electrophoresis was useful for acute diagnostic of chlamydophilosis (< 2 weeks), before seroconversion of the avian patient (CRAY and TATUM, 1998), underlining the fact that protein electrophoresis may be a valuable laboratory test to diagnose acute inammatory conditions. This study clearly demonstrates that A/G ratio decreases and acute phase proteins rise at least within 16 hours after the beginning of an inammatory condition, making plasma protein electrophoresis being reliable for early detection of an acute inammatory condition in birds. Monocyte counts appeared to be stable from the beginning to the end of the study, in accordance with what is generally accepted. Monocyte counts only rise in chronic pathological conditions in which chemotactic agents are released (like aspergillosis, chlamydophilosis, avian tuberculosis or massive tissue necrosis) (CAMPBELL 1995). Lymphocyte counts are generally thought to increase in chronic diseases, with chronic antigenic stimulation (CAMPBELL 1995). It was not therefore surprising that no signicant variation of lymphocyte counts were recorded from the beginning to the end of this study. However, the successive decrease and increase of these lymphocyte counts within 16 hours after surgery is quite difcult to understand and should be investigated in further studies. The dramatically increase of heterophil counts within the rst 3 hours postoperatively was probably due to massive endogen corticosteroid discharge in response to the surgical trauma (CAMPBELL 1995). The rise in the heterophil counts is therefore undoubtedly more rapid than protein in answer to the acute inammatory condition. Haematology appeared to be difcult to interpret in our case, because many immature cells were found in the blood stream in response to the massive consumption of granulocytes at the inammation site. Haematology and plasma protein electrophoresis are both reliable laboratory tests to diagnose acute inammatory conditions within the rst 16 hours, haematology being even far more precocious. However, haematology generally requires very experienced interpreters while plasma protein electrophoresis seems to be less prone to subjectivity. In return, at the present time, protein electrophoresis in birds

296

appears to be far less standardised than haematology and results clearly depend on the choice of serum instead of plasma, or in the choice of the equipment, the technique or the way of dening protein fractions.

5 AKNOWLEDGMENTS
To Sebia France for their generous gift of Hydragel protein 15/30 kits; to A SADRIN, C HOOTS, D VIDAL, F BURON for their English language proof-reading of this article.

6 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. 8. 9. BLANCO J M and HOFLE U. Plasma protein electrophoresis as a diagnostic and prognostic tool in raptors. Proc Eur Assoc Avian Vet, Tenerife 2003: 256 - 261. CAMPBELL T W. Avian haematology and cytology second edition. Ames, Iowa: Iowa State University Press/Ames 1995; 3 - 29. CRAY C and TATUM L. Application of protein electrophoresis in avian diagnostic testing. J Avian Med Surg 1998, 12: 4 - 10. DRESSEN PJ, WIMSATT J and BURKHARD M J. The effects of isourane anesthesia on hematologic and plasma biochemical values of American kestrels (Falco sparverius), J Avian Med Surg 1999, 13(3): 173 - 179. HOCHLEITHNER M. Biochemistries. In: RITCHIE B W, HARRISSON G H and HARRISSON L R. (ed): Avian medicine: principles and application. Lake Worth: Wingers Publishing Inc 1994; 237 - 238. LUMEIJ J T. Avian clinical biochemistry. In: KANEKO J J, HARVEY J W and BRUSS M L (eds): Clinical biochemistry of domestic animals. San Diego: Academic Press 1997; 859 - 861. MARGOLIN T. Normal electrophoretic values in cockatiels (Nymphicus hollandicus) and factors affecting these values. Proc Assoc Avian Vet, Nashville 1995: 65 - 66. OLSEN J H. Anseriformes. In: RITCHIE B W, HARRISSON G J and HARRISSON L R (eds): Avian medicine: principles and application. Lake Worth: Wingers publishing 1994; 1270 - 1272. WERNER L L and REAVILL D R. The diagnostic utility of serum protein electrophoresis. Veterinary Clinics of North America: Exotic Animal Practice 1999, 2: 651 - 662.

AUTHORS ADRESS Y. Roman, DVM, MsC. Le Parc de Clres Jean Delacour, Musum national dHistoire naturelle, 32 avenue du parc, 76690 Clres, France Email: yannick.roman@online.fr

297

Clinic for Birds, Ludwig-Maximilians-University Munich, Germany

DIGITAL SCANNING OPHTHALMOSCOPY IN BIRDS

R. T. Korbel, Prof. Dr. med. vet. Dr. med. vet. habil., Dip ECAMS, Cert Vet Ophthalmol

KEYWORDS
Digital scanning ophthalmoscope - Confocal scanning technique - Fluorescence angiography - Fundus oculi - Individual identication

ABSTRACT
Ophthalmoscopy is a routine ophthalmological procedure for examining ocular structures located behind the lens. The present study is focussing on ophthalmoscopy using a new scanning digital ophthalmoscope (SDO-A, Wild Medtec, Zrich, Switzerland) which allows rapid digital documentation of fundus. Investigation results in various raptor and psittacine species as well as pigeons show, that using an infrared device as an observation light allows documentation in pupil diameters as small as 4 mm, i. e. even in non-mydriatic pupils. Furthermore results show, that the new digital scanning ophthalmoscope includes major advances for avian ophthalmology such as short examination periods avoiding stress situations due to real-time image capture, rapid availability of single and sequence still images as well as video documentations, availability for combination with FAG and slit lamp examination and thus availability for daily routine follow ups of ophthalmological treatment procedures.

1 INTRODUCTION
Ophthalmoscopy is a routine ophthalmological procedure for examining ocular structures located behind the lens, i. e. the vitreous and the fundus oculi. Using ophthalmoscopy besides the vitreous the in birds anangiotic retina, the fovea, the pecten and at its basis the more or less obscured optic nerve head may be visualized. Depending on the pigmentation of the retinal pigment epithelium (RPE) the underlying vascular pattern of the choroid and most often due to minimal or absent pigmentation of the RPE in nocturnal species the sclera as the outer layer of the three ocular layers (retina, choroid and sclera) may be seen. In birds

298

ophthalmoscopy is indispensable in traumatised birds, while more than 35 % of all traumatised birds may develop intraocular haemorrhages, most often arising from the pecten oculi. These post-traumatic damages have to be diagnosed rapidly followed by effective therapy as visual impairment and blindness may be a possible result. Sophisticated diagnostic techniques based on ophthalmoscopy include uorescein angiography, as technique to diagnose fundus disorders associated to the vascular pattern. Furthermore visualising fundus structures will be of future importance for individual identication by pictorial documentation of individual vascular patterns and pecten structures, as these structures are like a ngerprint and may be used as a fraud proof method for individual markers without using rings or micro-transponders. Depending on the anatomy additionally structures like the ora serata, the pars plicata of the retina, structures of the lens including associated structures like the processus ciliares and even anterior eye chamber structures like the ltration angle with the pectinate ligamentum, iridal structures and more may be examined using an ophthalmoscope. For image documentation, the so-called fundus cameras may be used, whereas for avian ophthalmology and birds with a pupil diameter of more than 6 mm (most raptors, large psittacines) handheld systems like the KOWA RC 2 or KOWA Genesis for Animals and KOWAS Genesesis are suitable devices. Whereas for birds with a pupil diameter of less than 6 mm the special laboratory animal fundus cameras, like the KOWA Genesis for laboratory animals have to be used. Up to the most recent past, only analogous fundus camera systems (cameras using slide lms) were available, allowing only steady images including serial image documentation (for uorescence angiography) with up to 1 frames per second lacking the possibility for taking clips or movies. Most recently, digital cameras have been made available, enabling the examiner not only to take still photos but also videos. One of these systems is the newly available scanning digital ophthalmoscope (SDO-A, Wild Medtec, Zrich, Switzerland) which allows rapid digital documentation of the fundus.

2 MATERIAL AND METHODS

A scanning digital ophthalmoscope (SDO-A, Wild Medtec, Zrich, Switzerland) was used. For evaluation purposes this camera was used taking stills and videos in healthy diurnal raptors including common buzzards (Buteo buteo; n = 12), nocturnal raptors including great horned owls (Bubo bubo, n = 4), tawny owls (Strix aluco, n = 4), psittacines, including blue fronted amazons (Amazona aestiva, n = 12), macaws (Ara macao, n = 4) and racing pigeons (Columba livia, n = 12). Additionally, various raptor and psittacine species (total: n = 18) with diseased eyes were examined. For examination purposes of each individual, the left pupil was dilated using intracameral application of a 0.3 % d-tubocurine solution (0.01 0.03 cc) depending on the size of the anterior eye chamber, by performing paracentesis, while the right pupil was left un-dilated (KORBEL et al. 2000, KORBEL 2000). The pupil diameter of these birds examined covered a range down to 4 mm in diameter.

299

3 RESULTS
Investigation results in various raptor and psittacine species as well as pigeons showed, that using the new digital fundus camera allowed high resolution black and white as well as colour images. Able to document anterior as well as posterior fundus segments in all birds with a pupil diameter down to 4 mm, e.g. even in non-mydriatic pupils in raptors, real-time capture and video tracking, while combined with the ease of handheld and mobile operation procedure. Results have been documented producing jpeg and tiff les and will be documented within the presentation.

4 DISCUSSION
The results showed that the new digital scanning ophthalmoscope includes major advances for avian ophthalmology such as short examination periods avoiding stress situations due to real-time image capture, rapid availability of single and sequence still images as well as video documentations, availability for combination with FAG and slit lamp examination and thus availability for daily routine follow ups of ophthalmological treatment procedures by documenting anterior as well as posterior eye segment structures. Additionally, the digital camera may serve as an aid for identication of individuals by documenting the vascular pattern of the choroid rather as an alternative to rings or micro-transponders.

5 CITATION INDEX
1. 2. KORBEL RT, NELL B, REDIG PT, et al. Video uorescein angiography in the eyes of various raptors and mammals. Proc Assoc Avian Vet, Portland 2000: 89 - 95. KORBEL R. Diseases of the posterior eye segment. In: LUMEIJ JT, REDIG PT, REMPLE JD, et al. (eds): Raptor Biomedicine III. Lake Worth: Zool Educ Network 2000: 179 94.

AUTHORS ADRESS
R. T. Korbel, Prof. Dr. med. vet. Dr. med. vet habil., Dip ECAMS, Klinik fr Vgel, Ludwig-Maximilians-Universitt Mnchen, Sonnenstrasse 18, D 85764 Oberschleissheim, Germany Email: korbel@lmu.de

300

Loro Parque Tenerife Canary Islands, Spain

IMAGING TECHNIQUES IN AVIAN OBSTETRICS

L. Crosta, DVM and L. Timossi, DVM

KEYWORDS
Birds - Obstetrics - Endoscopy - Radiology - Ultrasound

ABSTRACT
Obstetrics is a relatively unexplored eld of avian medicine and radiology is still the most used diagnostic tool in birds. The paper presents common and unusual avian obstetrics cases, in which the different imaging techniques (endoscopy, radiographs and ultrasounds), have been effective in reaching a diagnosis, and/or useful for the resolution of the case.

1 INTRODUCTION
Although disorders of the reproductive apparatus represent an important part of domestic animal medicine, there is still a gap between knowledge and understanding of reproductive disorders in avian species and mammals (CROSTA et al. 2003). Some specic diseases of the female and male reproductive system have been described (BOWLES 2002, CROSTA et al. 2003), but still the diagnosis and subsequent treatment protocol of several female bird conditions are not well established. Egg retention and dystocia are among the most frequent obstetric disorders of birds (CROSTA et al. 2003). They are also the most frequent avian emergencies. Egg retention may be described as an egg which is not laid within the normal time (CROSTA 2000). Dystocia can be described as the mechanical obstruction of the egg passage in the caudal part of the oviduct (uterus and vagina) (GERLACH 1998). Causes of egg retention are multifactorial and involve: oversized or misshapen eggs, thin eggshell, excessive egg laying, calcium deciency, Vitamin A, E and selenium deciency abdominal hernia, dysfunction of the oviduct, obesity, torsion of the uterus, neoplasia, infectious diseases of the oviduct and genetic predisposition (CROSTA 2000, GERLACH 1998, OROSZ et al. 1997)

301

The diagnosis of egg retention should be based on clinical signs, physical examination, radiography and/or ultrasonography. Suspicion should arise if the clinician is presented with a female patient, with distended coelom and tenesmus. Other signs might include lameness, leg paralysis or respiratory difculty (OROSZ et al. 1997). The presence of an egg should be ascertained by radiographs or ultrasound, as coelomic palpation is not sensitive enough. In the case of egg retention with a soft egg with non-calcied egg, ultrasound is the preferred diagnostic tool (CROSTA 2000). Therapy depends on the physical status of the patient and the severity of the clinical signs (OROSZ et al. 1997). Patients which are still able to pass faeces and urine should be given antibiotics, uids, vitamin E/selenium, Vitamin D and calcium gluconate (HARRISON et al. 1992). The birds should be placed in a warmed and humidied cage (CROSTA et al. 2003). In minimally depressed birds, the egg will pass without further treatment (JOYNER 1994). To promote oviduct contractions Prostaglandin E2-gel can be inserted in the cloaca, at the utero-vaginal sphincter level (GERLACH 1998). In birds which are not able to pass faeces and urine, and run the risk of kidney damage and intestinal impaction, more aggressive therapy should be considered. After supportive therapy with uids, calcium and antibiotics, ovocentesis should be performed, through the cloaca or by transcoelomic access (OROSZ et al. 1997). The shell fragments should pass within a few days. In some cases, such as eggshell fragments are not delivered, or the removal through the cloaca is not possible, or if the adhesions between the egg and the uterus are severe, a coeliotomy might be necessary (JOYNER 1994). To prevent subsequent egg laying, which is extremely risky for the recently egg-bound hen, megestrol acetate can be given (CROSTA et al. 2003). Recently some publications have appeared about endoscopy of the oviduct (CROSTA et al. 2004) and also several improvements have been done using ultrasounds to scan the avian female reproductive system.

2 CASE REPORTS
Three example cases are taken into consideration for this study. Every case is involving a female bird that became egg bound. Since there were several differences, either because the birds belonged to different species, or there were soft shelled eggs, or even because more than one egg was involved in the problem, different techniques had to be used for the diagnosis and for the resolution of the problem. Case No 1 A 3 years old female emerald-collared parakeet (Psittacula calthorpae) was found resting in the bottom of her cage. At clinical examination a suspect diagnosis of egg retention was made, and it was conrmed radiologically. The egg was considered too large to be easily delivered and the bird was extremely weak and judged at risk for any invasive treatment. Also, due to the patient conditions, blood samples were not collected, but the cloaca was emptied and lubricated. SQ uids, antibiotics and diluted calcium gluconate were administered. Further, a PGE-2 gel was inserted

302

into the cloaca. The bird was then put in warmed cage and food and water were provided. After 12 hours, the egg had not been delivered yet, and the birds condition was clearly worsening: patient was not eating, but, most important, no faeces, nor urine were passed. The parakeet was quickly anaesthetized with isoorane, by face mask, The cloaca was carefully emptied, and an attempt to visualize the eggshell through the left oviduct-cloacal sphincter, was done. Since this was not possible, it was decided to perform a trans-abdominal ovocentesis: the abdomen was cleaned and disinfected, and a 18-G needle, connected to a 5ml syringe was inserted into the egg, passing through the abdominal and oviduct walls. During the aspiration of the egg content, the eggshell collapsed with no need for a manual compression, giving the impression of a relatively soft-shelled egg, even if this was not apparent at x-ray. Immediately after recovering from the minor procedure the bird looked better. This was likely due to the relief of the intra-abdominal pressure, for the presence of the oversized and retained egg. 6 hours after the procedure, the egg had not been delivered and the patients conditions were worsening again. It was thus decided to have a thoroughly endoscopic examination of the cloaca, and eventually be ready to perform a hysterotomy to retrieve the collapsed eggshell. The bird was anaesthetized again and entubated. The cloaca was carefully emptied again, using gently manoeuvred cotton swabs, and a cloacoscopy, was started. The cloaca was clearly inamed, but the oviduct-cloacal sphincter could easily be located. The endoscope, together with its examination sheath, was carefully introduced into the distal oviduct, and the collapsed eggshell could be visualized. The endoscope was retrieved and inserted into an operating sheath with an instrument channel and the whole system was inserted again into the cloaca. The eggshell was located again and it was grasped with the small exible 5-Fr grasping forceps. Unfortunately the 5-Fr grasping forceps were too small for a good grip, and only small amounts of eggshell were retrieved in this way. Nevertheless it was possible to pull the eggshell further distally through the oviduct, to a point where it could be visualized without the endoscope, but simply opening the cloaca with a small speculum. Small curved mosquito forceps were then inserted and the eggshell could be rmly grasped and fully retrieved. The parakeet recovered uneventfully from the surgery. The following day the bird received enrooxacin and meloxicam PO. Furthermore, oral megestrole acetate was added to treatment. Case No 2 A 9 years old female gentoo penguin (Pygoscelis papua), was presented for abdominal distension that was lasting since several days. Whole body X-rays were taken as well as blood for haematology and blood chemistry and microbiological samples. X-ray was pointing out the presence of two eggs, one of which, in a more distal position, was apparently poorly calcied. After several days the bird was still doing well, but there was no sign of egg laying. On the other hand the use of drug that may induce the contraction of the oviduct (oxytocin, PGE-2), was contraindicated, and a transabdominal ovocentesis was also to be excluded, because one egg was very difcult to locate, since it was soft shelled.

303

It was decided to have the eggs out surgically. A laparotomy technique, for rescuing dystocial eggs has been described (JENKINS 2000, ECHOLS 2002), but it has never been published in penguins. The patient was anaesthetised and maintained with isoorane and the oviduct was easily identied after entering the coeloma. As soon as the oviduct was opened the soft shelled egg was spotted. The oviduct was xed with two stay sutures and the egg content was gently aspirated with a 20ml syringe. Once the egg was emptied, it was retrieved with a small forceps. The second egg was in a very proximal position, so it could not be visualized, but it could be manually extracted. To facilitate this manoeuvre, a helper was needed to hold the penguin in a 45 position, so that the egg would naturally drop more distal in the coeloma. Oviduct and abdominal wall were sutured as described in other avian species. A long term course of antibiotics and megestrole were used for the penguin. Also Meloxicam was administered for a week, and the bird was not allowed to swim for 10 days after surgery, when the wound was healed and the bird was returned to the exhibit. Case No 3 A female blue and gold macaw (Ara ararauna), was presented for abdominal distension lasting several days. X-ray revealed the presence of a large egg. The bird was given Calcium and vitamins and, since its conditions were good, it was decided to wait 24 hours. The egg wasnt laid after this time and PGE-2 were administered intracloacally. The egg was then laid after 30 minutes. The day after ultrasounds ascertained the presence of a large soft shelled egg. The bird was monitored for few days, but since the clinical situation was not evolving, the egg content was aspirated by guiding the needle with the help of ultrasounds. A treatment with megestrole was started, but it was apparently non effective. After one week ultrasounds showed an oviduct that was still dilated. For this reason leuprolide acetate was administered and repeated at day 15 and 30.

3 RESULTS
Case No 1. The patient overcame the procedure with no apparent problems. She was treated for few days after surgery, to limit possible postoperative infections and help reducing temporarily the size and inammation of the oviduct. The patient has been ne since then, and is still with her cage mate in a large aviary where we are waiting for her to breed. Case No 2. The penguin recovered uneventfully from the surgery, and has been in very good clinical conditions since then. Exactly one year after surgery, she laid a normal egg. Case No 3. The bird was doing ne immediately after the procedure, but a new treatment was needed to have a complete recovery. The patient, a macaw used for a show, was reincorporated into the program and has been displaying during the show since then.

304

4 DISCUSSION
Obstetrics is still a relatively unexplored eld of avian medicine and radiology has historically been the most used diagnostic tool in birds. This especially because normally calcied eggs are well radio-opaque. While X-ray remains the main imaging technique in avian obstetrics, this paper shows how some of the techniques widely used in other animals are very promising even in birds. Endoscopy is one of the most used techniques in bird medicine, but its application to obstetrics has been very limited, to date. This likely because entrance to the oviduct is not easy to identify and special skills and very good instruments are needed to access this organ. Also, the relative large size of the avian egg may limit the application of endoscopy in avian obstetrics. Case No 1 is a good example of how endoscopy has few limits in birds: once an egg has been imploded, but does not come out in a natural way, the use of the endoscope may solve the problem in a relatively non-traumatic way. Ultrasounds have been already advocated as the only diagnostic tool able to conrm the presence of non-calcied eggs, but the authors showed that they have an application also in the treatment of egg-binding, when a hen is egg-bound with a soft shelled egg. Case No 3 is a clear example of how ultrasounds are a great help rst in the diagnosis, and then in the resolution of unusual obstetric cases. Finally, there are cases in which endoscopy and ultrasound do not nd a good application, but radiology is still a great diagnostic help. The gentoo penguin in Case No 2 is giving an example. This bird has been diagnosed to be egg-bound with, at least, one soft-shelled egg and a normally calcied one. Ultrasounds could not spot the eggs, probably because of the feathering of penguins that is too thick and ultrasound window that is too small. On the other hand, the endoscope was not able to reach the soft shelled egg. This is because, even if the egg was very low in the abdomen, still it was too far in the long oviduct of penguins for the endoscope. So the only option was to go for a standard surgery and rescue the eggs. It is obvious that these are simple examples of a very complicated eld, in which many different presentations (species, size and age of the patient; number, quality and relative size of the eggs) can lead the clinician to prefer one technique instead of another. The authors want to stress the need of a multidisciplinary approach to the diagnosis of the diseases of the avian female reproductive tract. Experience and constant practice are needed to become procient in the use of sophisticated techniques, such as radiology, endoscopy and ultrasonography, but the access to good quality tools and a creative mind may lead to discoveries also in this eld.

305

5 CITATION INDEX
1. CROSTA L, GERLACH H, BRKLE M and TIMOSSI L: Physiology, diagnosis and diseases of the avian reproductive tract. Vet Clin North Am: Exotic Animal Practice 2003; 6(3): 57 - 83. 2. BOWLES HL: Reproductive Diseases of Pet Bird Species. Vet Clin North Am: Exotic Animal Practice 2002; 5(2): 489 - 506. 3. CROSTA L: Patologie della Riproduzione e dellApparato Riproduttore. In: Proc Corso SIVAE (Societ Italiana Veterinari per Animali Esotici) di Medicina e Chirurgia Aviare, Cremona, Italy, 2000. 4. GERLACH H, Eibildende Organe, Erkrankungen. In: Wiesner E (Hrsg.): Handlexikon der Tierrztlichen Praxis. Stuttgart, Ferdinand Enke, 1998, 196/o-196/za. 5. OROSZ SE, DORRESTEIN GM and SPEER BL. Urogenital disorders. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM, et al. (eds): Avian Medicine and Surgery. Philadelphia: WB Saunders, 1997; 614 - 644. 6. HARRISON G, et al: Roundtable discussion: Emergency medicine. J Assoc Avian Vet 1992; 6(1): 10 - 15. 7. JOYNER KL: Theriogenology. In: RITCHIE BR, HARRISON GJ and HARRISON LR (eds.): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing 1994, 748 - 804. 8. CROSTA L, BRKLE M and TIMOSSI L: Endoscopy-assisted Resolution of Egg Binding in an Emerald-collared Parakeet. Exotic DVM 2004; ( 6.1): 19 - 22. 9. ECHOLS SM: Surgery of the Avian Reproductive Tract. Seminars in Avian and Exotic Pet Medicine 2002: 11(4): 177 - 195. 10. JENKINS JR: Surgery of the Avian Reproductive and Gastrointestinal Systems. Vet Clin North Am: Exotic Animal Practice 2000; (3:3): 637 - 692.

AUTHORS ADRESS
Lorenzo CROSTA DVM Veterinary Director Loro Parque & Loro Parque Foundation Avda. Loro Parque s/n 38400 Puerto de la Cruz Tenerife, Spain Email: lorenzo_birdvet@yahoo.com

306

Division of Zoo Animals and Exotic Pets, Vetsuisse Faculty, University of Zurich, Switzerland

QUANTITATIVE COMPUTED TOMOGRAPHY FOR THE ASSESSMENT OF BONE DENSITY IN BUDGERIGARS : A PILOT STUDY
I. Fischer, Dr. med. vet., A. Liesegang, Dr. med. vet., M. Hssig, Dr. med. vet., J.-M. Hatt, Prof. Dr. med. vet

KEYWORDS
Computed tomography - Bone mineral density - Bone mineral content - Budgerigar

ABSTRACT
Peripheral quantitative computer tomography (pQCT) is a non-invasive technique commonly used to measure bone mineral content (BMC) and bone mineral density (BMD). PQCT is used diagnostically in humans and has been adapted to measure bone quality in vivo in poultry. We evaluated 10 adult budgerigars (Melopsittacus undulatus) (5 females and 5 males) housed under identical conditions and identical feeding regime for BMC and BMD. The measurements were carried out on the tibiotarsus with peripheral quantitative computer tomography under general anaesthesia. BMC and BMD values were higher in the female group compared to the male group. These results demonstrate that pQCT may be used for in vivo to evaluate bone quality in budgerigars.

1 INTRODUCTION
The bone parameters commonly used to investigate bone mineral quality are bone ash, bone breaking strength and atomic absorption spectrophotometry (KORVER et al. 2004). The main disadvantage of these methods is that they are invasive. Peripheral quantitative computer tomography (pQCT) is a non-invasive technique commonly used to measure bone mineral content (BMC) and bone mineral density (BMD) in vivo. PQCT is used diagnostically in humans to assess osteoporosis (FORMICA et al. 1998) and has been adapted to measure bone quality in vivo in poultry (KORVER et al. 2004, LIESEGANG et al. 2004). To our knowledge QCT has not been described in psittacine birds.

307

2 MATERIAL AND METHODS

We evaluated 10 adult budgerigars (Melopsittacus undulatus) (5 females and 5 males) housed under identical conditions and identical feeding regime. The budgerigars received a commercial seed mixture (90 Hammer, SWV English budgerigar-food, Jordi AG.Signau, Switzerland) plus carrots and a mineral supplement (Quikon, Firma Quiko, Bocholt, Germany). Total bone mineral density (BMD) and content (BMC) were measured in the right tibitotarsus with peripheral quantitative computer tomography (pQCT) (Stratec XCT 2000 bone scanner, Stratec Medizinaltechnik GmbH, Pforzheim, Germany). The budgerigars were anaesthetized with isourane and positioned in dorsal recumbency with wings in normal position. The right leg was xated in extended position. Statistical data were analyzed using ANOVA (Systat, IL).

3 RESULTS
Bone mineral density and bone mineral content were evaluated in the tibiotarsus of a budgerigar with three measurements. The values were reproducible. The values of BMD and BMC were similar within the male or female group, respectively. Signicant differences were seen in BMC (p<0.0001) and BMD (P = 0.0003) between males and females. Mean values of BMD (xfemale = 509.82 mg/cm3 ; xmale = 373.66 mg/cm3) and BMC (xfemale = 1.65 mg/cm ; xmale = 1.04 mg/cm) were higher in the female compared to the male group. Table 1 Bone mineral density and bone mineral content of the tibiotarsus of budgerigars evaluated with peripheral quantitative computed tomography. M= male, F= female N 1 2 3 4 5 6 7 8 9 10 Sex M M M M M F F F F F BMD mg/cm3 427.1 389.5 391.5 335.9 330.3 414.3 444.9 713.4 382.3 594.2 BMC mg/cm 1.03 0.95 1.23 0.94 1.04 1.44 1.55 2.49 0.93 1.86

308

4 DISCUSSION
The BMD and BMC values of this study show a similar distribution within the same gender group and we were able to reproduce the values with several measurements in the same bird. The difference between male and female group may be due to calcium storage in the bones for eggshell formation. These results demonstrate that pQCT may be used for in vivo to evaluate bone quality in budgerigars. We encourage the evaluation of pQCT to investigate the inuence of different diets on bone quality, to evaluate the calcium metabolism or to control fracture healing. Before appliance in other psittacine birds the pQCT has to be evaluated.

5 CITATION INDEX
1. 2. KORVER D, SAUNDERS-BLADE J and NADEAU K. Assessing bone mineral density in vivo: quantitative computed tomography. Poult Sci 2004; 83: 222 - 229. LIESEGANG A, MESSIKOMMER R and WENK C. Einuss von durch Hitzebehandlung des Futters reduzierter Phytaseaktivitt auf die Phosphorversorgung und Knochenparameter von Broilern. Tagungsbericht, Schriftenreihe aus dem Institut fr Nutztierwissenschaften, ETH Zrich, 2004: 220 - 223 FORMICA C, NIEVES, J, CROSMAN F, et al. Comparative assessment of bone mineral measurements using dual x-ray absorptiometry and peripheral quantitative computed tomography. Osteoporos Int 1998; 8: 460 - 467.

3.

AUTHORS ADDRESS
Isabelle Fischer, Dr. med. vet. Division of Zoo Animals and Exotic Pets, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland Email: ischer@vetclinics.unizh.ch

309

Great Western Referrals, Swindon United Kingdom

BIOSECURITY OF THE AVIAN HOSPITAL FACILITY, BY DESIGN, PROTOCOLS AND PROCEDURES

N. Forbes BVetMed CBiol MIBiol DipECAMS FRCVS S. Smith BVetMed MRCVS.

KEYWORDS
Pathogens Hospital Biosecurity Fogging - Disinfection

ABSTRACT
Environmental sampling was conducted in two equally busy wards dealing with avian patients. Ward A was disinfected in a traditional manner for the rst week, whilst Ward B was disinfected by fogging. In the second week, both wards were disinfected in a traditional manner. In the third week, Ward A was disinfected by fogging, whilst Ward B was disinfected in the traditional manner. In the third week, Ward A was disinfected by fogging, whilst Ward B was disinfected in the traditional manner. Microbiology samples were taken on weekdays of each week. Surface samples and settle plates were employed to evaluate the levels of surface and air borne contamination, following conventional surface disinfection as opposed to F10 fogging. The results will be presented and discussed at the conference and will be published in the near future elsewhere.

1 INTRODUCTION
Avian and Exotic Animal Clinicians are faced with seriously pathogenic and on occasions fastidious pathogens on a daily basis. These pathogens present a risk to staff, all patients and the biosecurity status of the facility. The principles of modern hospital design and standards, which aims to minimise surfaces which may facilitate pathogen build up, designs in surfaces which are more readily cleaned and disinfected are well documented and will not be discussed here. In the human medical eld where design and material cost limitations may be less restrictive, the recent dramatic increase in methicillin-resistant Staphylococcus aureus (MRSA), which has led to a signicant number of deaths and endangered many thousands of other patients, serves to remind all those charged with maintaining hospital facilities, of the critical importance of optimal biosecurity procedures.

310

FRENCH et al (2004), sampling a London Teaching hospital environment, before and after traditional cleaning and disinfection procedures, documented MRSA levels of 74% and 66% respectively. In effect traditional cleaning techniques reduced the MRSA levels by only 11%. Those authors then compared these results collected after conventional terminal cleaning (i.e. after a patient has vacated an area), with hydrogen peroxide vapour disinfection (Bioquell PLC). The latter method reduced the contamination level by 98.4%. Within the avian and exotic animal setting, the clinician is commonly faced with a range of highly pathogenic organisms (Salmonella spp, Mycobacterium spp., Cryptosporidia, Campylobacter spp., Pseudomonas spp., Herpes virus, but those of greatest concern to the authors are Chlamydophila spp., Circovirus and the causative agent of Psittacine Proventricular Dilation Syndrome. These pathogens are highlighted as they are commonly present in apparently normal birds, such that the clinician is not aware of a potential contamination risk. BENDHEIM and DAVIDSON (2005) in a recently published survey of prevalence of circovirus in breeding establishments in Israel found a 20% level, whilst the incidence of Chlamydophila in similar situations has previously been reported to range from 10-60%. Chlamydophila and Circovirus are typically spread in feather or faecal dust, i.e. air borne, whilst (Circovirus) is also extremely persistent in the environment. Hospitals should be designed with the very clean areas (theatres) and more contaminated areas (isolation ward, general ward, autopsy room) positioned at dead-ends. In this way the risk of contamination of them, or from them respectively, is minimised. Within the ward cross contamination can be minimised by maintaining birds in their own discrete individually air extracted space, such that the risk of infection from one bird to another is minimised. Overalls and shoes used in contaminated areas can be banned from the cleanest areas (theatres). However having taken these steps, the greatest risk is then posed by consulting areas as well as central procedure or treatment areas, through which many different patients will pass in a day. In a typical avian practice it is likely that over 50% of these will appear ill, whilst a further signicant percentage will appear well but be carrying potentially contagious diseases. Such areas tend to be busy and are likely to contain signicant amounts of equipment. Where ever possible equipment and disposables should be enclosed such that air borne contamination risk is minimised. However it is recognised that the cleaning of complex environments containing medical equipment, soft furnishings, irregular hard surfaces is difcult and considered that the problem may be solved by using gaseous decontamination methods (FRENCH et al 2004). The aim of this trial was to compare the efcacy of two cleaning modalities, with the intention of then applying an improved protocol to all clinical areas. The trial was to be conducted in the common ward area of two wards (i.e. excluding patient areas which benet for discrete individually air extracted space), as this is a more controlled environment than a treatment or procedure area, and a cross over trial cold be readily performed. Circovirus has previously been shown to be controlled in an aviary situation, by the use of the disinfectant F10 (STANFORD personal communication), by fogging application. The active constituents of F10 Super Concentrate Disinfectant (F10) include quaternary ammonium and biguanide compounds, edetate and non-toxic

311

ampholytic surfactants, with the exact formulary remaining a commercial secret. F10. is said to be virucidal, bactericidal, fungicidal and sporicidal (VERWOERD 2001). Aerosol therapy with F10 has been successfully used in cases of lower respiratory tract infections at a dilution of 1:250 and is well tolerated by patients (CHITTY 2002). Biguanide compounds and quaternary ammonium compounds act via interference with cytoplasmic membrane function, causing, amongst other actions, coagulation of cytoplasmic constituents. Additionally, quaternary ammonium compounds cause increased permeability of the outer member in Gram negative bacteria (MAILLARD 2002). EDTA disrupts cell wall integrity by chelating metal ions (FOSTER and DEBOER 1998). The authors wished to evaluate fogged F10, as a routine disinfectant procedure, compared to more traditional veterinary hospital cleaning methods (see below).

2 MATERIALS AND METHODS

This investigation was conducted in the avian and exotic animal ward facility of the authors veterinary facility. Environmental sampling was conducted in two equally busy wards dealing with avian patients. Ward A was disinfected in a traditional manner for the rst week, whilst Ward B was disinfected by fogging. In the second week, both wards were disinfected in a traditional manner. In the third week, Ward A was disinfected by fogging, whilst Ward B was disinfected in the traditional manner. Samples were taken on weekdays of each week. Surface samples and settle plates were employed to evaluate the levels of surface and air borne contamination, following conventional surface disinfection as opposed to F10 fogging. 2.1. Environmental surface sampling and microbiological methods Cotton tipped sterile swabs, were moistened in sterile saline and used to sample pre determined surfaces of approximately 25cm2 by standardised swabbing in two directions at right angles, at ve sites (door furniture, oor wall interface, wall, patient cage fronts, shelving at a height of 1 metre), on 5 consecutive days. Cleaning was performed at the end of each working day and samples were collected some 12 hours later, prior to the start of the next day. Minimal human and patient movements occurred in the area in the interim. The air conditioning system was inactivated between disinfection and the time when sampling was completed. Settle plates contained blood agar (BA) (Oxoid, Basingstoke, Hampshire, UK), were placed in each ward on a daily basis, 12 hours after disinfection and left open for 60 minutes. After collection, swabs were re-suspended by agitation in 2ml of sterile saline for 30 seconds and ten left to stand for a further 5 minutes. Prior to plating the saline containing the swabs were re-agitated and the swabs were then used to inoculate Blood Agar and Maconkey Agar Plates (Oxoid, Basingstoke, Hampshire, UK), which were then incubated at 37oC for 36 hours. Growth was recorded semi-quantitatively: If growth remained in the area of primary culture, this scored 1, whilst if the growth extended beyond the area of initial inoculation, following streaking out, this was scored 2. Conventional Cleaning/

312

Disinfection: the conventional cleaning technique used in the ward area has been wet wiping with 1:250 concentration F10, which is left to dry naturally. F10 Fogging Disinfection: 1:250 dilution of F10 was delivered using a commercial poultry house fogger (RL Corporation Model 1037BR Pro-ULV) for a 15 minute period.

3 RESULTS AND DISCUSSION

The authors regret that due to technical and mechanical difculties the results are unavoidably delayed and not available at the time of going to press. The results will be presented and discussed at the conference and will be published in the near future elsewhere.

4 CITATION INDEX
1. BENHEIM U and DAVIDSON I. Molecular Identication and prevalence of psittacine circovirus in Israel. Proc Eur College Avian Med Surg, Arles 2005. 2. CHITTY J. A novel disinfectant in psittacine respiratory disease. Proc Assoc Avian Vet, Monterey 2002: 25 - 27. 3. FOSTER AP and DEBOER DJ. The role of Pseudomonas in canine ear disease. Comp Cont Ed 1998; 20(8): 909 - 918. 4. FRENCH GL, OTTER JA, SHANNON KP, et al. Tackling contamination of the hospital environment by methicillin-resistant Staphylococcus aureus (MRSA): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination. J of Hospital Infection (2004): 57: 31 37. 5. MAILLARD JY. Bacterial target sites for biocide action. J Applied Microbiol Symp Suppl. 2002: 92:16S - 27S. 6. VERWOERD D. Aerosol use of a novel disinfectant as part of an integrated approach to preventing and treating aspergillosis in falcons in the UAE. Falco 2001; 17: 17 - 18.

AUTHORS ADDRESS
Great Western Referrals, Unit 10, Berkshire House. County Park Estate, Shrivenham Road, Swindon, SN1 2NR, United Kingdom Email: n.forbes@gwreferrals.co.uk

313

Department of Pathobiology, Section of Pet Avian, Exotic Animals and Wildlife Division, Utrecht University, The Netherlands

CONSEQUENCE OF SEVERE DYSPNOEA FOR ORGAN FUNCTION

G. M. Dorrestein, DVM, PhD

KEYWORDS
Dyspnoea - Kidney failure - Liver pathology - Toxoplasmosis - Birds

ABSTRACT
Severe respiratory dyspnoea is a common clinical symptom in birds. The normal approach is to provide oxygen and attempting to remove the causative insults. In this presentation the changes in the lungs and the effects of hypoxaemia on other organs, e.g. liver and kidneys are illustrated. This will be done by presenting details of an acute outbreak of toxoplasmosis in crowned pigeons (Goura scheepmakeri sclaterii). The pathophysiological cascade of a hypoxaemia will be demonstrated and based on the changes a clinical approach to counter the effects of these changes is discussed.

1 INTRODUCTION
Severe respiratory dyspnoea is a common clinical symptom in birds. Acute pulmonary damage is often a non-specic reaction to injury of respiratory epithelial and endothelial cells from a variety of acute insults. This results in decreased lung compliance, hypoxaemia, and multiple organ dysfunction. In this presentation the pathology of an outbreak of toxoplasmosis in crowned pigeons (Goura scheepmakeri sclaterii) will be used to demonstrate the acute pulmonary lesions and the consequences for other organs. Toxoplasma gondii is a species of coccidian protozoa comprising intracellular parasites in many organs and tissues of birds and mammals, including humans. The only known complete hosts are cats and other Felidae (GARELL 1999, RATCLIFFE and WORTH 1991). The diagnosis is normally made at necropsy by demonstrating the Toxoplasma-cysts and pseudocysts by light microscopy using immunohistochemical staining. In acute cases lesions can be seen in many organs, including necrotic lesions in liver, spleen, intestines, kidneys and lungs (POELMA and ZWART 1972, TACKAERT-HENRY and KAGERUKA 1977). The acute changes in the lungs may play a crucial role in the acute and high mortality. The normal

314

approach in acute respiratory distress is providing oxygen and trying to remove the causative insult. Based on the changes a clinical approach to counter the effects of these changes is discussed.

2 CASE REPORT
Unexpectedly, 4 Scheepmakers crowned pigeons (Goura scheepmakeri sclaterii) housed in the same enclosure in a zoo died within 4 days. The birds, 2 juvenile and their parents were housed in a mixed exhibit with two yellow-tailed black cockatoos (Calyptorhynchus funereus funereus), two blue-winged kookaburras (Dacelo leachii) and two wattled brush turkeys (Aegopodius arfakianus). One bird was found moribund, the other three birds were found dead in the morning without previous clinical symptoms. At post mortem all birds were in good to excellent condition. The most prominent macroscopically recognisable alteration in all pigeons was seen in the lungs. All lungs showed dark red, marbled areas with oedema and a foamy content in the bronchi. Other areas of the lung were emphysematous. Changes of other organs were less consistent, but in general liver, spleen and kidney were degenerated (pale) and swollen. On histological examination peracute changes were seen in the lungs including hyperaemia and vascular damage leading to transudate and haemorrhages. Excessive cellular responses led to severe pulmonary damage. This cellular component existed of lymphocytes, heterophils and macrophages. In liver, kidney and spleen acute to subacute inammations with necrosis were seen. In the intestinal wall and pancreas a cellular inltration without necrosis was seen. In the brain no lesions were found. In all examined organs immunohistochemically positive organisms for antibodies against Toxoplasma gondii were demonstrated.

3 DISCUSSION
3.1 Acute changes in the lungs The acute reaction in the lungs is an excellent example to demonstrate what is going on in the body of the infected bird. It is a complex response to the invasion of the Toxoplasma parasite, involving many systemic responses. It is an overlapping series of events that form a continuum. Through the microscope we see all the typical local manifestations of an acute inammation. The rst reactions are haemodynamic changes. There is arteriolar dilatation resulting in hyperaemia. The increased permeability of the microvasculature is demonstrated by uid in the extravascular tissues resulting in oedema and uid in the parabronchi. A peripheral margination of white blood cells can be seen in capillaries and venules. In time the permeability increases and the uids change from watery transudate (oedema) to protein-rich, cellcontaining exudate. Direct damage or a scala of mediators like vaso-kinins, complement fragments, leukotrienes, prostaglandins, some cytokines and trombocyte-activating factors induce this increased vascular leakage (BOCHSLER and SLAUSON 2002). All acute changes lead to local stasis of the circulation, increased distance between air (O2) and blood (CO2), leading to an arterial hypoxaemia and a decreased supply of oxygen in all other tissues. This effect will decrease the potential for reaction and effective action in these organs. It will also support the development of local necrosis.

315

3.2 Effects on other organs In the liver the infection with the parasite resulted in an acute to subacute inammation. However, the low oxygen supply leads to ischemic necrosis of the centrolobular hepatocytes. The centrolobular zone, referred to as zone 3 in the functional concept of the hepatic acinus, is most distal to the blood supply from the portal tract and normally has a low oxygen tension. Microscopically, coagulative necrosis of centrolobular hepatocytes is accompanied by frank haemorrhage. Acute inammatory cells accumulate in the necrotic zones. In the kidney are the tubular epithelial cells, with their high rate of energy-consuming metabolic activity and numerous organelles, particularly sensitive to hypoxia and anoxia, which cause rapid depletion of intranuclear ATP in the tubular epithelium. This leads to acute tubular necrosis, a severe but potentially reversible impairment of tubular epithelial function, which results in acute renal failure (JENNETTE and SPARGO 1999). Mostly no pathological changes are seen in the glomeruli or blood vessels. Tubular injury is focal and is most pronounced in the proximal tubules. 3.3 Discussion of a clinical approach Although inammation fundamentally is a defensive reaction, inammations can be harmful as well. In many situations, and maybe also in this lung reaction, the host may suffer more from tissue damage as a result of an inammatory reaction than from damage caused by the initiating stimulus had there been no inammatory response at all. An example is seen in calves: if neutropaenia is induced by experimental means in calves with an induced pneumonic pasteurellosis, the calves are partially protected against the development of severe pneumonia and the resultant hypoxia (BOCHSLER and SLAUSON 2002). In the case with the pigeons the intensive inammatory reaction in the lung with tissue damaging contributions of the inammatory host-defence mechanisms (hyperaemia, brin, exudate and so forth) may be worse than tissuedamaging contributions of the original Toxoplasma infection. The arterial hypoxaemia at this stage cannot be reversed by simply increasing the oxygen tension of the inspired air. The inammatory process itself is aimed at maximising normalcy as an endpoint. Ideally this can be accomplished by removal of the initiating stimulus (in this case: Toxoplasma organisms) and regenerative repair of the injured tissue. However, because of the vital function of the lung for the O2/CO2 exchanges all therapeutic measures, in addition to providing extra oxygen, should be aiming at: Preventing free uids to come into the respiratory tissue (drowning effect) by using anti-inammatory drugs to limit, control or otherwise modify inammation (NSAIDs?). Remove extravascular uids from the lung as soon as possible to allow minimal distance between air- and blood capillaries by stimulating kidney activity (diuretics?). This will also reduce the development of permanent scar tissue after recovery. When the diagnosis is made in time, even immunocompromised patients may benet from treatment with chemotherapeutic drugs (such as pyrimethamine plus sulfadoxine, or trimethoprim plus sulfamethoxazole) (DIRIENZO et al. 2002).

316

4 CITATION INDEX
1. 2. BOCHSLER PN and SLAUSON DO. Inammation and repair of tissue. In: SLAUNSON DO and COOPER BJ (eds): Mechanisms of Disease. St Louis: Mosby 2002; 141 - 245. DIRIENZO AG, VAN DER HORST C, FINKELSTEIN DM and FRAME P. Efcacy of trimethoprim-sulfamethoxazole for the prevention of bacterial infections in a randomized prophylaxis trial of patients with advanced HIV infection. AIDS Res Hum Retroviruses 2002; 18: 89 - 94. GARELL DM. Toxoplasmosis in Zoo Animals. In: FOWLER ME, MILLER RE (eds): Zoo & Wild Animal Medicine. Current Therapy 4. Philadelphia: WB Saunders Co 1999; 131 - 135. JENNETTE J C and SPARGO BH. The Kidney. Chapter 16. In: RUBIN E and FARBER JL (eds): Pathology. 3rd edition. Philadelphia: LippincottRaven 1999; 900 - 901. POELMA FG and ZWART P. Toxoplasmose bij kroonduiven en andere vogels in de Koninklijke Rotterdamse Diergaarde Blijdorp. Acta Zool Path Antverpensia 1972; 55: 29 - 40. RATCLIFFE HL and WORTH CB. Toxoplasmosis of wild birds and mammals. Am J Path 1991; 27: 655. TACKAERT-HENRY MC and KAGERUKA P. Une pizootie de Toxoplasmose parmi les pigeons couronns, Goura cristata Pallas et Goura victoria Frazer, du Zoo dnvers. Acta Zool Path Antverpensia 1977; 69: 163 - 168.

3. 4. 5. 6. 7.

AUTHORS ADDRESS
Gerry M. Dorrestein, DVM, PhD Faculty of Veterinary Medicine, Dept. of Pathobiology, Section Pet Avian, Exotic Animals and Wildlife, Utrecht University, Yalelaan 1, 3584 CL Utrecht, the Netherlands Email: g.m.dorrestein@vet.uu.nl

317

Zoo/Exotic Pathology Service Sacramento, California, United States of America

A REVIEW OF AVIAN ENDOVENTRICULAR MYCOSIS SUBMITTED DURING 1998-2004

D. Reavill, DVM, Diplomate ABVP, certied in avian practice, Diplomate ACVP

KEYWORDS
Birds - Yeast - Ventriculus - Koilin

ABSTRACT
Endoventricular mycosis is a proliferation of yeast-like organisms within the koilin of the ventriculus. Fifty three cases from 13,950 avian histology samples collected over six years were identied from the database of Zoo/Exotic Pathology Service. All cases were necropsy ndings and ante-mortem diagnosis and therapy in the live bird is not reported.

1 INTRODUCTION
The lesion of endoventricular mycosis is a common one of the ventricular koilin in nch and nch-like birds (HUBBARD 1985). It is also described in ratites, psittacines, and galliformes (GRAHAM 1994). The reported clinical signs range from unexpected death to weight loss and passing intact seeds in the droppings. Various conditions such as recent shipping, crowded housing, reproductive activities, and mixed species aviaries are common recent stresses. Although antibiotic therapy is typically associated with secondary yeast infections, this is not a frequently reported occurrence with endoventricular mycosis (HUBBARD 1985).

2 MATERIALS AND METHODS

From 13,950 psittacine biopsy samples submitted, 53 cases of endoventricular mycosis were identied. Tissues were submitted xed in 10% buffered formalin. They were trimmed, embedded in parafn, sectioned at 5 m, and stained with hematoxylin-eosin. Follow-up information was obtained by mailing an inquiry form to referring veterinarians or by telephone conversation. The information requested was

318

for the species, sex, age, duration of clinical signs, any ancillary tests, survival, and any other therapies.

3 RESULTS
The most common birds with a diagnosis of endoventricular mycosis were passerines (n = 38) composed of nches (species not provided) (n = 7), Gouldians (Poephila gouldiae) (n = 7), cordon bleu (Uraeginthus bengalus) (n = 5), purple Grenadier (n = 2), orange cheeked waxbill (Estrilda melpoda) (n = 2), white-headed munia (Lonchura maja) (n = 2), red-tail laughing thrush (n = 2), mockingbird (n = 2) and one each bronze-winged mannikin (Lonchura cucullatus), canary (Serinus canaria), owl nch, zebra nch (Poephila castanotis), red-crested cardinal (Paroaria coronata), goldenfronted leafbird (Chloropsis aurifrons), bird of paradise, honeycreeper and starling. The nine psittacines were cockatiels (Nymphicus hollandicus) (n = 3), macaws (Ara species) (n = 3), and one each Amazon (Amazona species), conure (Aratinga species) and parrotlett (Forpus species). Smaller number of other species included blacksmith plover (n = 2), and one each amingo, trumpeter swan, guinea fowl and a quail (Coturnix coturnix japonica). There were 12 males and 13 females when the sex was known or reported. The age range was from 3 days to 24 years with an average of 4.35 years. Of the 50 survey responses, only 1 case reported blood work that included anemia and decreased serum protein. The common clinical signs reported included signicant weight loss (n = 21) and whole seeds passing in the droppings (n = 3). In only four birds were gross lesions noted in the proventriculus of dilation. Many birds (n = 31) had concurrent diseases. Nematode infections were most common (n = 9). Other parasitic infections included protozoa (coccidia, agellates, and cryptosporidia) (n = 5). Amyloid that was recognized only in the nches (n = 5), megabacteria or gastric yeast (n = 4), fungal pneumonia (n = 3), and proventricular dilatation disease (n = 2) were the next most common diseases. One case each of malignant lymphoma, cytomegalovirus, and systemic mycobacterium were recognized. From the responses and histopathology, eight of the nine psittacines had other serious diseases and conditions. Two macaws had proventricular dilatation disease, one conure had chronic cloacal papillomatosis, one cockatiel had chronic crop stasis, the Amazon had systemic mycobacterium, and three birds (a cockatiel, parrotlett, and macaw) were less than one week old with systemic diseases. Based on follow-up of the other avian species (42/44), there were 20 nches from aviary collections, 16 birds were from zoo collections, and three were wildlife rescues. Three of the nch aviaries reported using multiple water-based drug therapies. As the disease, endoventricular mycosis, was not identied ante-mortem in any of the birds, no specic therapies were attempted.

319

4 DISCUSSION
Endoventricular mycosis is seen in a variety of pet birds and is especially common in nches. Fungal organisms (usually Candida sp.) are found in the koilin layer and occasionally in the mucosa. Inammation is usually mild, and gross changes are rarely seen. The only gross lesion noted in our submissions was of proventricular dilation. Histologically, fragmentation of the koilin was a common associated lesion of yeast infections of the ventriculus. It is this fragmentation of the koilin and possible gastric dysfunction that may have resulted in the proventricular dilation, although two of the birds had proventricular dilation disease. These lesions, like those of bacterial infections of the koilin, may bleed causing anemia or rarely a fatal blood loss. Only one bird had ante-mortem blood work and it was anemic. Ante-mortem blood work was uncommon in these birds as many were nches in large collections or zoo birds, where individual testing was not performed. Many of these birds were under signicant stress; either induced by other diseases or husbandry situations (large aviaries, zoological displays, or wildlife rehabilitation). Known factors predisposing avian species to yeast infections include: prolonged antibiotic therapy, hypovitaminoses (especially Vitamin A), feeding spoiled, stale, or sour foods, a stressful environment with moist oors, dirty nests, and fecal contamination, malnutrition, and co-existing bacterial or viral infections. Although antibiotic therapy is typically associated with secondary yeast infections, many of our cases received no specic therapy. Only three nches came from aviaries reporting multiple water-borne drug therapies. It is interesting that the majority of endoventricular mycosis cases were in nches, whereas yeast infections in psittacines are more commonly isolated to the ingluvies.

5 CITATION INDEX
1. 2. GRAHAM D. Endoventricular mycosis: an avian pathologists perspective. Proc Assoc Avian Vet, Reno 1994; 279 - 282. HUBBARD G, SCHMIDT R, EISENBRANDT D, et al. Fungal infections of ventriculi in captive birds. J Wildl Dis 1985; 21: 25 - 28.

6 ACKNOWLEDGMENTS
Drs. Burke, Dazen, Douglass, Fisher, Fiskett, Froneeld, Fudge, Govers, Lane, Littlehale, MacCabe-Laduke, Mauroo, Nye, Okimoto, Orosz, Puzio, Rich, Riggs, Ristich, Roose, Sitinas, Stonebreaker, Swerida, and Tippit.

AUTHORS ADDRESS
Drury Reavill, DVM, Diplomate ABVP, certied in avian practice, Diplomate ACVP, Zoo/Exotic Pathology Service, 7647 Wachtel Way, Citrus Heights, CA 95610, United States of America Email: Dreavill@zooexotic.com

320

Department of Animal Science1,University of Camerino; Private practitioners2; Department of Animal Pathology3, University of Pisa; Department of Animal Production4, Epidemiology and Ecology, University of Turin, Italy

THE FIRST ITALIAN OUTBREAK OF POLYOMAVIRUS INFECTION IN MACAWS

S. Pesaro1, R. Ceccherelli2, C. Tarantino3, S. Scoccianti2, E. Bert4, G. Rossi1 KEYWORDS Avian polyoma virus - Outbreak - Macaws - Immuno-electron microscopy Polymerase chain reaction

ABSTRACT
Avian polyomavirus (APV) is one of the most signicant viral pathogens of cage birds. Macaws are susceptible to APV infection and disease is seen up to approximately 14 weeks of age, after which infection is asymptomatic. The mortality peak in macaws occurs from 4 to 8 weeks of age. In the literature descriptions of juvenile and adult macaws APV infections are infrequent compared to budgerigar and lovebird cases, and each report involves one or few subjects. In addition, until now, no reports of macaw deaths related to APV infection are described in Italy. We present the description of an outbreak of APV infection in three different breeding farms of the central area of Italy, characterized by high mortality in embryos, in juvenile birds (3 to 12 weeks of age), and in some adults (1.5 to 3 years of age). In total 10 Ara chloroptera (9 chicks and an adult), 13 Ara ararauna (12 chicks and an adult), 3 Ara spp.(chicks of 2 weeks of age), and 11 Ara spp. (eggs containing embryos of different age), were analysed. Macro- and microscopic lesions were typical of APV infection and, in all cases, viral presence was revealed by immunohistochemistry, transmission electron microscopy and immunogold techniques. In most cases PCR conrmed these tests and was the only test performed in eggs/embryos. No concurrent PBFD infection was detected in analysed macaws.

1 INTRODUCTION
Polyomaviruses and papillomaviruses are members of subfamilies of the Papovaviridae. Avian polyomaviruses (APV) that infect budgerigars, other psittacine birds and nches appear to be morphologically and, in some cases, antigenically

321

similar. However, evidence suggests that the genome of the virus that infects various psittacine birds and nches partially differs. Additionally, the clinical presentation, distribution of lesions and epizootiology of these viruses differ dramatically among susceptible species (CLUBB et al. 1984, RITCHIE et al. 1991). With regards to polyomavirus infections in mammals, characterized by apathogenic - subclinical infections in the natural immunocompetent host, the acute nature of some avian polyomavirus infections is typical of bird infection. The rst acute, generalized infection associated with APV was described in young budgerigars and was called budgerigar edgling disease (BFD) (LEHN et al. 1986). Because of the differences in the way avian polyomavirus affects budgerigars versus larger psittacine birds, the extensive information derived from studies in budgerigars may not be completely applicable to other psittacine birds (RITCHIE et al. 1991). Antibody surveys indicate that most infections in non-budgerigars psittacine birds are sub-clinical, and the immune response that occurs in these birds prevents the acute form of the disease. The most important information about APV infection in macaws derived from experimental infections and only sporadic descriptions of natural APV infection, characterized by per-acute death with no premonitory signs or following a 12-48-hour period of clinical changes (i.e. depression, anorexia, delayed crop emptying, diarrhoea, bleeding under the skin, polyuria and dyspnoea) are reported (RITCHIE et al. 1991). In addition, until now, no reports of outbreak of macaws death related to APV infection are described in Italy.

2 MATERIALS AND METHODS

During 2003, in a period of six months, 10 (Ara chloroptera) (9 chicks and an adult), 13 (Ara ararauna) (12 chicks and an adult), 3 (Ara spp.) (chicks of 2 weeks of age), and 11 (Ara spp.) (eggs containing embryos of different age) were analysed in our laboratory (Dept. of Veterinary Sciences, Camerino, Italy). These animals belonging to three different breeding farms of large psittacine birds of the central area of Italy, in which an outbreak characterised by high mortality in embryos, in juvenile birds (3 to 12 weeks of age), and in some adults (1.5 to 3 years of age) was observed. All birds, recently dead, were necropsied to identify the aetiology of the outbreak. Fresh smears of intestinal content were made and examined both for parasites and bacteriological tests. For the latter purpose, the faecal material was inoculated onto Desoxycholate Agar (Difco Laboratories, Detroit, Michigan, USA), and onto Campylobacter Kit Blaser (Difco Laboratories); some stool samples were enriched using Selenite Broth (Difco Laboratories) and then inoculated onto SSA (Salmonella-Shigella Agar) and BGA (Brilliant Green Agar) (Difco Laboratories). Blood smears were stained with May Grnwald - Giemsa for haemoprotozoan examination. Samples of liver, spleen, kidney, intestine, bursa, pancreas, lungs, heart, central nervous system and skin were collected from all birds, xed in 10% buffered formalin, and embedded in parafn; 3 m sections were stained using haematoxylin and eosin and periodic acid Schiffs reaction (PEARSE 1985) for histological examination. Sections were also mounted on positively charged glass slides (Superfrost Plus, Fisher, Pittsburgh, PA), routinely deparafnized and then immersed in distilled water containing 15% H2O2 for 30 min at room temperature in order to inactivate endogenous peroxidases. The slides were

322

rinsed in phosphate-buffered saline (PBS) three times for 2 min each, followed by a proteinase K (DAKO Corporation, Carpinteria, CA) treatment at room temperature for 6 min. In order to minimize non specic background staining, the sections were incubated in a serum blocking solution (10% non-immune horse serum in 1% bovine serum albumin dissolved in PBS) for 1 hr at 37 C. The sections were then incubated with primary anti-VP-1 antibody (KHAN et al. 2000) diluted 1:500 in PBS overnight in a 4 C moist chamber, rinsed in PBS and incubated with a biotinylated anti-mouse secondary antibody for 45 min at room temperature. After three rinses in PBS, sections were incubated with enzyme conjugate (ABC-peroxydase complex, Vector, Burlingame, UK) for 45 min at room temperature, followed by incubation with 3,3diaminobenzidine solution containing H2O2 as substrate-chromogen for 5 min. The slides were rinsed in distilled water, rapidly counterstained with haematoxylin and examined microscopically. Negative controls included normal bird tissues and positive controls included psittacine bird tissues infected by APV. In addition, portions of liver, spleen and kidney were immediately xed in phosphate buffered 0,1M 2% glutaraldehyde pH 7.4, postxed in phosphate buffered 1% OsO4 and, after dehydration, embedded in Epon/Araldite (Polyscience Inc., Warrington, Pa). Semi-thin sections were stained with methylene blue and Azure II while for immuno-electron microscopy, ultra thin sections (70nm) were placed on 200-mesh nickel grids supplied with formvar-carbon lm (Agar Scientic Ltd., Stansted, UK), etched with sodium metaperiodate for 30 min, and oated on drops of 10% normal goat serum, 0.2% saponin, and 0.1% bovine serum albumin (BSA) plus 0.05% Tween 20 in PBS (BTP). Sections were then incubated for 24 hr at 4C with either anti-VP-1 mouse monoclonal antibody (6), or non immune mouse ascitic uid (both diluted 1:10 in BTP containing 1% normal goat serum and 0.2% saponin). Finally, the sections were incubated with goat anti-mouse immunoglobulin G gold conjugated, 10nm (Sigma, Chemical Co., St. Louis, Missouri) diluted 1:50 in BTP containing 1% normal goat serum and 0.2% saponin for 60 min. Grids were then xed in 1% glutaraldehyde and stained with uranyl acetate and lead citrate and examined with a JEOL 1200-EX transmission electron microscope (JEOL, Peabody, MA). Control procedures included the use of a known polyomavirus infected tissue. Tissues and embryos at different stage of development were also sampled and utilised for PCR examination. For this purpose DNA extraction: DNA was extracted from animal tissues using the Wizard genomic DNA purication kit (Promega USA) with some modications. Amplication for BFDV: the amplication was carried out with primers n2 and 4 described by YPELAAR et al. (1999), that amplied a 717 bp tract of the BFDV ORF1. Primer sense (n2): AAC CCT ACA GAC GGC GAG. Primer antisense (n4): GTC ACA GTC CTC CTT GTA CC. The PCR amplication was carried out in a total volume of 25 l. The nal reaction conditions were as follows: 50mM KCl; 10 mM Tris-HCl pH9; 1.5mM MgCl2; 200mM of each dNTP; 100 ng of each primer and 0.15 units of Taq polymerase (Promega USA). PCR was performed in a GeneAmp PCR System 2400 (Applied Biosystem -USA) thermal cycler. An initial denaturing step at 94C for 2 min was followed by 32 cycles of 60C for 30s, 72C for 1 min and 94C for 1 min. A nal run of 72C for 7 min completed the program. PCR products were separated by

323

electrophoresis for 45-60 min at 7-10V/cm in a 2% agarose gel stained with ethidium bromide. Amplication for APV: The amplication was performed using primers which have been published by PHALEN et al. (1991), according to the procedure suggested by the authors. 3 RESULTS Similar lesions were observed in all necropsied birds; all birds had abdominal distension with spleno- and hepatomegaly with irregular red and yellow mottling of the liver. Haemorrhages were also observed in spleen, heart, meninges and, in some cases, in skin and skeletal muscles. In the most of affected birds, kidneys were pale and swollen and hydropericardium was also evident. The lungs were generally congested and contained an exudate. A large number of birds showed severe thickening of the intestinal walls with haemorrhage. No bacterial organisms were isolated from cultures prepared with material collected on necropsy. Histological examination evidenced in all birds various tissue lesions characterized by the presence of typical karyomegalic, amphophilic intra nuclear inclusions, suggestive of an APV infection. Especially in the liver, the presence of intra nuclear inclusions was accompanied by multi-focal hepatocellular necrosis and haemorrhages. In some cases inammatory cell inltrates, consisting in multi focal heterophilic to lympho-plasmacitic cells, surrounded the areas of hepato-cellular necrosis, strictly associated with hyper-plastic aspect of the Kupffer cells. Similar aspects of variable degrees of epithelial necrosis, accompanied by interstitial inammatory inltrates, were also observed in renal tubular epithelial cells, but in these cells intra-nuclear inclusions were less frequently observed. In the spleen as well as in the bursa, severe aspects of depletion and lymphocytic apoptosis phenomena were observed; in the same tissues inclusion bodies were present within intimate cells of arterioles. In addition, various degrees of myocardial necrosis were detected in of necropsied birds. Ultra-structural examination of affected tissues demonstrated the presence of aggregates of a great number of 42- to 49 nm viral particles inside intra-nuclear inclusions. Using monoclonal VP-1 specic antibody, immuno-histochemistry documented APV infection in damaged tissues belonging to all dead birds, which demonstrated typical endo-nuclear inclusions. The positive reaction was characterized by intense brown chromogen deposition in large inclusions, indicating the antibody recognition of VP-1 specic of APV. All negative control tissues were devoid of chromogen deposition. Similar results were obtained in immuno-electron-micropic tests, in which endo-nuclear aggregates of viruses were labelled by gold particles. PCR results demonstrated the presence of DNA of polyomavirus in different organs of chicks and in embryos.

4 DISCUSSION
Acute and chronic epornitics of avian polyomavirus (APV) infection have been observed in various genera and species of juvenile and adult psittacine birds, especially budgerigars (Melopsittacus undulatus), lovebirds (Agapornis spp.), cockatiel (Nymphicus hollandicus), Amazon parrots (Amazona spp.) and African

324

gray parrots (Psitacus erithacus), blue and gold macaws (Ara ararauna), white bellied caiques (Pionites leucogaster), painted conures (Pyrrhura picta), moluccan cockatoos (Cacatua moluccensis), quaker parakeets (Myiopsitta monachus), graycheeked parakeets (Brotogeris pyrrhopterus), rose-ringed parakeets (Psittacula krameri), splendid parakeets (Neophema splendida), eclectus parrots (Eclectus roratus) and pionus parrots (Pionus spp.) (RITCHIE et al. 1995). In previous reports, different Authors describe clinical signs, necroscopic ndings, and histological lesions observed in one or few animals during spontaneous episodes of APV infection. In macaws, the most important observations are performed during experimental infections and very little is known about the possibility of APV embryos infection. In addition, no reports regarding APV outbreak in macaws are described in Italy. Our report, conrms the susceptibility of macaws to APV infection and the fact that the disease up to approximately 14 weeks of age, and the peak mortality occurs from 4 to 8 weeks of age, as reported by different authors (PHALEN et al. 1992, 1993, RITCHIE et al. 1991). According to the literature (PHALEN et al. 1992, RITCHIE et al. 1995) the most relevant lesions observed in necropsied birds were represented by vasculitis (related to diffuse haemorrhages) with presence of inclusion bodies in the walls of blood vessels, membranous glomerulopathy with focal necrosis (and rare inclusion bodies), depletion and death of lymphocytes in the bursa, necrosis of skeletal and myocardial muscular cells, necrotic foci in spleen parenchima and hepatic foci of centrilobular necrosis with abundant, IHC-positive inclusion bodies. Polyomavirus persistent infections are frequently described in mammals and in birds, particularly in budgerigars (PHALEN et al. 1993). Several studies suggest that these subclinically infected birds, when stress or concomitant diseases intervene, shed virus resulting in outbreaks of disease (PHALEN et al. 1993). In addition, when various causes provoke a similar deciency in the immune system, this might be responsible for the occasional APV-induced death in adult persistently infected psittacine birds or the frequent deaths that occur in some born infected young birds (RITCHIE et al. 1995). Therefore, this is the rst report of an acute APV outbreak in embryos and young macaws in Italy, and conrms that young macaws were very susceptible to avian polyomavirus acute and symptomatic infection. On the basis of the macro and microscopic ndings, we can conclude that polyomavirus-induced lesions suggested an immune-mediated disease, and the histological ndings are related to high prevalence of immune-complex production during the disease. It is possible to explicate the tissue lesions represented by membranous glomerulonephritis, generalized vasculitis, and immune-mediated hepatitis.

5 CITATION INDEX
1. 2. CLUBB SL and DAVIS RB. Outbreak of papova-like viral infection in a psittacine nursery retrospective view. Proc Assoc. Avian Vet, Boulder 1984: 121 129. KHAN MS, JHONE R, BECK I, et al. Development of a blocking enzymelinked immunosorbent assay for the detection of avian polyomavirus-specic antibodies. J Virol Methods 2000; 89; 39 48.

325

3.

LEHN H and MULLER H. Cloning and characterization of budgerigar edgling disease virus (BFDV), an avian Polyomavirus. Virology 1986; 151; 362 - 370. 4. PEARSE AGE. Histochemistry, theoretical applied. Analytical technology, 4th edition, Edinburgh: Churchill Livingstone Inc, 1985; 735 - 832. 5. PHALEN DN, WILSON VG and GRAHAM D.L. Polymerase chain reaction assay for Avian Polyomavirus. J Clinical Microbiol 1991; 1030 - 1037. 6. PHALEN DN, WILSON VG and GRAHAM DL. Avian polyomavirus infection and disease: a complex phenomenon. Proc Assoc. Avian Vet, New Orleans 1992: 5 - 10. 7. PHALEN DN, WILSON VGH and GRAHAM DL. Avian polyomavirus biology and its clinical applications. Proc Eur Assoc Avian Vet, Utrecht 1993: 200 - 216. 8. RITCHIE BW and CARTER K. Avian viruses: function and control. MATTEWS J., HUDELSON S.K., and HUDELSON P. (eds). Lake Worth: Wingers Publishing Inc, 1995; 136 - 167. 9. RITCHIE BW, NIAGRO FD, LATIMER KS and DAVIS RB. Avian polyomavirus: an overview. J. Assoc. Avian Vet. 1991; 5: 147 - 153. 10. YPELAAR I, MR BASSAMI, GE WILCOX and RAIDAl SR. A universal polymerase chain reaction for the detection of Psittacine Beak and Feather Disease Virus. Vet Microbiol 1999; 68: 141 - 148.

AUTHORS ADDRESS
G. Rossi, DVM, PhD Department of Animal Science, University of Camerino, Via della circonvallazione 93/95 62014 Matelica MC, Italy Email: giacomo.rossi@unicam.it

326

Angell Animal Medical Center, Boston, United States of America

CLOACAL DISEASE AND DISORDERS IN THE AVIAN PATIENT

T. K. Ritzman, DVM, Dipl ABVP (Avian)

KEYWORDS
Cloaca Avian Coprodeum Urodeum Proctodeum Cloacoscopy Cloacotomy

ABSTRACT
The cloaca is a complex structure in the bird. It functions as a holding and processing area for products of the gastrointestinal, reproductive and urinary systems. Due to the complexity of the cloaca and the pertinent anatomical structures associated with it, the health of this structure is essential to the well being of the avian patient. The anatomy and physiology of the cloaca will be discussed as well as the diagnosis and treatment of different cloacal conditions. Cloacal disorders discussed will include cloacal infection and inammation, prolapse, obstruction and neoplasia. Specic case examples are included for each of these categories.

1 INTRODUCTION
The cloaca is comprised of three areas: the coprodeum, the urodeum and the proctodeum. The coprodeum is the connection between the distal colon and the cloaca. The urodeum contains the openings of the ureters and the genital ducts. A fold within the cloacal lumen called the uroproctodeal fold separates the urodeum and the proctodeum. The proctodeum communicates with the outside of the birds body through the vent. The opening and closing of the vent is controlled by striated sphincter muscle. The Bursa of Fabricius is a diverticulum of the dorsal wall of the urodeum. The bursa is involved with the immune function of the bird and is the location of B-lymphocyte differentiation. The bursa reaches its largest size at eight to twelve weeks of age and decreases in size as the bird ages. In male birds of some species, a copulatory organ may be housed within the cloaca. Anseriformes, ratites and an occasional psittacine species (Vasa parrot, Coracopsis vasa) have phallic bodies housed within the cloaca. The coprourodeal fold prevents faecal contamination of the urodeum and proctodeum and during defecation protrudes through the vent to serve this function. During the egg laying process the coprourodeal fold will protrude

327

in a similar manner. Most birds reabsorb water from the urine held in the urodeum. Urine deposited from the ureters into the urodeum often moves retrograde into the rectum where this reabsorption takes place (SKADHAUGE 1968). Stress-induced polyuria can occur if the bird releases the cloacal contents too quickly for normal urine processing and water reabsorption to occur. Common causes of cloacal disease The causes of cloacal disease can be related to any of the systems associated with this structure. Causes of cloacal abnormalities include bacterial or fungal infection, inammation, faecalith or urolith formation, retained eggs, prolapse, or neoplasia. Cloacaliths, with multiple lamellar layers, can develop into large concretions of several centimetres in size causing severe dilatation and obstruction of the cloaca. Masses can develop from the cloacal mucosa or associated structures. Cloacal papillomatosis can produce proliferative lesions of the cloacal mucosa (SUNDBERG 1986, PHALEN 1997). The author diagnosed a case of complete cloacal obstruction in an umbrella cockatoo (Cacatua alba) from a fungal granuloma. Cloacal neoplasia is an infrequent diagnosis but should be included as a differential for cloacal wall thickening or luminal masses (ANTINOFF 1997). Cloacal lymphoma was diagnosed by the author in an Indian ring-necked parakeet (Psittacula krameri) from cloacal mucosa biopsies obtained via cloacoscopy. Vent stricture and subsequent cloacal obstruction can occur in avian patients secondary to distal cloacal and external vent sphincter abnormalities. Diagnosis of Cloacal Disease Diagnosis of cloacal disease is often possible during the initial examination of the bird. A thorough anamnesis should include information on age, gender, reproductive status and activity, diet and management. The avian patient with a blockage of the cloaca will present with clinical signs of obstructive disease (RITZMAN 1999). Clinical signs of cloacal disease can vary but include tenesmus, haematochezia, decrease or change dropping appearance and production, diarrhoea, atulence, soiling of vent area, lethargy, anorexia, change in perching posture, inability to breed or produce eggs normally, prolapse of cloacal mucosa or other cloacal structures, or mass effect in the caudal abdominal region. If cloacal disease is due to a mass effect within the cloacal lumen, respiratory signs may be apparent due to impingement of the abdominal airsacs. Routine haematology including a complete blood count and chemistry prole will often be normal. With cloacal inammation or infections a change in the protein electrophoresis pattern may occur. Some birds with cloacal infection may develop a heterophilic leukocytosis. If the cloaca becomes obstructed congestion of the ureters can occur, causing possible renal failure and haematological changes such as an elevation in uric acid values. A thorough cloacal examination should be performed on every bird as part of the routine physical assessment. The vent, or cloacal orice, should be examined prior to performing an internal cloacal exam. Change in vent size or symmetry, tone or sensation may be present. After the vent has been evaluated, an internal cloacal exam can be performed. A cursory examination can be performed in the restrained

328

bird. A sterile, lubricated, appropriately sized swab should be gently inserted into the cloaca. Intraluminal masses and mucosal inammation or ulceration can be detected with this swab technique. The distal cloacal mucosa can then be gently everted for examination. The mucosa should appear moist and pink with a uniform appearance. Any masses or changes in mucosal consistency should be further evaluated. Papillomatosis of the cloaca can cause proliferative mucosal changes (SUNDBERG 1986, PHALEN 1997). Application of dilute acetic acid (vinegar solution) to the cloacal mucosa has been reported to increase suspicion of papillomatous changes by causing a white discoloration of the affected mucosal tissue. A denitive diagnosis of papillomatosis is made from histopathologic examination of a biopsy (SUNDBERG 1986, PHALEN 1997). Swab samples for bacterial or fungal cultures can be collected. A Grams stain should be performed on a faecal sample collected from the cloaca. Cloacal cytology can be useful in diagnosing disorders of the lower intestinal tract, reproductive tract, urinary tract or cloaca itself. Cell samples collected from the cloaca may originate from any of these organ systems and additional diagnostics are required to localize abnormalities. Normal cloacal cytology often includes epithelial cells (noncornied squamous or columnar), urate crystals, extracellular bacteria, plant and faecal material and other background debris. Direct visualisation of the cloacal lumen is the most effective way to diagnosis cloacal disease. The internal visual cloacal exam can be performed in several ways. The size of the patient dictates equipment potential. In small birds, it may not be possible to safely insert anything into the cloaca. In medium to large-sized birds, however, there are several options. An otoscope with a sterile cone tip can be used but has limited range of view. Infusion cloacoscopy is an ideal diagnostic tool for internal cloacal visualisation and described by Dr. Michael Taylor (TAYLOR and MURRAY 2002). A 2.7 mm rod lens endoscope with an instrumented sheath system works well for an internal cloacal examination (TAYLOR and MURRAY 2002). Internal cloacal structures such as the ureteral openings, oviductal orice and distal colon can often be examined. Insufating the cloaca with an infusion of a liquid such as sterile saline will aid in visualisation (TAYLOR and MURRAY 2002). Cloacal mucosal biopsies may be taken at the time of the endoscopic examination. Care must be taken to choose a biopsy site devoid of pertinent anatomic structures, and full-thickness biopsies are contraindicated in most cases. Radiography is a useful diagnostic modality in the diagnosis of obstructive cloacal disease. For initial assessment, survey radiographs can be taken. A whole body ventrodorsal and lateral view should be taken to allow for evaluation of the entire gastrointestinal tract, reproductive organs and urinary systems. Obstructive conditions in the cloaca can cause subsequent dilatation of the distal intestinal tract or changes in other related organ systems. If survey radiographs indicate any abnormalities of the gastrointestinal system, contrast radiography can then be utilised to further delineate structures. A contrast agent such as dilute barium suspension can be administered by tube into the proximal gastrointestinal tract (crop). Timed radiographs are then taken after contrast administration. For most cases, 15 minute, 30 minute and 60 minute views are taken. The number and timing of the radiographs

329

must be adjusted according to the patient size and condition. Endotracheal intubation should be performed, if possible, to prevent potential aspiration of the contrast agent. Gastrointestinal emptying times vary according to species and patient size, and references should be consulted when evaluating the contrast lms. Fluoroscopy can be utilised to evaluate the gastrointestinal motility and function of the bird. A localized contrast study can be performed on the cloaca itself. This diagnostic test, although useful, is rarely necessary for the diagnosis of cloacal obstruction since direct internal visualisation is often diagnostic. For this study, a exible catheter can be inserted into the cloaca, and the contrast agent (usually 3- 6 ml) can be directly infused, into the cloacal lumen. After the contrast agent has been infused an immediate radiograph can be taken. Any masses within the cloacal lumen will be outlined with the contrast agent. Retroperistaltic movement of the contrast agent into the colon is normal and will often be noted with this study. Treatment Options Cloacal conditions can either be managed medically, surgically or with a combination. The optimal treatment program is dependent on the underlying cause of the condition. Because many cloacal disorders are caused by a prolapse or mass effect within the cloaca, surgical intervention if often required. Cloacoscopy can be utilised for the biopsy of internal cloacal masses and the treatment of papillomatosis and debridement of cloacal lumen masses. A technique is being developed utilizing diode laser application via cloacoscopy with uid insufation by Dr. Stephen HernandezDivers at the University of Georgia, College of Veterinary Medicine. There is potential in the future for development of a bipolar cautery through the rigid endoscope that may replace the diode laser technique and be more readily available to the veterinary clinician. Laser ablation therapy via cloacoscopy was utilised at the University of Georgia, College of Veterinary Medicine to treat a case of cloacal adenocarcinoma (HERNANDEZ-DIVERS 2004). For maximum visualisation of the cloacal lumen and removal or resection of large masses, a cloacotomy may be required (DVORAK et al. 1998). A cloacotomy allows for direct visualisation of the cloacal lumen and facilitates removal of masses (e.g. cloacaliths or eggs) or resection or biopsy collection of abnormal tissues. A surgical technique has been described for the treatment of cloacal papilloma via a cloacotomy and cloacal mucosal stripping (ANTINOFF 2000). For the cloacotomy procedure, the patient is anesthetised and placed in dorsal recumbency. The caudal abdomen is prepared and an incision is made over the cloaca. The shape of the incision made depends on the size of the patient and the exposure required. For maximum exposure, an L-shaped incision can be made. After the skin is incised, the subcutaneous tissues are gently dissected. The thin abdominal muscle wall and pleuro-peritoneum must be incised, and in most birds, there is a subcutaneous layer of fat that must be reected prior to encountering the cloacal serosa. Once the cloacal serosa is exposed, a fullthickness incision is made through the cloacal wall. Care should be taken to avoid normal anatomic structures of the cloaca such as the ureteral openings or reproductive structures. Once the cloacal lumen is exposed, a complete visual examination can be

330

performed and any masses gently lifted out of the cloaca or resected. Biopsy samples can be collected, or in the case of papillomatosis, resection of affected tissue can be performed. Because the cloacal mucosa is often inamed or infected, haemorrhage of the tissue is quite common. Gentle tissue handling techniques will minimize mucosal bleeding, and absorbable sponges (Gelfoam, Upjohn, Kalamazoo, MI) can be placed within the cloacal lumen to control haemorrhage and promote haemostasis. If cloacal prolapse has occurred, cloacapexy can be performed at the time of the cloacotomy. Closure of the cloacal wall is made with an interrupted or continuous inverting pattern with absorbable suture. The body wall and skin are closed with a simple continuous or interrupted pattern. The condition of the vent should also be assessed at the time of surgery. Cloacal prolapse, egg binding and other obstructive cloacal conditions can lead to vent stretching and atony. If indicated, ventplasty surgery can be performed in order to reduce vent size and prevent future prolapse. Care should be taken to maintain adequate vent diameter to allow for normal defecation and reproductive activity. If there is a history of cloacal obstruction due to egg binding in a female bird not intended for breeding, medical or surgical prevention of egg laying may be indicated. The patient should be supported post-operatively with analgesia, uids and general supportive care. Inammation and infection of the cloacal mucosa can lead to ascending infections of the gastrointestinal tract, reproductive tract, or ureters; antimicrobial and/or antifungal medications should be given if indicated. Nutritional support should be initiated as soon as the patient can be fed. A readily digestible diet such as an extruded commercial avian food is recommended as the base of the diet. If the bird normally eats a seed-based diet, a gradual conversion to the formulated diet is recommended.

2 CONCLUSION
Cloacal disease is a common clinical entity in the avian patient. Regular cloacal examinations are recommended for all avian patients, and those diagnosed with cloacal disease may require more frequent evaluations. Cloacal assessment is recommended at regular three to six month intervals for the rst year post-cloacotomy and at least annually for subsequent years. Clients can be informed on how to monitor their birds for cloacal dysfunction, which will allow for early diagnosis of any problems and prevention of obstruction. Close monitoring of the droppings and the external vent area as well as regular examinations will aid in the prevention and early diagnosis of cloacal disease.

3 CITATION INDEX
1. 2. ANTINOFF N, HOEFER HL, ROSENTHAL KL, et al. Smooth muscle neoplasia of suspected oviductal origin in the cloaca of a blue-fronted Amazon parrot (Amazona aestiva). J Avian Med Surg 1997; 11(4): 255 - 259. ANTINOFF N. Treatment of a cloacal papilloma by mucosal stripping in an Amazon parrot. Proc Assoc Avian Vet, Portland 2000: 97 - 100.

331

3. 1. 4. 5. 6. 7. 8. 9.

DVORAK LR, BENNETT RA and CRANOR K. Cloacotomy for excision of cloacal papillomas in a Catalina macaw. J Avian Med Surg 1998; 12 (1): 11 - 15. 4.. HERNANDEZ-DIVERS S, Personal communication, Department of Small Animal Medicine & Surgery, College of Veterinary Medicine, University of Georgia, 2004. LUMEIJ JT. Gastroenterology. In: RITCHIE B, HARRISON G, and HARRISON L (eds): Avian medicine: principles and application. Lake Worth, FL: Wingers, 1994; 509 - 512. PHALEN DN. Viruses. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM, and QUESENBERRY KE (eds). Avian medicine and surgery. Philadelphia: WB Saunders, 1997; 310 - 312. RITZMAN TK. Obstructive cloacal conditions in the avian patient. Proc Assoc Avian Vet, New Orleans 1999: 87 - 91. SKADHAUGE E. Cloacal storage of urine in the rooster. Comp Biochem Physiol 1968; 24: 7 - 18. SUNDBERG JP, JUNGE RE, OBRANION MK, et al. Cloacal papillomas in psittacines. Am J Vet Res 1986; 47(4): 928 - 932. TAYLOR M. and MURRAY MJ. The psittacine cloaca: a clinical review. Proc Assoc Avian Vet, Monterey, 2002: 265 - 269.

AUTHORS ADDRESS
Tracey K. Ritzman, DVM, Dipl ABVP-Avian Practice Angell Animal Medical Center-Boston, 350 South Huntington Ave, Boston, MA 02130, United States Email: tritzman@angell.org

332

Clinique Vtrinaire de lArche, Valence, France

AURICULAR DISEASES IN BIRDS

F. Rival, DVM

KEYWORDS
Birds - Ear - Otitis - Tympanic membrane

ABSTRACT
This paper intends to describe the anatomy and physiology of the ear and the most common diseases as seen in private practice. The avian ear, an organ of hearing and balance, is composed of an external part, often hidden by specialised feathers, a middle part with an air lled tympanic cavity and an inner part, a complex structure with the membranous labyrinth. Diseases of the ear are not very common in birds, but the practitioner must be familiar with and be able to recognise them when encountered.

1 INTRODUCTION
The ability to listen and vocal communication play an important role in the natural life of birds. Birds possess a highly evolved auditory system and their hearing performance is not necessarily inferior to those of most mammalian species.

2 ANATOMY AND PHYSIOLOGY OF EARS

The avian ear, an organ of hearing and balance, is composed of an external part, often hidden by specialized feathers, a middle part with an air lled tympanic cavity and an inner part, a complex structure with the membranous labyrinth. In birds, the outer or external ear, unlike many mammals, have no pinna. The short canal (external acoustic meatus) leading to the tympanic membrane (eardrum) is covered by special feathers that lack barbules and do not obstruct sound transmission. They are called ear coverts or auriculars. The tympanic membrane, at the end of the external acoustic meatus, protudes cone-like into the meatus. This is caused by the processes of the extracolumellar cartilage. This can be observed during endoscopy examination of the canal.

333

2.1. Middle ear The middle ear is an air lled tympanic cavity containing a muscle, ligaments, the tympanic membrane, the cochlear (round) window (accessory tympanic membrane) and a rod-like bone (ossicle) known as the columella. This single ossicle consists of two segments: a cartilaginous extracolumella and the bony columella proper. The columella picks up sound vibrations from the eardrum and transmits them by the footplate at the proximal end to a membranous oval window in the inner ear. Birds differ from mammals in that they have only one bony ossicle (the columella is equivalent to the stapes) while mammals have three (malleus, incus, and stapes). The infraorbital sinus in most birds opens dorsally into the middle and caudal nasal conchae and is connected by small canals in the tympanic cavity with the cervicocephalic air sacs (KRAUTWALD 1998). 2.2. Inner ear The inner ear is the sensory receptor for both sound and equilibrium. It consists of the cochlear organ and a vestibular organ. The cochlear organ contains the basilar papilla as a sensory epithelium for hearing. The cochlea is not coiled as in mammals but slightly curved.

3 SOME AVIAN CHARACTERISTICS

3.1. Hair cells Loss or damage of sensory or hair cells in the inner ear results in hearing impairment. These cells can be damaged by overexposure to intense sounds. In mammals, once these hair cells are lost, they are not replaced, so hearing loss is permanent. Birds, however, have the remarkable ability to regenerate and replace the hair cells of the inner ear following damage or loss (DOOLING and DENT 2001). Twenty eight days after an exposure to a chemical (kanamycin) that destroys hair cells the number of hair cells in the regenerated bird ear had nearly returned to normal levels (DOOLING and DENT 2001). 3.2. Hearing ranges of birds The structure of a birds ear is simpler than in humans, but their hearing seems to be about the same. The range of frequencies (pitch) is similar, though the sensitivity to loudness is reduced. For most birds, hearing is best at frequencies of 1 - 4 kHz, but some birds are able to detect much higher frequencies (up to 10 - 12 kHz). The ability of birds to discriminate differences in the frequency of sounds and to detect gaps between sounds is generally similar to humans.

However, birds appear less sensitive to higher and lower vocal tones as compared to humans. Their ability to differentiate various sounds is ten times faster than humans. In other words, a canarys song would have to be slowed down ten times before the

334

human ear could identify all the notes that are learned by a canary chick. Most birds, including parrots, do not hear ultrasonic vibrations. 3.3. Sound localization Birds have good hearing but they hear things differently to humans. For birds it is important to localize the vocalisations or other sounds (and therefore a prey). Some birds can echolocate by producing specic sounds. There is considerable evidence that birds can and do localise the vocalisations (or other sounds) of their own and other species. Many owls are extremely capable of localising the source of sounds (and therefore, prey).

They can locate a mouse by its sound. While most sounds can be distinguished quite well with one ear alone, pinpointing where sounds are coming from requires a complex process called binaural fusion, where the brain compares information received from each ear and translates subtle differences into a perception of a single sound coming from a particular location. The ability to identify where sounds are coming from based on auditory cues is common to all hearing creatures, but owls excel at this task. These birds exhibit such extraordinary sound localization abilities that they are able to hunt in total darkness. Barn owls (Tyto alba) can locate prey by sound alone and this remarkable sensitivity and accuracy is achieved by having: A facial disc (like a satellite dish) that funnels sound to the eardrums. Ear openings that are asymmetrical, i.e. one opening higher than the other, which means sounds appear louder in one ear than the other and helps to determine the distance and direction of the source of the sound. Flight feathers that have soft rounded edges allowing the owl to y silently. This also means that they do not hear their own wing beats.

3.4. Echolocation Echolocation occurs in 2 families of birds: Steatornidae (oilbirds) and Apodidae (cave swiftlets). Used for orientation in dark caves. Birds (and other animals) echolocate by producing clicking sounds and then receiving and interpreting the resulting echo. Penguins also appear to hunt by echolocation while underwater. 3.5. Ear opening The outer ear canal membranes are open at hatching in cockatoos and king parrots. In eclectus parrot (Eclectus roratus) hatchings, the ear canals are covered by a thin membrane that has tiny openings evident by day 2. The ear canals of macaws are also covered at hatching, but do not begin to open until approximately 23 days. In many macaws and eclectus parrots the ear membrane opens very slowly creating pinpoint holes that dilate fully in four to ten days.

335

4 AURICULAR DISEASES
Ear problems are not very common in birds. There are disorders specic to the ear and other more generalised diseases that may affect the ears. 4.1. Specic auricular diseases 4.1.1. Otitis externa Otitis externa is an inammation of the external ear. It may by caused by bacterial or fungal organisms. Pruritus may be present causing the bird to scratch the ear or rub the head on the perch. The ear opening may be swollen and red and the feathers may be matted with a discharge. A serous to purulent discharge may be noted. If there is a bacterial infection, the tissues become hyperaemic and swollen and the opening is no longer clear. It has been reported (KERN 1994) that the ear openings may also appear hyperaemic in birds with sinusitis. The infraorbital sinus has rostral and caudal extensions near the ear. Infraorbital sinusitis can involve the ear. The most frequent bacteria isolated include Pseudomonas aeruginosa, Klebsiella sp, Enterobacter sp and Kocuria kristinae (DESMARCHELIER 2004 personal communication) a human opportunist bacteria. Lovebirds seem to be more prone to external ear infections (otitis externa) than other species of birds. 4.1.2. Haemorrhage from the ear Trauma can occur and a haemorrhage in the external ear is usually due to a cranial injury. Acute or chronic bleeding from the external meatus may be present, with or without signs of vestibular dysfunction. Deafness is a possible sequel. Benign neoplasia of unspecied type was reported to be the cause of haemorrhage in one Amazon parrot. 4.1.3. Occlusion of the external openings may occur in macaws. Macaws hatch with a thin membrane covering their ear canals. This membrane should start to open in 12 - 35 days. If it does not, it must be opened surgically. 4.1.4. Foreign body It is possible to nd foreign bodies in birds ear, e.g. grass lawn seed. 4.1.5. Parasitic diseases Some parasites are seen near or in the ear of birds such as y larvae, hypoboscis, Cnemidocoptes pilae or C. mutans. The ea Echidnophaga gallinacea, is often localized on the head and near the ears (BOURDEAU 2004 personal communication). Harvest mites of the family Trombiculidae may enter the ear. 4.1.6. Neoplasia Neoplasia can affect the ears. Carcinoma of the ceruminous gland of the ear, squamous cell carcinoma (KERN 1997) are the most common neoplasm and must be differentiated from chronic infection. However, squamous cell cancers are much more aggressive than basal cell cancers.

336

4.1.7. Balance problem Birds with balance problems and regurgitate after rubbing on their toy, may have ear infections. 4.1.8. Non specic diseases TELL et al. (2001), described a mycobacterial infection in a pionus parrot (Pionus menstruus) with weight loss, lethargy, and a mass protruding from the left ear. ABRAMS et al. (2001), described an Aspergillus blepharitis and dermatitis in a falcon with generalized feather loss. This loss was present with thickened, crusty lesions extending around eyes, over the top of the head and invading the ear. Fibroepithelial hyperplasia is a rare lesion of the external acoustic meatus. We have seen this in a PBFD positive kakariki (Cyanoramphus sp). Viral agents such as paramyxovirus (PMV) can cause otitis interna, resulting in loss of balance and torticollis. Clinical signs associated with PMV infection in companion avian species are dependent on the strain of virus and the age, species and condition of the bird affected. Neurological signs may range from depression, ataxia, hyperexcitability, torticollis, opisthotonus, unilateral or bilateral paralysis of the legs and wings, circling and seizures. Histopathologic lesions from PMV within the central nervous system are usually noted in the cerebellum, cerebrum, brain stem and spinal cord, more than in the ear. In pigeons, a condition loosely described as vertigo has been reported quite commonly and whilst this is a non-specic term which may be applied to a variety of pathologies, e.g. PMV1 paratyphoid or polyneuritis, the possibility of otitis should not be overlooked nor should the ears be overlooked during post-mortem examination. Otitis media and interna may result from septicaemia. Pseudomonas aeruginosa infection was reported in an African grey parrot (Psittacus erithacus). Chronic Pasteurella multocida infection is a frequent cause of otitis media and interna in poultry (KERN 1997). Vascular or cardiac disease with associated anaemia, have also been incriminated. Ostriches (Struthio camelus) can develop poxvirus-induced lesions on the eyelids, face, feet and around the opening to the external ear

5. CLINICAL EXAMINATION AND DIAGNOSIS

Early in the illness, many birds have no obvious clinical signs, and it is important to note if any of these signs are present: Pruritus. Skin changes around the ear. Crusts, ulcers or proliferative (excessive growth) tissue. Bleeding. Odour (rare). Discharge. Nodular masses. Large growths lling the ear canal. Vestibular (balance) signs. The opening is usually rounded but can vary in diameter from small (2.0 to 15.0 mm in passerine and psittacine birds) to very large. A 1.9 mm telescope is often needed to explore the deeper aspects of the canal. In birds, ears are hidden from

337

view. To nd the ears, look behind and below the eye. The feathers directly over the ears have a slightly different texture and appearance. Part them and the ears will be apparent. The clinical examination consists of a visual inspection, radiography or endoscopy. Cultures and cytology may also be necessary to conrm a diagnosis. The vestibulocochlear nerve, cranial nerve VIII, provides information on the threedimensional position of the head in space as well as hearing (loss of function would result in deafness, head tilt and/or nystagmus, circling, and/or loss of balance). Cochlear function is tested by examining the patients response to a sudden loud noise. Usually these animals are more difcult to rouse. With vestibular disease, it is important to determine whether the lesion is central or peripheral. Central disease involves the brainstem and carries a poorer prognosis than peripheral disease. Central lesions are often associated with changes in mental status, postural reaction decits are on the ipsilateral side of the lesions. Possibly other cranial nerve decits may be present. Vertical nystagmus is associated with brain stem lesions. Peripheral vestibular signs are often restricted to horizontal or rotatory nystagmus that does not change with changes in the position of the head. KRAUTWALD et al. (1998) had shown that the suitability of the radiographic projections to demonstrate the anatomic structure of the external auditory meatus is different in the Psittacifomes compared to the Accipitridae, Falconidae and Strigiformes. In the Psittaciformes, dorsoventral and lateral projection are good while in the others dorsoventral projection is not helpful and lateral projection is of limited use. The ear canal should be swabbed for cytology and Grams staining of the exudate. A sample should be taken for bacterial and fungal culture and sensitivity testing. If a mass is present, submit a biopsy sample for histopathology. A haemogram and skull radiography is necessary when the bird has clinical signs of otitis media or interna. Immunosupression is frequently associated with otitis and a PBFD PCR should be performed.

6 TREATMENT
6.1. Medical Treatment is by cleaning and the application of topical antibiotics. An antibiotic or antifungal solution can be applied two to three times daily into the ear canal. Ophthalmic antimicrobial preparations without steroids are convenient to use. Consider broad spectrum systemic antibiotic therapy for suspected bacterial otitis media and interna and provide appropriate supportive care. Corticosteroids may also be helpful in reducing inammation in the external part of the ear. Corticosteroids are given topically and not systemically. No particular problem has been encountered in our experience. 6.2. Surgical Surgery is necessary for foreign body removal, occlusion of the external openings, or neoplasia. Electrosurgery is the preferred technique.

338

7 CONCLUSION
Auricular diseases are uncommon in birds but the avian veterinarian must include the ear in the clinical examination. Consider that immunosupression is frequently associated with otitis and perform a PCR for diagnosis of PBFD.

8 CITATION INDEX
1. ABRAMS et al. Aspergillus blepharitis and dermatitis in a peregrine falcongyr falcon hybrid. J Avian Med Surg 2001; 15(2): 114 - 120. 2. DOOLING RJ and DENT ML. New studies on hair cell regeneration in birds. J Acoustical Soc Japan 2001; 22: 93 - 100. 3. HARRISON GJ and RITCHIE. Making distinctions in the physical examination. In: HARRISON GJ. HARRISON LR and RITCHIE BW (ed) Avian Medicine: principles and application. Lake Worth: Winger Publishing Inc 1994: 144 175. 4. KERN TJ. Disorders of the special senses, the ear. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM and QUESENBERRY K. (ed): Avian Medicine and Surgery. Philadelphia: WB Saunders 1997; 583 - 589. 5. KRAUTWALD-JUNGHANNS ME, KOSTKA VM and DRSCH B. Comparative studies on the diagnostic value of conventional radiography and computed tomography in evaluating the heads of psittacine and raptorial birds. J Avian Med Surg 1998; 12 (3): 149 - 157. 6. NECKER R. The Avian ear and hearing. In: Whittow GC (ed): Sturkiess Avian Physiology, 5th edition. San Diego: Academic Press 2000; 21 - 30. 7. PESEK L. The avian ear: part I and II. Winged Wisdom, Pet Bird Magazine, May - June 2003 8. SCHUBOT M, CLUBB KJ and CLUBB SL. Psittacine Aviculture, Perspectives, Techniques and Research. Psittacine Neonatal Development, ABRC Publications 1992; 12 17. 9. TELL LA, WOODS L and CROMIE RL. Mycobacteriosis in birds. In: Rev Sci Tech Off Int Epiz., 2001; 20(1): 180 - 203

AUTHORS ADDRESS
F. Rival, DVM, Prsident du GENAC, CES dOphtalmologie Clinique vtrinaire de lArche, 192 Avenue de Romans, 26000 Valence, France Email: franck.rival@wanadoo.fr

339

Meadow Lane Veterinary Centre, Loughborough, Leicestershire, England United Kingdom

THE NUTRITIONAL IMPLICATIONS OF PRODUCING THE OPTIMAL EGG IN CAPTIVE BIRDS

B. C. Stockdale BVM&S, MRCVS

KEYWORDS
Nutrients - Avian embryo - Dead-in-shell - Oxidative stress - Antioxidants

ABSTRACT
The success of the avian reproductive cycle from the induction of follicle formation through ovulation, fertilization, egg production, and the subsequent rearing of the young, is dependent on adequate and appropriate nutrition. Because all the nutrients required for embryonic development and survival must be placed within the egg prior to shell formation, a heavy onus is placed on the nutritional status of the female bird. As egg nutrient provision is funded from both endogenous protein reserves and exogenous dietary sources, a nutrient complete diet both pre and during the ovulatory period are essential in ensuring that the requisite essential nutritional components are available for optimal egg formation. This paper provides an overview of some of the nutritional dynamics involved in the production of the optimal egg. The implications of inadequate egg nutrition are reviewed and it is proposed that nutrition in captive birds may play a more prominent role in reproductive failures, characterised by deadin-shell than is currently appreciated.

1 INTRODUCTION
Avian embryonic development takes place in a semi-closed environment, the egg, where only exchanges of gas and water take place. The egg composition is designed in such a way that all nutrients necessary for the development of the future embryo are accumulated within the egg yolk, white, and shell. Poor maternal state at the start of breeding affects the female birds ability to produce and incubate eggs, and recycle. It also inuences their willingness to provide parental care and ability to rear chicks up to edging. Poor egg quality affects chick hatch size, vigour, early feeding behaviour, and the immune status of the chick. The future fecundity of female nestlings has also been linked to the nutrition received both from the egg and immediately post-hatch

340

(GORMAN and NAGER 2004). Maternal nutrition can therefore be considered as the major determinant in the development and health of the avian embryo and viability in early post hatch life. Very little work has been done on the specic role of nutrition in achieving improved reproductive performance in birds kept as avicultural specimens. A wealth of literature does exist, however, showing how dietary manipulation can achieve higher production in terms of fertility, hatchability, postnatal survival, and weight gain in domestic poultry. Whilst care must be observed when discussing levels of specic nutrients, and overinterpreting the parallels between precocial chicks and altricial nestlings, this should not detract from the hens worth as a model for understanding the consequences of inadequate embryo nutrition in other avian species. The biochemical and biological nature of the deciencies and excesses within the egg should still hold true.

2 OVERVIEW
During reproduction, the nutritional needs of laying birds include the nutrients required for general maintenance, a rapid increase in tissue mass of the female reproductive tract, and those required for egg synthesis. The additional nutritional needs may be met either directly from the diet; from nutrients stored in anticipation of egg synthesis; from nutrient reserves, which may include stores or any tissue component that can be drawn upon to reallocate endogenous nutrients when daily intake falls below needs; or any combination of the above (MURPHY 1994). Among different nutrients that can signicantly affect chick embryo development and subsequent viability, proteins, lipids, and antioxidants are specically indicated and the following sections highlight their respective roles in the production of the optimal egg. 2.1. Protein as a limiting factor Egg production is a protein-demanding process. Birds laying eggs require protein for maintenance, development of the oviduct and accretion of egg proteins. The growth of the oviduct and the synthesis of several yolks are mostly complete before the rst egg is laid. Consequently, the females requirements increase at least a week prior to her rst oviposition. In most species, egg albumen is synthesised in the oviduct during a 24h period before ovulation, thus dietary amino acid requirements are especially high on the day preceding each oviposition. The quality of these proteins is reected by the quality of the daily dietary protein levels (MURPHY 1994). Estimates of the additional protein requirements of several avian groups range from 86-232% of their minimum maintenance requirements (ROBBINS 1981). These estimates assume that the dietary protein is of satisfactory quality, i.e. that the pattern of the amino acids in the dietary protein is supplied more or less, in proportion to the birds needs. The requirements for specic essential amino acids for egg production, however, are often far in excess of those required for maintenance. Lysine, methionine, and cysteine are commonly cited as limiting amino acids, with, for example, levels of lysine 4.1 times greater than maintenance being required for egg production in the budgerigar.

341

Where dietary protein levels fall below the required levels for reproductive performance, body plundering occurs and use is made of endogenous reserves. A signicant loss of muscle weight at the time of egg formation is documented in 21 of 29 species studied (HOUSTON 1995), suggesting that the use of tissue protein to assist with egg production may be widespread in birds. Overnight, all birds rely at least partially on amino acids derived from tissue proteins to synthesize egg proteins, even if they restore tissue proteins the next day. During egg laying, protein removal from the sarcoplasm occurs over a range of different proteins. All tissues need to be considered as potential protein sources, but the pectoral muscle mass provides the main supply. This is a costly process for the female bird as the balance of amino acids in skeletal muscle is sufciently different from that in egg protein. The individual amino acids are released more or less in proportion to their occurrence in proteins breaking down, not in proportion to the needs for synthesis. Eggs are enriched with methionine and cysteine relative to skeletal muscle and so greater volumes of endogenous proteins must be used to provide for those proteins in the egg. This can have a serious knock-on effect on the overall health of the female, on the levels of amino acids she is able to fund for the next clutch of eggs and her ability to both incubate and rear her nestlings. 2.2. Lipids and polyunsaturated fatty acids (PUFAs) The production of a clutch of eggs requires the deposition of large amounts of yolk lipids, mostly during the several days prior to ovulation. Yolk lipids and proteins are synthesised in the liver under the inuence of oestrogen and progesterone and are transferred through the blood to the ovarian follicles. Lipids in the yolk are of two main types: specic yolk-targeted very low-density lipoproteins (VLDLy) and vitellogenins (VTN). Lipids comprise about 10% of the total weight of an egg and 33% of the yolk weight. Almost all the lipids in the yolk are present as lipoprotein complexes with an overall lipid:protein ratio of about 2:1. Deposition of egg yolk is completed 24h before its ovulation. The importance of lipoproteins for chick embryo development is unequivocal (SURI 2002). About 90% of the energy required for the developing chick is supplied from egg yolk by fatty acid oxidation. Approximately 80% of the entire lipid content of the yolk is mobilised and absorbed into the embryonic tissues over the last few days of development and the remainder, in the form of residual yolk, is sufcient for the adequate maintenance of the chick for several days post hatch. During embryonic development, lipids, as well as providing an energy supply, play a crucial role in providing the developing embryo with a source of important constituents for the production of biological membranes and biologically active substances. The importance of PUFAs in cell metabolism is difcult to overestimate (SURI 2002). 2.3. Dietary requirements of essential fatty acids The fatty acid composition of the maternal diet and subsequent inappropriate fatty acid provision within the yolk, has been shown to affect embryonic development, viability, and hatchability, and is recognised as a cause of increased chick mortality (VILCHEZ 1990).

342

The basic building block fatty acids that are deemed to be essential dietary requirements for birds are linoleic (LA) and linolenic (LNA) acids. Little work has been done other than with gallinaceous birds into the ability of avian species to convert these fatty acids and it is suggested that given the variability among other vertebrate taxa, that diets for birds should also contain arachidonic acid and eicosapentanoic acid (KLASING 1998). Natural foods contain differing proportions of these fatty acids. Green leafy vegetables, linseed and rape oils are good sources of LNA and grains and plant oils such as corn, and sunower are rich sources of LA. As well as having sufcient levels of essential fatty acids in the maternal diet, it is important that they are supplied in approximately equal proportions. LNA is the parent for n-3 fatty acids and LA the parent for the n-6 series of fatty acids and they are not inter-convertible. Both of these groups compete for the same modifying enzyme systems and a preponderance of one affects the levels of the other. 2.4. Antioxidant systems and avian embryo development The animal body is under constant attack from free radicals formed as a natural consequence of the bodys normal metabolic activity within its tissues and antioxidants provide protection against their damaging effects. The most important effect of free radicals on the cellular metabolism is in their participation in lipid peroxidation. Prime targets for peroxidation are the PUFAs that are integral in the structure of cellular membranes, and preservation of their integrity and function. Failure to prevent lipid peroxidation within the egg will potentially result in excessive tissue damage and embryonic death. At hatching, the embryonic liver contains high levels of PUFAs derived from the yolk that require a considerable degree of antioxidant defence against peroxidation. The antioxidant system of the newly hatched chick includes the fat-soluble antioxidants, vitamin E and carotenoids; the water soluble antioxidants. ascorbic acid and glutathione; as well as antioxidant enzymes. The main fat-soluble antioxidants, vitamin E and carotenoids, cannot be synthesised de novo by animals, so must be derived from the diet. 2.5. Vitamin E. Vitamin E is a generic term which encompasses eight main tocol and tocotrienol derivatives (SURI 2002). It is considered to play a central role in antioxidant production during embryogenesis and low levels have been associated with decreased hatchability and slow embryonic development. Vitamin E is transported from the maternal diet to the hen birds liver and further to the developing oocyte as a component of VLDLy and is subsequently deposited within the lipid rich fraction of the yolk. In the chicken it has been shown that producing each egg requires the release of twice as much vitamin E as is stored in the liver (SURI 2002) indicating that the liver is not a good reservoir of vitamin E in the laying hen. Exclusion of vitamin E from the diet can therefore cause a rapid decrease in tocopherol concentration in the egg yolk. The main site of vitamin E accumulation in the avian embryo is the liver. Yolk lipid droplets containing vitamin E are taken up by the yolk sac membrane and processed into lipoprotein particles that are released into the embryo circulation. These lipoproteins

343

are then metabolised by various tissue lipoprotein lipases and the resultant remnants taken up by the embryo liver. The concentration of tocopherol in the liver tissue increases 3-5 times in the last week (chicken) of incubation reaching a maximum just after hatching. Vitamin E is particularly important at hatching, when the onset of pulmonary respiration results in increased oxygen tension and increased free radical production. This can result in high levels of pipping death. Liver levels of vitamin E in chickens drop dramatically over the rst few weeks post-hatching, indicating that dietary intake is insufcient to maintain reserves. This may be explained as a result of low pancreatic lipase activity and restricted bile production in the developing avian alimentary tract. The passerine nestling also shows a similar lack of enzyme activity (CAVIEDES-VIDAL 2001). Maternal diet, therefore, plays a crucial role in maintaining physiological levels of vitamin E in the chicken tissue during the rst ten days of postnatal development. 2.6. Carotenoids Carotenoids play a specic role in avian embryonic development and there are some indications that carotenoids are extremely important elements in maintaining the chick immune system. They also function as important visual indicators of offspring health. The bright gape anges of nestlings act as a temporal stimulus eliciting parental care and feeding. The intensity of the colour is thought to be a true reection of the quality of the nestlings health - pale gapes being a consequence of insufcient provision or diversion of carotenoids to combat disease. The most common carotenoids in seeds and plants are lutein, zeaxanthin and carotene, which occur in varying levels. Absorption follows a similar pattern to that of vitamin E; digestion of the food matrix is followed by the formation of lipid mixed micelles which are taken up into intestinal mucosal cells and delivered to the plasma via lymph system. As the lymphatics are not well developed in birds, it is possible that lipoproteins are released directly into the portal vein and transported straight to the liver. Plasma levels vary signicantly depending on dietary provision and assimilation. During the rapid growth of the ova, carotenoids are deposited in a dose dependant manner, as opposed to a set level deposition, and the levels in eggs respond very quickly to dietary level changes. The main storage sites within the body are the liver and, surprisingly, the toe web. The gradual loss of pigment from the shank and beak of certain birds as egg production continues, can be explained by the mobilization of carotenoid stores for incorporation into the yolk. Although fat deposits contain a high level of carotenoid pigments, imparting its characteristic yellow colouration, they are considered to be sinks rather than stores with the carotenoids being unavailable for use except during fat metabolism. Studies are limited, but species specicity in absorption and transfer to the egg would seem to occur. Those wild birds studied having a much higher egg yolk carotenoid level than those commercially reared. This is probably a direct effect of dietary manipulations - access to a wider range of variable carotenoid containing

344

food substances, but may indicate a more prominent role for carotenoids in embryo protection in certain species. It may also be relevant that the antioxidant activity of carotenoids is mainly expressed at the low oxygen tensions that prevail in embryonic tissues.

3 DISCUSSION
The production of eggs is a nutritionally demanding process for many birds, particularly small passerines, some of which lay a clutch of eggs weighing more than the females own body weight. As reproduction is a life-compromising activity there is inevitably a conict between female and egg. Conservative diets aimed at providing maintenance levels of nutrients, with no additional provision for the nutritional demands of egg laying, may be contributing to reduced reproductive potential in captive birds. Reviews of captive bird diets focus principally on maintenance levels of nutrients and generally fail to address the issue of additional reproductive nutritional needs. Feeding levels for essential nutrients (as far as these are known) are usually linked to energy based formulae. These tend to be misleading, as energy expenditure and thus demand, in captive birds is much lower than wild birds. Protein levels geared to energy requirements of wild birds will grossly under-fund captive birds protein needs. Reproduction puts an even greater strain on a limited amino acid pool. Japanese quail (Coturnix coturnix japonica), for example, require a maintenance level of protein around 5.5%. This increases to over 23% at breeding without a substantial increase in energy requirements. Whilst the proximate factor in initiating breeding in birds from temperate regions is photoperiodic change, for opportunistic breeders from neotropical regions (where a large number of our avicultural species originate), food availability is a much stronger cue. The protein component of ripe cereal seeds has long been recognized as having the potential to be nutritionally limiting for birds, the results of a poor match between the amino acid balance of seed and the requirements for synthesis and renewal of tissue proteins. Biological mechanisms exist within wild birds to time breeding to coincide with the production of ripening seeds. These have a much higher essential amino acid percentage than dried seeds (ALLEN and HUME 1997). When protein is the limiting factor in cereal-based diets, the deciency is in quality rather than quantity. Nevertheless, dry seeds still make up the main ingredients of many captive bird diets. The provision of a range of foods whilst offering a wider amino acid prole may not necessarily correct the problem. Foraging birds can nd foods containing protein of various quality to satisfy their amino acid needs by either choosing only those foods that contain a suitable array of essential amino acids protein shift, (a move in breeding granivorous birds to also consume high protein legume seeds and invertebrates), or by choosing foods in amounts that permit complementation of constituent amino acids. Small birds appear, however, to have limited ability to actively exploit dietary amino acid complementation. Their dietary habits are xed more by their mechanical ability both to husk and consume a seed type in the most expedient time, regardless of content. Whilst mobilization of body tissues occurs in many species to aid with the

345

amino acid needs of reproduction, the relative costs are high. Whether by preferential selection or chance consumption, wild birds are able, to a greater extent, balance their amino acid intake. Captive birds are limited by the very nature of their captive diets, in their ability to manipulate their protein intake. More onus is placed, therefore, on endogenous reserves. Supplementation of diets with foods high in limiting amino acids or the use of formulated diets may achieve a solution. Adequate provision of antioxidants is essential for successful embryonic development and hatching. To date there is little information available relating to the tocopherol content of the egg yolk of different avian species. Less is known about the features of vitamin E metabolism and accumulation in the egg yolk of wild birds. Species-specic differences in the metabolism of vitamin E may inuence yolk levels but perhaps more importantly for captive birds, the level of vitamin E in the diet will have a direct inuence. Independent of later nutrition, individuals experiencing a short period of low quality nutrition during the nestling period have a two-fold reduction of these antioxidants at adulthood. In the zebra nch, over 70% of the females daily intake of tocopherol and carotenoids are deposited in the egg yolk. The deposition of these antioxidants declines with laying sequence, suggesting that the decline in provisioning may be a consequence of resource limitation (SURI 2002). It is suggested that low-quality neonatal nutrition results in a long-term impairment in the capacity to assimilate dietary antioxidants, resulting in a reduction of both reproductive and actual longevity. As vitamin E is poorly stored, sufcient carry over from diet to egg on a daily basis must be made in breeding birds. Listed maintenance levels for vitamin E need to be increased, therefore, to accommodate the provisioning of the egg. With many avicultural species, especially psittacine birds, vitamin A levels may also be marginal. Low dietary vitamin A can have a direct adverse effect on reproduction and a knockon effect on the levels of carotenoids availability. Articially high dietary vitamin A levels can equally have an adverse effect on vitamin E levels. A lot of attention has been focused on vitamin A levels in avian diets but little on the levels of carotenoids. As more evidence accumulates showing that the maternal diet can have a profound effect on the health status, growth and development of the avian embryo and newly hatched chick, perhaps it is time that we reassessed the role of nutrition in reproductive management of captive birds. Failure to breed, dead in shell, failure to incubate, nestling mortality, and nestling desertion may all have a greater nutritional basis than currently appreciated. Diets for breeding birds need to better reect their reproductive needs. Food quantity should never be an issue in captive bird management, whereas food quality, expressed in terms of available nutrients, can be.

4 CITATION INDEX
1. 2. ALLEN LR and HUME ID. The importance of green seed in the nitrogen nutrition of the Zebra Finch. Austr J Ecology 1997; 22: 412 - 418. CAVIDES-VIDEL E and KARASOV WH. Developmental changes in digestive physiology of nestling house sparrows. Physiol Biochem Zoology 2001; 74: 769 - 782.

346

3. 4. 5. 6. 7. 8. 9.

GORMAN H and NAGER R. Prenatal developmental conditions have long-term effects on offspring fecundity. Proc R Soc London B 2004; 1923 - 1928. HOUSTON DC et al. Changes in the muscle condition of female zebra nches during egg laying and the role of protein storage in bird skeletal muscle. Ibis 1995; 137: 322 - 328. KLASING K. Comparative avian nutrition. CAB International 1998: 171172. MURPHY M. Amino acid composition of avian eggs and tissue: Nutritional implications. J Avian Biol 1994; 25: 27-38. ROBBINS C.T. Estimation of the relative protein costs of reproduction in birds. Condor 1981; 83: 177. SURI P. Natural antioxidants in avian nutrition and reproduction. Nottingham Press. 2002 VILCHEZ C et al. The inuence of supplemental corn oil and free fatty acids on the reproductive performance of Japanese quail (Coturnix japonica). Poultry Sci 1990; 69: 1533 - 1538

AUTHORS ADDRESS
Brian C Stockdale BVM&S MRCVS Meadow Lane Veterinary Centre, 9 Meadow Lane, Loughborough, Leicestershire, LE11 1JU, England Email: the.stockdales@dial.pipex.co.uk

347

Birch Heath Veterinary Clinic Cheshire United Kingdom

THE EFFECT OF UV-B RADIATION ON CALCIUM METABOLISM IN GREY PARROTS

M. Stanford BVSc MRCVS

KEYWORDS
Grey parrot Ultraviolet radiation Hypocalcaemia Vitamin D- Ionised Calcium

ABSTRACT
This study investigates the effects of exposing healthy captive grey parrots to articial UV-B (315-285nm) radiation for 12 hours daily. Two groups of grey parrots were fed different diets for 12 months whilst being exposed to UV-B radiation from uorescent tubes. A signicant increase in the serum concentration of 25 hydroxycholecalciferol and ionised calcium was found independent of the diet fed after UV-B radiation exposure.

1 INTRODUCTION
The grey parrot (Psittacus e. erithacus) is wide spread throughout equatorial Africa. It is the second most commonly traded psittacine bird in the world pet trade desired mainly due to its excellent ability to mimic (DEL HOYO 1997). Captive breeding of the grey parrot has increased dramatically in the last decade and it has become a common patient for veterinary surgeons worldwide. Hypocalcaemia is a commonly recognised syndrome in grey parrots in captivity although the aetiology is still unconrmed (ROSSKOPF et. al. 1985). Affected adult birds present clinically with a variety of neurological signs, ranging from slight ataxia to seizures, which respond to calcium or vitamin D therapy (HOCHLEITHNER 1989). In captive bred grey parrots osteodystrophy is a common clinical sign in young birds with deformity of the long bones easily identied radiographically (HARCOURT-BROWN 2003). It is thought that the syndrome could be due either to a primary hypoparathyroidism or a secondary hyperparathyroidism due to inadequate husbandry. Seed based diets are commonly fed to grey parrots in captivity containing low levels of calcium and vitamin D3.These diets also contain high levels of phosphorus that form phylate complexes with calcium reducing the bio availability of the mineral. It has therefore been

348

postulated that poor diet contributes to a nutritional secondary hyperparathyroidism (Klasing 1998) but this does not explain why other psittacine species despite being fed the same diets, rarely develop clinical signs of hypocalcaemia (RANDELL 1981). In captivity grey parrots are usually kept indoors with limited access to ultraviolet light. A lack of adequate ultraviolet light (UV-B spectrum 285-315nm) in captive birds may lead to a functional vitamin D deciency and subsequent problems with calcium metabolism. This might suggest that they require higher ultraviolet light supplementation than other psittacine birds. The role of UV-B (285-315nm) radiation in the control of vitamin D metabolism has not been researched in psittacine birds at the present time. Vitamin D is supplied to birds from a combination of endogenous synthesis and dietary supply. The skin has been established as the organ for vitamin D3 production as in mammals. Birds secrete 7-dehydrocholesterol (Provitamin D3) onto the featherless areas of skin (Koch et al., 1941). Recently it has been shown that there is thirty times more Provitamin D3 on the featherless leg skin than the back indicating the importance of this area for vitamin D3 metabolism (TIAN et al. 1994). Provitamin D is converted to cholecalciferol (Previtamin D3) by an ultraviolet light (285-315nm wavelength) dependent isomerisation reaction. Cholecalciferol is a sterol prohormone which undergoes a temperature dependent isomerisation reaction to form vitamin D3 (HOLICK, 1989). After translocation into the circulation vitamin D3 is transported bound to a specic globulin binding protein. There is a time delay between vitamin D3 production on the skin and its translocation into the circulation (TIAN et al. 1994). Cholecalciferol can be stored in adipose tissue but to be physiologically active it must be metabolised by a 2-stage hydroxylation process (HOLICK 1995). Cholecalciferol is initially metabolised to 25 hydroxycholecalciferol in the liver (BLUNT et al. 1968). 25 hydroxycholecalciferol is transported to the kidney via carrier proteins and converted to either 1, 25 dihydroxycholecalciferol or 24, 25 dihydroxycholecalciferol, the active metabolites of cholecalciferol in the domestic fowl. The most signicant active metabolite of vitamin D3 in domestic chickens is 1, 25 dihydroxycholecalciferol controlling both bone development and intestinal calcium absorption. Failure to provide adequate UV-B light in poultry kept indoors will produce symptoms of vitamin D3 deciency. Studies have been conducted in poultry to demonstrate the requirement for cholecalciferol kept in the absence of ultraviolet light indicating (EDWARDS et al. 1994). If ultraviolet light was excluded and dietary cholecalciferol concentrations fell below 400ICU/kg symptoms of rickets were seen with concurrent low plasma ionised calcium concentrations. The measurement of 25 hydroxycholecalciferol has been shown to be a useful indicator of the UV-B exposure of an individual. The ultraviolet light required for endogenous vitamin D synthesis can either be supplied naturally from full spectrum sunlight or using articial lamps manufactured to provide UV-B radiation. The maximum conversion of provitamin D to Previtamin D occurs at the 297+/- 3nm wavelength (HOLICK et al. 1982). Once sufcient previtamin D3 has been formed additional solar radiation transforms the provitamin D to biologically inactive compounds lumisterol and tachysterol (HOLICK 1994). This explains why hypervitaminosis D has never been reported from excessive exposure to UV-B light. Fluorescent tubes that provide some UVB radiation in addition to visible light are available commercially mainly designed for captive reptile exhibits (LOGAN 1969). In one study none of the commercial lamps produced signicant amounts of UVB wavelength in the vitamin synthesis spectrum

349

so clearly clear should be taken in lamp selection (BERNARD 1995). The aim of this study was to investigate the effects of articially supplied UV-B radiation on 2 groups of healthy grey parrots on different dietary regimes.

2 MATERIALS AND METHODS

The main population consisted of 100 healthy sexually mature grey parrots housed indoors as 50 pairs. All the birds were purchased from a single source as wild caught imports from Guyana. Each bird in the main population had been examined clinically prior to purchase in 1998 using a standard format. Faecal samples taken from each individual had been subject to parasitology, microbiology and gram stain examinations. Blood samples taken from each bird were subject to routine haematological and biochemical analysis including circovirus, polyoma virus and chlamydophilia PCR tests. Each bird was examined by laparoscopy to conrm sexual maturity and gender. On the basis of these tests only healthy birds were included in the main population. Each pair was housed in an identical individual aviary measuring 2m by 2m of identical brick and wire construction. Each aviary had a wooden nest box measuring 40cm by 30cm of shoebox design. All the aviaries were positioned in a single span farm building of brick and slate roof construction with no exposure to natural ultraviolet light. 40 birds were selected from the main population using a simple randomisation process to form the study group. The group of 40 birds was randomly allocated into 2 groups of 10 pairs of grey parrots (n=20 birds per group: 10 male and 10 female). The birds were kept in the same building under the same conditions as the main population. One group was fed Harrisons High Potency Course pellet diet (Harrisons International Bird Foods, Nebraska, US); the other group was maintained on an unsupplemented mixed seed diet (Tidymix, John Heath, Hull, UK). After a year of no exposure to UV-B radiation during the annual health examination additional blood was taken from the study group to assay 25 hydroxycholecalciferol and ionised calcium in grey parrots with the informed consent of the owner. All of the birds were subsequently placed under articial ultraviolet light (UV-B 285315) for 12 hours daily whilst keeping the diets constant. The light was supplied using 1200mm 36W FB36 Arcadia bird lamps (Arcadia, Arcadia House, Cairo New Road, Croydon, UK) suspended directly above each pair of birds. Two tubes were supplied to each pair of birds. When perching each birds would be a maximum distance of 0.5 metres from the tubes. A reector (Arcadia ALR36) was mounted behind each tube to direct light towards the birds maximising the amount of UV-B that each bird received. An Elsec UV-B light monitor 763 (Littlemoore Scientic Engineering, Railway Lane, Oxford UK) was used to demonstrate an increase in the ultraviolet light levels experienced by all the birds in the third year. The monitor uses two photodiodes to detect UV-B radiation in the 285-400nm wavelengths expressed as mW/M2. The tubes were replaced after 6 months according to the manufacturers instructions. After 12 months exposure to articial UV-B lighting further blood tests were taken during the annual health examination to assess the effects of UV-B radiation on calcium metabolism in grey parrots.

350

All the blood samples were taken under isourane anaesthesia. A 2ml blood sample was taken from each bird split equally into heparin and EDTA eperndorph tubes. The EDTA sample was centrifuged for 15 minutes at 1600/s and the plasma titrated into another eperndorph tube. The plasma sample was immediately cooled to 70 Celsius for subsequent analysis for parathyroid hormone. The blood testing was performed on September 1st each year outside the breeding season to minimise the effects of both oestrogen and seasonality on both vitamin D3 and calcium levels (BENTLEY 1998).

3 RESULTS
3.1 Descriptive statistics 25 hydroxycholecalciferol (nmol/l)
Year of Study Dietary group Seed Year 1 Pellet Seed Year 2 Pellet Mean 71.47 130.77 139.66 115.44 SD 90.01 108.23 69.22 16.56 SE 21.22 24.83 17.31 4.14 Median 35.25 118.40 122.45 112.85 95% of mean 26.71-116.23 78.61-182.94 102.78-176.55 106.72-124.37

3.2 Descriptive statistics ionised calcium (mmol/l)

Year of Study Year 1 Dietary Group Seed Pellet Seed Year 2 Pellet Mean 1.11 1.20 1.23 1.24 SD 0.06 0.07 0.05 0.05 SE 0.01 0.02 0.01 0.01 Median 1.10 1.19 1.24 1.24 95% of mean 1.08-1.13 1.16-1.23 1.20-1.25 1.22-1.27

3.3 Effect of UV-B radiation on each dietary group

Parameter Ionised calcium 25 hydroxycholecalciferol Dietary group Seed Pellet Seed Pellet Mean year 1 1.11 1.20 71.47 130.77 Mean year 2 1.23 1.24 139.66 115.44 Wilcoxon W statistic 2.0 27.5 18.0 123.0 P value 0.0001 0.0053 0.0038 0.2753

351

3.4 Comparison between dietary groups after UV-B supplementation.

Parameter Dietary group Seed Pellet Seed Pellet Mean year 3 1.23 0.19 1.24 139.66 115.44 0.37 0.5446 0.6620 Kruskal-Wallis ANOVA statistic P value

Ionised Calcium

Vitamin D

4 DISCUSSION The study suggested that articial UV-B radiation could be used in grey parrots to inuence calcium metabolism. The ionised calcium concentration was signicantly increased in both the pellet and seed fed groups after the provision of UV-B radiation. The vitamin D concentration was signicantly increased in the seed fed group but not the pellet fed group. This might suggest that the pellet fed group already had adequate stores of vitamin D in the form of 25 hydroxycholecalciferol. The additional UV-B light would not lead to vitamin D toxicity due to the natural feedback mechanisms to prevent this. At the beginning of the study there was a signicant difference between the serum ionised calcium and vitamin D concentrations between the 2 dietary groups. This was due to the higher vitamin D and calcium content in the pellet over the seed diet. After the provision of UV-B light for 12 months there was no signicant difference in 25 hydroxycholecalciferol or ionised calcium concentrations in the 2 dietary groups. The ndings are potentially signicant in a breed known to commonly suffer from disorders of calcium metabolism. As most captive grey parrots are either kept indoors or live in Northern latitudes they would not be expected to receive similar ultraviolet light levels compared with equatorial Africa. Furthermore the birds are traditionally fed a diet with a low calcium and vitamin D content. This may well explain why grey parrots are so susceptible to symptoms of hypocalcaemia. It is proposed that grey parrots should be provided with UV-B radiation as part of their husbandry. Preferably this should come from solar radiation due to potential problems with supplying articial UV-B with light both from the performance of the lamps and the practicalities of keeping the bulbs close to the birds. The author now recommends the provision of 2.5% UV-B uorescent lighting to all captive grey parrots kept indoors. Future research should determine whether calcium metabolism in grey parrots differs from other species of psittacine birds concentrating particularly on the effect of ultraviolet light within different species.

352

5 REFERENCES
BENTLEY, P.J. Comparative Vertebrate Endocrinology. Cambridge: Cambridge University Press 1998, 269 - 301. 2. BERNARD JB. Spectral irradiance of special uorescent lamps and their efciency for promoting vitamin D synthesis in herbivorous reptiles. PhD dissertation, Michigan State University 1995. 3. BLUNT JW, DELUCA HF and SCHNOES HK. 25 hydroxycholecalciferol. A biologically active metabolite of vitamin D3. Biochemistry 1968; 7: 3317 - 3322 4. DEL HOYO J, ELLIOT A and SARGATAL J. eds: Handbook of the Birds of the World. Vol 4. Sandgrouse to Cuckoos. Barcelona: Lynx Editions 1997. 5. ROSSKOPF W.J., WOERPEL R.W. and LANE R.A. The hypocalcaemic syndrome in African Greys: An updated clinical viewpoint. Proc Assoc Avian Vet, 1985; 129 - 132. 6. EDWARDS HM JR, ELLIOT MA, SOONCHARERYNING S and BRITTON WM. Quantitive requirement for cholecalciferol in the absence of ultraviolet light. Poult Sci 1994; 73(2): 288 - 294. 7. HARCOURT-BROWN N. Incidence of juvenile osteodystrophy in hand reared parrots (Psittacus e psittacus). Vet Rec 2003; 152: 438 - 439. 8. Hochleithner, M. Convulsions in African Grey parrots in connection with hypocalcaemia: Five selected cases. Proc Euro Symp Avian Med Surg, 1989; 44 - 52. 9. KLASING, K.C. Comparative Avian Nutrition. New York: CAB International 1998, 290 - 295. 10. KOCH EM and KOCH FC. The provitamin of covering tissues of chickens. Poultry Science 1941; 20: 33 - 35. The full list of references is available from the author 1.

AUTHORS ADDRESS
Michael D Stanford BVSc MRCVS Birch Heath Veterinary Clinic Birch Heath Road, Tarporley, Cheshire, CW6 9UU United Kingdom Email: stan_vet@btinternet.com

353

The Animal Emergency Center1, Glendale, WI; Avian and Exotics2, Miami, FL; Angell Memorial Animal Hospital3, Boston, MA; Medical College of Wisconsin4, Milwaukee, WI; Avian and Wildlife Laboratories5, University of Miami, Miami, FL; Virginia Regional College of Veterinary Medicine6, United States of America

RESPONSE TO FLUID RESUSCITATION AFTER ACUTE BLOOD LOSS IN MALLARD DUCKS (ANAS PLATYRHYNCHOS)
M. Lichtenberger1, DVM, Dipl ACVECC, W. Chavez2, DVM, C. Cray5, PhD, C. Orcutt3, DVM, Dipl ABVP-Avian, D. DeBehnke4, MD, FACEP, E. Stumpp1, DVM, S. Diehl1, DVM, B. F. Feldman6, DVM, Ph.D, C. Page1, BSc, CVT, L. Mull1, BSc, CVT, L. Inman1, BSc, R. Kirby1, DVM, Dipl ACVIM, Dipl ACVECC

KEYWORDS
Hypovolemic shock Resuscitation Crystalloids - Colloids, Polychromasia

ABSTRACT
The pathophysiology of haemorrhagic shock is poorly understood in avian species. Acute blood loss of 30-40 % of blood volume has been shown to result in 50% mortality (LD50) in mammals. Blood loss is better tolerated in birds than in mammals. The LD50 for acute blood loss in ducks has been shown to be 60% of the total blood volume. No studies have been reported in birds documenting changes in heart rate and blood pressure after acute blood loss and uid resuscitation. Reticulocytosis, or polychromasia, is the hallmark of intensied erythropoiesis, allowing classication of anaemias into regenerative or non-regenerative types. Reticulocyte release from the bone marrow in mammals commonly occurs 5 days after acute blood loss and rarely can occur as early as 2-4 days. No studies have been reported in birds after acute blood loss showing regenerative changes documented by absolute numbers of polychromasia, haematocrit and haemoglobin levels. The purpose of this study in mallard ducks (Anas platyrhynchos) was threefold: a) to demonstrate the heart rate and blood pressure response to acute blood loss at the predetermined LD50, b) to demonstrate if birds respond to acute blood loss with a baroreceptor response as seen in man and mammals and c) to document the efcacy and safety of smallvolume crystalloid resuscitation alone or in conjunction with colloids or Oxyglobin after acute blood loss; and to demonstrate the time required for a regenerative red blood cell response after acute haemorrhage by documenting an increase in absolute polychromasia, haematocrit and haemoglobin concentrations. Twenty-seven mallard ducks were included in the study. Phlebotomy was performed on each duck to a LD50 of 60% of their total blood volume. The birds were randomized into three groups

354

to receive crystalloids alone, hetastarch and crystalloids, and Oxyglobin and crystalloids. Indirect blood pressure measurements and heart rates were recorded throughout the study. The birds were recovered and observed for mortality over 96 hours. Blood was collected from 3 birds in each of the 3 uid resuscitation groups at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h and 96 h. The blood was evaluated for polychromatic changes, red blood cell count, haematocrit, and haemoglobin concentration.

1 INTRODUCTION
Acute haemorrhagic shock is a syndrome resulting from reduction of the effective circulating volume, and in which impairment of the circulation steadily progresses until it eventuates into a state of irreversible circulatory failure (WIGGERS 1975). This denition of haemorrhagic shock was described a hundred years ago, but remains valid today with the outcome optimised by uid resuscitation (MARINO 1997). The most common causes of blood loss in birds include traumatic injury and haemorrhagic lesions of internal organs, such as ulcerated neoplasms and proventricular/ventricular ulcerations (FELDMAN et al. 2000). Signicant blood loss can occur during surgical procedures. Heavy infestation with blood-sucking ectoparasites or gastrointestinal parasites, coagulopathies associated with toxicities or severe liver disease are less common causes of blood loss anaemia in birds (FELDMAN et al. 2000). The concept of haemorrhagic shock is poorly understood in avian species. Acute blood loss of 30-40 % of blood volume has been shown to result in 50 % mortality (LD50) in mammals (MARINO 1997). Blood loss is tolerated better in birds than in mammals. The LD50 for acute blood loss in ducks has been shown to be 60% of their total blood volume (LICHTENBERGER et al. 2002). Acute blood loss of greater than 40 % of the blood volume in mammals usually marks the onset of hypovolemic shock with a subsequent increase in heart rate due to a strong baroreceptor response. A high level of sympathetic tone commonly occurs and is characterised by pale mucous membranes, prolonged capillary rell time, poor pulse quality and tachycardia. It has been stated that in the chicken, acute blood loss is not associated with a carotid sinus baroreceptor response as it is in mammals (JENKINS 1997, (STURKIE 1986). In mammals, optimal uid resuscitation subsequent to acute blood loss results in a decrease in heart rate and increase in blood pressure (WIGGERS 1975). This study will investigate if birds have a similar baroreceptor response to shock as seen in mammals. No studies have been done in birds documenting changes in heart rate and blood pressure after acute blood loss and uid resuscitation. When blood loss is severe, the priority of resuscitation is to expand the vascular space and improve cardiac output. Resuscitation implies an urgent need to restore tissue perfusion and oxygenation. The goal is to give the least amount of uids to reach the desired endpoints of resuscitation. The endpoints of resuscitation are a normal blood pressure and heart rate. Crystalloids such as lactated Ringers solution are popular uid therapy for shock as they are inexpensive, and readily available. However, in

355

shock resuscitation, only about 25% of the infused volume of crystalloids remains in the intravascular space by the end of infusion; therefore, it is of limited value as a plasma volume expander unless given repeatedly in very large volumes (TAKORI and SAFAR 1967). Large molecular weight colloids, such as hetastarch (HES) (Abbott Laboratories, North Chicago, IL, USA) and Oxyglobin (Biopure Corporation, Cambridge, MA, USA), given via the intravenous or intraosseous route, remain primarily within the vascular space and do not freely pass into the interstitial space as do crystalloids. Thus colloids should be more effective than crystalloids for rapid, small-volume uid resuscitation subsequent to haemorrhagic shock (MARINO 1997). After acute blood loss, mammals are dependent on red blood cell (RBC) regeneration to maintain oxygen delivery to the tissues. In response to tissue hypoxia, erythropoietin stimulates RBC production by the bone marrow. Reticulocyte release from the bone marrow in mammals occurs rarely after 2-4 days and most commonly longer than 5 days after acute blood loss (FELDMAN et al. 2000). Reticulocytosis, or polychromasia, is the hallmark of intensied erythropoiesis, allowing classication of anaemias into regenerative or non-regenerative types (FELDMAN et al. 2000). A previous study in acute blood loss in the duck documented a early regenerative response shown by presence of polychromasia at 36 hours after blood loss (LICHTENBERGER et al.2002). The relatively short life span of the red blood cell (28-45 days) and presence of a nucleated red blood cell (RBC) may account for a birds ability to mount a very early regenerative response. No studies have been done in birds with acute blood loss, showing regenerative changes documented by absolute numbers of polychromasia, changes in haematocrit (HCT), and haemoglobin (Hb) concentrations. The purpose of this study in mallard ducks (Anas platyrhynchos) was threefold: to demonstrate the heart rate and blood pressure response to acute blood loss (at the predetermined LD50) as well as the response to uid resuscitation; to demonstrate the efcacy and safety of small-volume colloid resuscitation (HES and Oxyglobin) with crystalloids as compared to small-volume crystalloid resuscitation after acute blood loss; and to demonstrate the time required for a regenerative red blood cell response after acute haemorrhage by documenting an increase in absolute polychromasia, HCT, and Hb concentrations.

2 MATERIALS AND METHODS

Mallard ducks were used in the uid administration studies. The uid resuscitation studies were performed at the Medical College of Wisconsin research facility and were approved by the Medical College of Wisconsin Animal Care and Use Committee. All birds were housed at the Medical College of Wisconsin research facility during the study period and were fed a commercial pelleted ration. Nine of the 29 birds were housed in pairs in cages. The other 18 birds were housed together in large pens. Twenty nine mallard ducks (15 males and 15 females, less than 1 year of age) were initially included in this study. Physical examination ndings and weight were recorded. Heart rates using an electrocardiogram (Escort, Medical Data Equipment, Arletta, CA, USA) and indirect blood pressure monitoring (Park Medical Electronics

356

Inc., Aloha, OR, USA) were recorded after anaesthesia induction and intubation, after phlebotomy, and after uid resuscitation. A 19-gauge buttery catheter was placed in the jugular vein, and 6 ml/100 gm body weight (determined as the LD50 for acute blood loss) was withdrawn over 10-20 minutes. Two birds were eliminated from the study, because they died after blood collection and prior to uid resuscitation. Subsequently, the remaining 27 birds were randomized (9 birds in the crystalloid group, 9 birds in the HES/crystalloid group and 9 birds in the Oxyglobin/crystalloid group) to receive either 15 ml/kg of an isotonic crystalloid, 10 ml/kg of a crystalloid with 5 ml/kg of HES, or 10 ml/kg of a crystalloid with 5 ml/kg of Oxyglobin over a 5-minute period. Anaesthesia was turned off after the uid resuscitation. Heart rate and blood pressure monitoring were recorded after uid resuscitation and until the bird woke up from the anaesthesia. The birds were recovered in a cage. The birds were observed for mortality over the following 96 hours. Any surviving birds were then euthanised. Blood was collected from 3 birds in each of the 3 uid resuscitation groups at 0 h (before phlebotomy, after phlebotomy and after uid resuscitation), 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h. A blood smear was made and the rest of the sample was placed in a microtainer (Microtainer Brand, Becton Dickinson Vacutainer Systems, Franklin Lakes, New Jersey, USA) with EDTA anticoagulant after a blood smear had been made. All blood smears and samples were sent to the Avian and Wildlife Laboratories (University of Miami, Miami, FL, USA) for evaluation within 24 hours from collection. The HCT was evaluated at 0 h, 24 h and every 12 hours until completion of the study. The haemoglobin concentrations were evaluated at 0 h, 48 h, and every 12 hours until completion of the study. The absolute degree of polychromasia (polychromatic cells/100 RBC cells with an average of 300 cells counted) was determined at 0 h and every 12 hours until completion of the study. Death rate between the three uid resuscitation groups was statistically analysed using chi-square analysis. Polychromasia, RBC counts, haemoglobin (Hb), and HCT were evaluated using paired, 1 or 2 tailed T test.

3 RESULTS AND DISCUSSION

Haemorrhagic shock is poorly understood and not well studied in avian species. During the uid resuscitation study, there was a trend of decreased mortality in the Oxyglobin group; however, we found no statistical difference in mortality rate among crystalloids, HES and Oxyglobin used for resuscitation from acute blood loss. The oxyglobin group resuscitated quickly to normal haemodynamic parameters and awoke without signs of weakness. The HES and crystalloid group did not recover as well as the oxyglobin group. This may show that Oxyglobin may be a superior resuscitation uid for acute blood loss, when compared to HES or crystalloids. Further studies are needed to monitor the haemodynamic response to acute blood loss as well as to various protocols of uid resuscitation in the avian species. Also, future studies need to be done using a larger number of birds and use of different volumes of uids to a resuscitation endpoint.

357

All birds in the study became tachycardic with a decrease in blood pressure immediately post phlebotomy, and hypotensive and tachycardic when the amount exceeded 2030%. The heart rate and blood pressure returned to normal after uid resuscitation in most of the birds. These responses may indicate that birds have a baroreceptor response to intravascular uid volume changes as is seen in mammals. In this study, birds started to show a RBC regeneration starting at 12 hours after acute blood loss and peaking at 60 hours, as measured by the absolute number of polychromatic cells, Hb concentration and HCT. This response is much earlier than that seen in mammals. Further studies need to be done evaluating erythropoietin levels and bone marrow changes after acute blood loss.

4 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. WIGGERS CI. Hemodynamic and metabolic alterations in peripheral tissue during haemorrhagic shock. Ann Surg 1975;11: 696 - 703. MARINO PL. Hemorrhage and hypovolemia. In: MARINO PL (ed): The ICU Book. Philadelphia, PA: Williams & Wilkins ; 1997; 207 - 227. FELDMAN BF, ZINKL JG and JAIN NC. Red blood cell regeneration. In: FELDMAN BF, ZINKL JG, JAIN NC (eds): Schalms Veterinary Hematology. Baltimore: Lippincott Williams & Wilkins; 2000; 445 - 467. LICHTENBERGER M, ORCUTT C, DeBEHNKE D, et al. Mortality and response to uid resuscitation after acute blood loss in mallard ducks (Anas platyrhynnchos). Proc Assoc Avian Vet, Pittsburgh 2002: 65 - 70. JENKINS JH. Avian critical care and emergency medicine. In: ALTMAN AB, CLUBB SL, DORRESTEIN GM and QUESENBERRY K (eds): Avian Medicine and Surgery. Philadelphia: WB Saunders 1997: 839 - 863. STURKIE PD. Body uids: Blood. In: STURKIE PD (ed): Avian Physiology. New York, NY: Springer-Verlag 1986: 102 - 121. TAKORI M and SAFAR P. Treatment of massive hemorrhage with colloid and crystalloid solutions. Studies in dogs. J Am Med. Assoc 1967; 199: 297.

AUTHORS ADDRESS
Marla Lichtenberger, DVM, DACVECC The Animal Emergency Center 2100 West Silver Spring Drive Glendale, WI, 53209 United States of America Email: AECMARLA@AOL.COM

358

Clinic for Birds, Meppel, The Netherlands

PARROTS DONT BITE CHILDREN

J. Hooimeijer, DVM CPBC

KEYWORDS
Parrots - Behaviour - Biting - Children

ABSTRACT
An impressive beak is one of the salient features of a parrot. It is also often the source of considerable anxiety for parents who associate a beak with biting and are concerned about the damage that could be inicted on their children. It is therefore important to realize that a parrot does not use its beak in the wild in order to injure or kill, but for climbing, eating, preening, feeding and defending.

1 INTRODUCTION
The beak of a parrot, a cockatoo or a macaw is an imposing instrument that many bird owners regard with a certain amount of awe and anxiety. The power behind a parrots beak is well known to everyone. In the wild, beaks are used to crack open hard nuts and strong seed coverings. Nesting holes in trees are enlarged using this same powerful tool. In captivity proffered tree branches are turned into matchsticks, nuts that have been fastened with a wrench are loosened from their bolts, and toys and furniture are reduced to fragments, all by these same beaks, and seemingly without effort. The amount of power that a parrot can exert with a lightweight skull and a lightweight beak is exceedingly impressive. By combining strong muscles and the hinge construction of the upper beak, parrot beaks can be as effective as a pair of strong sharp pliers. Apart from eating and nest building, the beak also has many other important nonaggressive functions. It is used as a third foot when the birds are climbing to keep them steady. It is used to hold objects so that the sensitive tongue can investigate them. The beak is also the instrument that is used to care for the birds own feathers and for those of his or her partner. Young birds are also cared for using the beak.

359

Biting is a frequently cited reason for getting rid of a pet parrot, who then disappears into the cycle of sale and re-sale, or is dumped in a rescue centre. The arrival of a baby in the house often coincides with the departure of the parrot because of the new parents fear that their offspring will not be safe around their pet. That beak, after all, what damage that could do to little ngers, little toes, little ears, or little nose!!!

2 BITING OTHER PARROTS IN THE WILD

It is striking that there is no signicant data to support the idea that parrots inict serious or fatal bite wounds on each other in the wild. On the contrary, all evidence indicates that deliberately wounding or killing their fellows is not part of the natural behaviour of parrots. Although they are equipped with a built-in lethal weapon that could easily maim or kill another bird of their own kind, such behaviour is practically unknown in nature. Debilitating members of the same species is not in the interest of the preservation of that species. Skirmishes certainly take place, but these are mostly displays and mock ghts in which real damage is seldom done. Parrots learn early to read the body language of their fellows and know precisely what is permitted and how far they can go in their combativeness. Playful romps with other youngsters are part of the learning and socialization process for every young parrot and seldom if ever lead to injury.

3 BITING OTHER PARROTS IN CAPTIVITY

In captivity biting problems are seen most notably among the cockatoos, where males have been known to seriously injure or even kill a female. Pet cockatoos are also prone to express frustration by mutilating themselves using their beak. Neither of these behaviours is known to occur in the wild. Limitations due to the size of the housing of birds in captivity often hinder avoidance behaviour or make it impossible for the birds to respond appropriately to body language that in the wild would elicit a retreat from a confrontational situation. Unable to ee, a bird becomes insecure and aggressive. Attacking or biting other birds can be regarded as unnatural behaviour due to the circumstances of captivity. In captivity birds of some species have been known to attack and even kill sick or wounded fellows. Serious head wounds have been observed in budgies, cockatiels and lovebirds when they are housed in same-species groups. It is not uncommon that the dead birds are then cannibalised. The author is not aware to what extent, if at all, this occurs in nature. We can regard biting in captivity as an expression of insecurity, and thus part of a behaviour problem. We observe insecurity in birds that are not treated with respect, as well as in periods of hormonal or sexual activity, and in instances of physical problems or sickness.

360

Birds with a strong attachment to their owner exhibit bonding behaviour, which in turn causes territorial behaviour. This territoriality is often considered aggressive or dominant behaviour although it is actually insecure and defensive. Away from his own territory, or when the partner/owner is absent, the bird behaves completely differently.

4 BITING PEOPLE IN CAPTIVITY

There is a constant stream of stories and anecdotes from parrot owners who report having been bitten by their pets. The number of instances where subsequent medical attention was necessary, however, remains exceedingly small. This is surprising considering the amount of damage a parrot beak could do if actually used with the intention to maim or injure. It is even more surprising that at the Clinic for Birds in the last 22 years we have not seen a single incident of a parrot biting a child. In addition to that, consistent inquiry by this author as to personal, anecdotal or media-covered experience of bitten children has not uncovered a single incident of a child being bitten by a parrot. Again, considering the actual capabilities of a parrot beak, and the size of a childs nger, nose, or ear, one might have expected to hear stories of severed or mutilated young appendages. In our experience, parrots react completely differently to children compared to the way they react to adults. Apparently parrots view children in much the same way as human adults do: they are not seen as threatening and therefore do not make the birds feel insecure. This is all the more striking when compared with the behaviour of dogs. When a dog owner is afraid that his pet might bite a child, his insecurity turns the child into a confusing factor in the dogs environment and increases the chances that the dog indeed will bite. In spite of the fact that most parents feel anxiety about the perceived risk that a parrot will bite a child, parrots do not respond to this by biting. At most, the bird plays a game in which he pretends to bite, but does not carry out the threat. The frightened reaction of the parents can be regarded as a reward for this undesired behaviour, thus reinforcing it. Even in situations where one could think that the parrot had every reason to bite, as when a child pets too hard, pulls a tail, or intentionally or inadvertently teases, parrots do not inict the expected wounds. At most, a blackand-blue mark may be the result, and this is most often caused by pulling back of the nger or hand that was being held in the beak. Apparently parrots have a natural inhibition when it comes to biting children. This is all the more reason to respect their natural behaviour. In spite of the many mistakes that are made in handling them, biting is not a normal parrot behaviour pattern.

5 CONCLUSION
Biting can be regarded as learned behaviour in parrots, behaviour that is often unintentionally rewarded. Parrots that bite are usually not approached and treated with respect. Allowing a parrot to see that a person is afraid of it, is a sign of disrespect. When a parrot sees that the owner has a problem, it becomes insecure and loses

361

its respect for the owner. It is striking to observe that children apparently cause no feelings of insecurity in parrots even when the parents make it clear that they do not trust the bird. There is every reason to have much respect for the intelligence and the normal behaviour of parrots. This is certainly the case when we realize that they adapt to extremely unnatural circumstances in order to integrate in captivity as house pets. It is even more impressive when we realize that parrots dont bite children in spite of the fact that all too many children are victims of child abuse by their own parents or are victims of biting dogs.

6 ACKNOWLEDGEMENTS
I would like to thank Gina Kornblith for helping with the translation of this paper.

7 CITATION INDEX
1. 2. 3. WILSON L. Biting and screaming in companion parrots. Proc Assoc Avian Vet, Portland 2000, 71 - 76 HOOIMEIJER J. Behavioral problems of cockatoos in captivity. Proc Assoc Avian Vet, New Orleans 2004, 271 - 281. HOOIMEIJER J. A Practical behavior protocol for dealing with parrots. Proc Assoc Avian Vet, Pittsburgh 2003: 177 - 181

AUTHORS ADDRESS
Jan Hooimeijer, DVM CPBC Clinic for Birds, Galgenkampsweg 4, 7942 HD Meppel, The Netherlands Email: jan.birds@worldonline.nl

362

Concurrent Master Classes Sessions

Department of Veterinary Pathology, University of Utrecht, The Netherlands

THE AVIAN RESPIRATORY SYSTEM : NORMAL AND PATHOLOGICAL ASPECTS

P. Zwart, DVM, PhD, Dipl ECVP, Em. Prof.

ABSTRACT
An extensice survey was conducted of pathological ndings in the lungs and air sacs of birds. Macroscopic and microscopic designations of types of lesions are given. These included the pathogenesis and aspect of the lesions.

KEYWORDS
Bird Lung - Respiratory system - Air sac - Pathology

1 INTRODUCTION
The respiratory organs of the bird reveal specic anatomical and physiological peculiarities, especially during clinical examination, diagnostic techniques and anaesthesia. These must be taken into consideration by the practitioner. The avian respiratory system is the most sophisticated structure in the Animal Kingdom. The lungs are relatively small, but very efcient. The total volume of the avian lungs is 50 % smaller than in mammals. Yet, the surface available for gas exchange is 10 times larger. In contrast to the general belief, the avian lung is elastic. This is clearly demonstrated in the following setup: at post mortem only one lung is removed, while the other lung remains in its position in the thorax. Both parts are xed. After xation the second lung is removed and the difference in volume of the two lungs is measured (personal observations). The trachea is provided with rings of cartilage. In some avian species, especially in cranes, the trachea winds subcutaneously over the breast muscles in a species-specic way. The main bronchus enters the lungs and opens caudally into the posterior thoracic and the abdominal air sac. From the main bronchus, the ventral bronchi, and more caudally, the dorsal bronchi branch. The dorsobronchi split into the parabronchi. These run mainly parallel. The inner surface of the parabronchi is covered with a network of muscle bundles. The end terminals of this network are the openings to the atria. The atria continue as infundibuli, which cross the respiratory tissue, to reach the outer boundary of the respiratory unit

365

(SMITH et al. 1986). The delicate air capillaries branch from the infundibuli. The air capillaries anastomose with each other and are surrounded by blood capillaries. The infundibuli, as well as the air capillaries, are covered by very thin pneumocytes. The basal membrane is covered on one side by the pneumocytes and on the other side by the capillary endothelium. At light microscopy, the pneumocytes are invisible as they only show up in electron microscopy. Between the pneumocytes type I, the granulated type II pneumocytes can be found. In the avian lung, surfactant is produced (SMITH et al. 1986). Blood supply to the lungs is via the arteria pulmonalis, which branches between the parabronchi. Short, delicate arterioles enter into the respiratory tissue to branch into the blood capillaries. The venous conveyance via primordial veins, starts near the inner surface of the parabronchial lumen. It is then collected in larger veins, running between the parabronchi. Lymph vessels surround the arteries. Small ganglia of nerve cells are scattered throughout the lung. A remarkable anatomy is seen in king penguins (Aptenodytes patagonica). In these birds, broad bands of connective tissue, rich in blood vessels, are situated between the parabronchi. Starting in these bands, blood vessels enter into the respiratory tissue. The air sacs consist of two thin layers of connective tissue, the bres of which run perpendicular to each other. There is an arterial blood supply, a capillary network and venous transport. The wall is covered with a very thin epithelium, the surface of which is mainly provided by microvilli. Near the entrances of the bronchi, there are scattered, small areas of taller cells provided with ciliae. The innervation of the air sac wall is via the rami pulmonales of the N. vagus. For further examination of air sacs at post mortem, a slide can be placed under an airsac wall and cut loose at the edges. A drop of physiological saline is added and the sample is covered with a cover glass. Especially in polarised light, the bres of connective tissue and abnormalities can be recognized. Pathology of the upper respiratory tract There are only a few, more extensive treatises on the pathology of the upper respiratory tract. GYLSTORFF and GRIMM (1998) recognised obliteration of the nares caused by hypertrophy of the cere as well as foreign or regurgitated material and necrotic and/or ulcerative processes. Trauma can cause marked lesions. They may be the result of picking at food outside the cage, ghts (pheasants, chicks, arassaris and birds of prey). In young pigeons, lesions around the nares may be the result of rank-order ghts. These may result in ulcerative or granulomatous lesions, which, however, rarely lead to clinical signs such as sneezing, accumulation of uid or respiratory distress. In pigeons and agapornids, pox can be recognised. In canary birds, Knemidocoptes pilae infection is a frequent aetiology of lesions around the nares. Vitamin A deciency may lead to a variety of secondary disorders of the respiratory system. However this problem is well known and is only mentioned here.

366

Aspiration Trachea: Main Bronchus: A seed (blocks the trachea). 1) A seed causes local inammation. 2) Pieces of peanuts results in local accumulation of fat (BRUNNER and MEINL 1976). 3) Eggs of Syngamus trachea: severe local inammation. 1) Food particles [starch granules]: (forced feeding) Distributed throughout the lungs. Acute death. 2) Pieces of plants etc: Inammation evt. inducing foreign body giant cell reaction. 1) 2) 3) Deposition in lungs 3) Soot (anthracosis) (originating from burned coal, oil, wood etc.): Accumulation of black, non-birefringent material in atrial walls. 4) Dust (pneumoconiosis) (from sand, earth, diatoms etc): Granulomas with greyish, birefringent material in atrial walls. 5) Oxalate-Crystals produced during A. niger infection in vivo (WOBESER and SAUNDERS 1975): causes a haemorrhagic necrotic pneumonia. 6) Iron pigment (pulmonary haemosiderosis): in case of haemolytic anaemia (YAMATO et al. 1996). 7) Amyloid in muscle-bundles of atrial walls (NAKAMURA et al. 1998). In cases of generalised amyloidosis. 8) In man: Lung carcinoma related to inhalation of dust from feathers? The trachea may be subject to a number of inammation types. Viruses, bacteria, Mycoplasms, parasites and irritating gases are known aetiologies. The character of the inammation may be serous, catarrhal, purulent or chronic proliferative changes. Occasionally, secondary lymphoid tissue may develop. We observed a severe proliferative reaction of the tracheal epithelium in Gouldian nches (Chloebia gouldiae), after inhaling a formalin-based disinfectant. The animals suffocated. Infectious aetiologies are: mites (esp. canaries), Syngamus sp., Trematodes (Tracheophilum sisowi in swans). Blood (effect of trauma). Yolk of ruptured egg follicle: severe catarrhal pneumonia. Stomach contents: Inammation; foreign body reaction.

Lungs

Retrograde aspiration

367

The effects of Vitamin A deciency on the trachea may be different in different birds. In Galliformes, cornied epithelial cells accumulate in the mucous glands and lead to the production of pinhead-sized whitish, elevating spots. In Psittaciformes, the mucous glands are lost and the tracheal mucosa changes into a uniform layer of stratied, cornifying epithelium leading to infections. In the syrinx, metaplasia of epithelium, may lead to a most severe respiratory distress, which can be accompanied by sounds. Tracheoscopy may loosen the mass, which can then be inhaled. Suffocation of the patient can be a consequence. Aspiration of the material can eventually alleviate the acute danger. Bleedings in the trachea, eventually followed by aspiration of blood may be consequences of trauma such as collision with cables and others. The main bronchi Chronic infections or irritations may lead to the production of Bronchiolar Associated Lymphoid Tissue (BALT). Inammatory changes are seen in case of cryptosporidiosis. Proliferation of the epithelium of the main bronchi may occur in canary pox infections (presence of an epidermal growth factor). The respiratory tissue Microscopically, cartilage and bone tissue can be found as a normal constituent of the avian lung. Especially in Galliformes, Psittaciformes and Accipitriformes, small pieces of these tissues are present (BORST and ZWART 1976). Aplasia Aplasia of part of a lung is a rare nding. Mechanical lesions of the lungs Perforation of the thoracic wall may occur. Bullets, arrows etc. can cause considerable damage. In case of lesions caused by claws of a cat, there is a danger of Pasteurella multocida infection. Generalised lesions These are: 1. 2. 3. 4. Inhalation of water (drowning). Inhalation of Teon fumes. Heat shock (high temperatures, keeping a bird for some period in the closed hand). Typical example are pigeons, waiting in their basket to be vaccinated. The subcutaneously situated venous plexus at the ventral side of the neck is overlled. The lungs are markedly hyperaemic and serous uid is extravasated into the respiratory capillaries and the parabronchi.

368

5. 6.

7. 8.

Emphysema. It is essential for the functioning of the lungs that a sufcient volume of air passes through the parabronchi. In case of pneumonia, at least part of the parabronchi dilates to ensure such a passage. In case of suffocation (narcosis in concentrated ether) there is an acute dilatation of the parabronchi. After 2-4 days of respiratory distress, the infundibuli dilate; the rst signs of disturbance of respiratory capillaries occur. With time, there is a progressive loss of respiratory tissue, resulting in a wide parabronchial lumina, a minimal amount of respiratory tissue, and increase in connective tissue. Most probably this situation has been studied separately (ZANDVLIET et al. 2001). Causes of emphysema are sub acute of chronic respiratory impairment such as chronic inammation due to mite infection, compression by tumours, pulmonary tuberculosis and/or mycosis etc. Technically perfect survey radiographs may reveal an indication of dilatation of parabronchi. Passive congestion of blood in lungs. This may occur in case of degeneration of the heart muscle. At microscopy, a characteristic aspect is the parallel arrangement of the blood capillaries. In a minah bird (Gracula religiosa), suffering from hydrops ascites, the congestion of lungs coincided with the production of haem-crystals.

Changes in blood vessels 1. 2. 3. 4. 5. Fat emboli (in 22 out of 49 psittaciformes (BRUNNER and MEINL 1976). Thrombi (causes: 1) vascular invasion by Absidia corymbifera; 2) diffuse intravascular coagulation, 3) bacterial thrombi). Thromboembolic lesions (in case of bacterial inammation such as bumble foot or endocarditis (GREENWOOD et al. 1996). Proliferation of Arteria pulmonalis branches in the lungs Calcium deposits in arteria.

Pneumonia Pneumonias are the most frequent cause of death in captive birds (IPPEN and SCHROEDER 1972). The classication given in this paper, in principle, is based on comparative pathology. However, this cannot be applied throughout, due to differences in anatomy and physiology. For instance, the caudal parts of the lungs are most frequent affected, which most probably is related to the retrograde ow of air through the lungs. At post-mortem it should be paid attention whether the parabronchi are lled with air or with uid or exudates. (in case of deep freezing there is a lot of free uid it is no longer xed in cells). At macroscopic examination, various types of lesions can be recognised 1. 2. Focal haemorrhagic-catarrhal pneumonias can be the result of viral (canary pox), bacterial (salmonellosis, pseudotuberculosis) and fungal (aspergillosis in weakened birds) infections. Cavernous pneumonias are rare. I have seen these in connection with foreign materials such as Silicium (pneumoconiosis) and plant material.

369

3.

Granulomatous pneumonias occur especially with bacterial (E. coli, Salmonella typhimurium, Y. pseudotuberculosis) and fungal infections (A. fumigatus).

According to the type of reaction, avian pneumonias can be differentiated in: 1. 2. 3. Serous pneumonia, with exudation of protein rich exudates and generally a marked hyperaemia. The stage of hepatisation was not recognized in birds; most probably they died before this stage has developed. Catarrhal pneumonia with exudation of heterophils and lymphocytes. Fibrinous pneumonia with exudation of brin. This can be a localised reaction, for instance around inammatory foci caused by bacteria of fungi.

Atrial walls are centres of reaction. They are involved in: 1. 2. 3. 4. Swelling and proliferation in case of an acute diffuse pneumonia. Eventually combined with a metaplasia to mucus producing cells. Exudation of macrophages (phagocytising fungal spores and other materials). Small nests of lymphocytes may occur in atrial walls. Organisation of exudates in parabronchial lumina, by broangioblastic tissue. Eventually in combination with phagocytising foreign body giant cells.

Pathologic changes in the respiratory tissue generally are very delicate. Only occasionally proliferation of the respiratory epithelium and/ or the capillary endothelium is recognised. Impressive changes were found in a white-fronted goose (Anser albifrons) suffering from leucosis. The respiratory epithelium of air capillaries was cubic (and the epithelium of the atria revealed polyp-like proliferations). The exudate in the parabronchi had produced hyaline membranes. Calcium deposition on the walls of respiratory capillaries may incidentally occur. Loss of respiratory tissue is seen in case of emphysema due to prolonged respiratory distress or chronic infections of low intensity. It can also be a consequence of the presence of parasitestages in the endothelial cells (Plasmodium infection in penguins). Fibrosis of lungs is very rare. Though some brosis generally occurs in case of emphysema (as a result of inammation), very few cases are known in which the volume of respiratory tissue has not distinctly diminished and reveals a diffuse increase in connective tissue. Pathologic proliferation of muscle bres in the tips of atrial walls occurs in cases of chronic irritation. Focal proliferation is seen near the adherence sites of lung mites. A more diffuse proliferation is occasionally seen, though the aetiology could not be dened. The lymph vessels around the artery can also be involved in pneumonias. In case of oedema, these lymph vessels may be overlled. They also may contain lymphocytes and/or heterophils, bacteria or parasites (tachyzoites of Toxoplasma). Tumours Primary lung-tumours are rare in birds (Table 1).

370

Table 1: Primary lung tumours listed in birds.

Common name Cockatiel Grey parrot Salmon crested cockatoo Scientic name Nymphicus hollandicus Psittacus erithacus Cacatua moluccensis Type of tumour Bronchial tumour Fibrosarcoma Cystadenoma Author Burgmann1994 Andr and Delverdier 1999 Powers et al. 1998

Metastasis of tumours to the lungs is known (Table 2). Table 2: Metastasis of tumours to the lungs listed in birds.
Common name Golden eagle Red-tailed hawk Eider duck Scientic name Aquila chrysaetos Buteo jamaicensis Somateria mollissima Type of tumour Bile duct carcinoma Bile duct carcinoma lipomatosis/ bromatosis Author Mikaelian et al. 1998 Hartup et al. 1996 Daoust et al. 1991

Tumours may cause compression of the lungs. This was seen in a chick (Gallus gallus forma domestica) suffering from an osteosarcoma of the ribs. It had resulted in extensive emphysema of the lungs. The air sacs Generally, lesions in the air sacs are related to these in the lungs. We could recognise catarrhal and chronic reactions. Rupture of air sacs Rupture of air sacs are mainly caused by trauma or marked respiratory distress (pneumonia, endocarditis). Un-infected wounds generally heal without further treatment. In dense colonies of terns (Sterna hirundo) and herring gulls (Larus argentatus), the frequency is 1.57 %. Most of these cases are the result of trauma and removal of accumulated air results in a rapid healing. In case uid is accidentally injected into an air sac, this may result in suffocation or pneumonia (check by redrawing the plunger). Inammation of air sacs Acute catarrhal lesions were characterised by hyperaemia and production of an exudate. At microscopic examination, hyperaemia was clearly recognised. In addition inltration of heterophils, lymphocytes and macrophages could be recognised. Chronic alterations were seen as a variable increase in connective tissue, inltration of inammatory cells, and local production of lymph follicles. At endoscopy examination of the air sacs, aspergillomas, mites or lariae may be found. Inammation of muscle bundles stretching from the ribs over the ventral surface of the lungs was recognised

371

in a macaw, suffering from a bacterial pneumonia. The muscles were hyaline and yellowish. In addition, the same muscles were invaded by sarcosporids. In case of rupture of the proventriculus (psittacine proventricular dilatation syndrome), perforation of the gizzard (sharp objects), food particles and even grit may be propelled into the air sacs. This may lead to inammation of the air sacs, showing up as thickening and (brownish) discolouration.

2 CITATION INDEX
1. ANDR JP and DELVERDIER M. Primary bronchial carcinoma with osseous metastasis in an African grey parrot (Psittacus erithacus). J Avian Med Surg 1999; 13 (3): 180 - 186. 2. BORST GHA and ZWART P. Bone structures in avian and mammalian lungs. Vet Path 1976; 13: 98 - 103. 3. BRUNNER P and MEINL M. Fat emboli in the blood vessels of the avian lung (Fettembolische Verschlsse in den Blutgefssen der Vogellunge). Vet Pathol 1976; 13: 17. 4. BURGMANN PM. Pulmonary brosarcoma with hepatic metastases in a cockatiel (Nymphicus hollandicus). J Assoc Avian Vet 1994; 8 (2): 81 - 84. 5. DAOUST P-Y et al. Multicentric intramuscular lipomatosis/bromatosis in free-ying white-fronted and Canada geese. J Wildl Dis1991; 27(1): 135 - 139. 6.GREENWOOD AG. et al. Vegetative endocarditis in a Waldrapp ibis (Vegetative endocarditis in a Waldrapp ibis). Avian Pathol 1996; 25(2): 387 391. 7. GYLSTORFF I and GRIMM F. Avian diseases (Vogelkrankheiten). 2nd Ed. Ulmer, Stuttgart 1998; 330 - 334. 8. HARTUP BK. et al. Cholangiocarcinoma in a red-tailed hawk (Buteo jamaicensis). J Zoo Wildl Med 1996; 27(4): 539 - 543. 9. IPPEN R and SCHROEDER H-D. A contribution to diseases of zoo-birds (Ein Beitrag zu den Erkrankungen der Zoovgel). Verhandl. Ber. Erkrk. Zootiere 1972; 14: 11 - 27. 10. MIKAELIAN I. et al. Metastatic cholangiocellular carcinoma and renal adenocarcinoma in a golden eagle (Aquila chrysaetos). Avian Pathol 1998; 27(3): 321 - 325. 11. NAKAMURA K. et al. Systemic amyloidosis in laying Japanese quail. Avian Dis 1998; 42(1): 209 - 214. 12. NORRMANN A. The diseases of the respiratory organs in the budgerigar (Die Krankheiten der Atmungsorgane beim Wellensittich). Kleintierpraxis 1971; 16: 169 - 179. 13. POWERS LV. et al. Axillary cystadenocarcinoma in a Moluccan cockatoo (Cacatua moluccensis). Avian Dis 1998; 42(2): 408 - 412. 14. SMITH BL. et al. Pneumoconiosis in the captive New Zealand Kiwi. Vet. Pathol 1973; 10: 94 - 101. 15. SMITH JJ. et al. Microscopic and submicroscopic anatomy of the parabronchi, air sacs and respiratory space of the budgerigar (Melopsittacus undulatus). Am J Anat 1986; 177(2): 221 - 242.

372

16. WELLS R. et al. Acute toxicosis of budgerigars (Melopsittacus undulatus) caused by pyrolysis products from heated polytetrauoroethylene: clinical study. Am J Vet Res 1982; 43(7): 1238 - 1242 and 1243 - 1248. 17. WISSER J. ZWART P. et al. Deposits in the air passages of zoo birds (Ablagerungen in den Luftwegen bei Vgeln aus zoologischen Grten). Verhandl. Ber. 32 Int. Symp. Erkrank, Zootiere Eskilstuna 1990; 137 - 142. 18. WOBESER G. and SAUNDERS, JR. Pulmonary oxalosis in association with Aspergillus niger infection in a great horned owl (Bubo virginianus). Avian Dis 1975; 19: 388 - 392. 19. YAMATO O. et al. Hemolytic anemia in wild seaducks caused by marine oil pollution. J Wildl Dis 1996; 32(2): 381 - 384. 20. ZANDVLIET MMJM. et al. Chronic pulmonary interstitial brosis in Amazon parrots. Avian Pathol 2001; 30: 517 - 524.

AUTHORS ADDRRESS
Peernel Zwart DVM., PhD., Emerit Professor. Dept Veterinary Pathology, University of Utrecht, Burg. V. d. Weijerstraat 16, 3981 EK BUNNIK, The Netherlands. Email: bibliozoo.p.zwart@wxs.nl

373

The Animal Emergency Center Glendale, WI., United States of America

SHOCK, FLUID THERAPY AND CPCR FOR THE AVIAN PATIENT

Marla Lichtenberger, DVM, DACVECC

ABSTRACT
Increasing number of birds are being kept as pets and owners want to receive high quality medical care for these pets. Many of these birds present in a state of hypovolemic shock due to trauma, gastrointestinal disease, surgical complications, coagulophaties and severe live disease. Treatment of hypovolemic shock and critical care monitoring in birds are complicated by small patient size, physiological diversity and lack of research and clinical data on their response to therapy. Despite these impediments, the same principles and techniques of monitoring used in domestic animals can be applied to the exotic patient. The goal of this presentation is to provide an in-depth discussion on the principles and pathophysiology of shock, types of uids, monitoring techniques, and shock resuscitation methods for use in birds. An understanding about uid therapy and blood pressure monitoring will be discussed. The message of clinical importance is that xed uid regimens (e.g., Lactated Ringers), xed volumes (e.g., ml/kg) and rules of thumb are in most instances outdated, inappropriate and often times inadequate. Appropriate uid therapy, combined with frequent patient evaluation and periodic blood pressure monitoring techniques, can produce astounding and at times miraculous results in avian medicine.

1 INTRODUCTION
Increasing numbers of birds are being kept as pets, and owners want to receive high quality medical care for these pets. Treatment of hypovolemic shock and critical care monitoring in birds are complicated by small patient size, physiological diversity and lack of research and clinical data on their response to therapy. Despite these impediments, the same principles and techniques of monitoring used in domestic animals can be applied to the avian patient. The goal of this article is to provide an in-depth presentation on the principles and pathophysiology of shock, types of uids, monitoring techniques, and shock resuscitation methods for use in birds. Principles of cardiopulmonary-cerebral resuscitation will also be discussed. Arterial blood pressure measurement is an important tool in the management of the critically ill bird. The message of clinical importance is that xed uid regimens (e.g., Lactated Ringers), xed volumes (e.g., ml/kg) and rules of thumb are in most instances outdated,

374

inappropriate and often times inadequate. Appropriate uid therapy, combined with frequent patient evaluation and periodic blood pressure monitoring techniques, can produce astounding and at times miraculous results. Most birds do not show signs of illness in the early stages of disease. Often, birds with chronic disease present as an emergency because of their ability to mask clinical signs of the disease until the condition is severe. In virtually all cases, I advise the receptionist to recommend the bird be brought in for an exam. If the owner is concerned enough to call, then the bird is probably very sick and needs to be seen. While all signs reported by the client can be of concern, sitting at the bottom of the cage, bleeding, respiratory distress, regurgitation and anorexia are considered true emergencies. The client should be instructed to bring the bird in a cage, if possible, otherwise instructs the owner about suitable alternatives (e.g. box, cat carrier). The water dish should be emptied but the cage should not be cleaned prior to travelling to the hospital. Once the bird has arrived, it is ideal to have a trained receptionist call for an immediate triage by a nurse. Prompt, accurate treatment is vital to a favourable outcome. The nurse should assess the condition of the bird in a room. In cases of bleeding, seizuring, head trauma and respiratory distress, the bird should be evaluated immediately by the veterinarian. If the bird is uffed, weak or sitting at the bottom of the cage, they should be placed in a warmed incubator and oxygen is administered. The optimum temperatures for ill birds are 85 to 90F (29-30C). Exam Initially the bird should be evaluated in its cage. Its posture, ability to ambulate and perch, respiratory status, interest in the environment, and ufng of the feathers are assessed. The cage can be examined for discharges and vomit and sources of lead and zinc. Examine the faeces (colour, amount or blood), urates (normally is white to off white), and urine (an increase or change in colour is abnormal) parts of the droppings. Following stress, birds frequently demonstrate polyuria. Perform a complete physical exam as the condition of the bird allows. When handling a bird, it is best to work in a small room with low ceilings, closed window and no fans. Some birds, particularly nches and canaries, should be picked up in a darkened room. Prior to picking up the bird, you should determine if it is safe to restrain the bird. Weak birds and birds in respiratory distress could die during handling. Sudden death is no uncommon with restraint of obese budgies fed an inadequate diet. Efciency is important when handling a bird. Setting up ahead of time for procedures to be performed will reduce the total restraint time. Prociency in handling birds is an important factor in gaining client condence. It is important to warn clients prior to picking up the bird what is going to happen. Most clients have never heard their bird scream or seen their bird struggle the way it does during restraint. It may be better to postpone a complete physical exam in a weak bird or bird in respiratory distress. A quick one minute exam can be performed on a bird while taking the bird out of the cage and placing in the incubator. The head should be examined for

375

oculonasal discharges and swellings. The oropharynx and choanal is examined for colour mucous and presence of blunted papillae. The beak is examined for bleeding, symmetry or fractures. Hydration is assessed by eyelid mobility, skin turgor and dry mucous membranes. The crop is palpated for presence of food or foreign bodies. Observation of rell time of the basilic (wing) vein will estimate perfusion status. Normal veins rell in 1-2 seconds after depression. The heart and lungs are ausculted. The respiratory rate and effort is evaluated. The pectoral muscle should be evaluated to judge the birds body condition. The abdomen is palpated for signs of masses or uid distension. In normal birds, it is difcult to palpate abdominal organs. An increase in the distance between the caudal end of the sternum and the pubis can suggest abdominal organomegaly, neoplasia or ascites. The vent should be examined for matting, redness or swelling. The grasp reex of the feet will help determine weakness. The bird is also weighed on a gram scale.

2 SHOCK IN THE AVIAN PATIENT

Shock is dened as poor tissue perfusion from either low blood ow or unevenly distributed ow. This results in an inadequate delivery of oxygen to the tissues. This denition applies to all species of animals. Recent studies done by the author on shock in birds, has provided in-depth knowledge of a birds response to hypovolemic shock. Fluid resuscitation of the patient in hypovolemic shock can be a challenge and the clinician should understand the basic pathophysiology of shock, principles of perfusion, have knowledge of the different types of uid and blood pressure monitoring techniques. Appropriate uid therapy, combined with frequent patient evaluation and blood pressure monitoring can produce favourable outcomes. The rst part of this paper will discuss general principles of hypovolemic shock, blood pressure monitoring and characteristics of uids. These principles will then be applied to treatment of hypovolemic shock in the avian species. The importance of pain management and nutrition will also be discussed. The second part of this paper will discuss principles of CPCR (Cardiopulmonary-Cerebral Resuscitation) and ARD (acute respiratory distress). Glucocorticoids The use of glucocorticoids in the treatment of shock is controversial. These drugs have been extensively investigated in the shock syndrome. The side effects of immunosuppression, increased risk of infection (i.e., aspergillosis, psittacosis), hyperglycemia and gastric ulceration may outweigh their benets. Their use in shock caused by haemorrhage and hypovolemia is not currently recommended.

376

Sodium bicarbonate The most important method of correction of severe metabolic acidosis is aimed at increasing the pH through increasing the extracellular uid pH. Crystalloid uids containing lactate, acetate, and gluconate (i.e., Plasma-Lyte, Normasol R, LRS) are considered an important means of increasing the alkalinity of the extracellular uid. Correction of acidemia initially begins with correction of the patients perfusion and hydration status through the use of uid therapy. Blood gas parameters must be evaluated before considering the administration of sodium bicarbonate. Since this is rarely possible in the avian patient, use of sodium bicarbonate in shock is not recommended.

3 HYPOVOLEMIC SHOCK-PATHOPHYSIOLOGY
Hypovolemic shock is caused by either an absolute or relative hypovolemia. Potential aetiologies of absolute hypovolemia would be any cause of haemorrhage, including trauma, coagulopathy, gastrointestinal bleeding, surgical mishaps or a ruptured neoplasia. Examples of relative hypovolemia would include severe dehydration from gastrointestinal loss, or extensive loss of plasma as in a burn patient, or loss into a third-body space such as the coelomic cavity, uterus or gastrointestinal tract. The most common cause of hypovolemic shock is haemorrhage. When an animal begins haemorrhaging, there is a decrease in blood volume and decrease in venous return to the right side of the heart. This causes a decrease in return to the left side of the heart and therefore a decrease in cardiac output. With a substantial hypovolemia, blood pressure decreases below a mean arterial pressure of 60 mm Hg or a systolic pressure of less than 90 mm Hg. The carotid and aortic artery baroreceptors detect a decrease in stretch due to the decrease in cardiac output. This sends a neural signal to the vasomotor centre in the medulla oblongata, which results in inhibition of vagal parasympathetic centre and stimulation of the sympathetic centre. This causes vasoconstriction of the veins and arterioles throughout the peripheral circulatory system and increases heart rate and strength of heart contraction. The humoral response is an increase in adrenal circulating catecholamines which in turn stimulates renin release via adrenergic receptors on cells of the juxataglomerular apparatus (specialized smooth muscle cells in the afferent arterioles). Renin causes release of Angiotensin 11, aldosterone, and antidiuretic hormone. There is a strong vasoconstriction and water retention, from their release causing an increase in extracellular uid volume and an increase in blood pressure. The pathophysiology of hemorrhagic shock is poorly understood in avian species. Acute blood loss of 30-40 % of blood volume has been shown to result in 50% mortality (LD50) in mammals. Blood loss is better tolerated in birds than in mammals. The LD50 for acute blood loss in ducks has been shown in a recent study to be 60% of the total blood volume.

377

A recent hemorrhagic shock study in mallard ducks (Anas platyrhynchos) documented an increase in heart rate and decrease in blood pressure following acute blood loss. That study may show that birds have a baroreceptor response to shock similar to that seen in mammals.

4 HYPOVOLEMIC SHOCK-COMPENSATORY RESPONSE

a. Early or compensatory The early or compensated stage of shock occurs as a result of the baroreceptormediated release of catecholamines. Blood pressure increases due to the increase in cardiac output and systemic vascular resistance. A hypermetabolic state results from the humoral release of epinephrine. This is the stage seen commonly in birds with blood loss less than 20% of their total body weight. Clinical signs in the bird include an increase in heart rate, normal or increased blood pressure, and normal or increased ow (bounding pulses and capillary rell less than 1 second). The increased heart rate and normal or increased blood pressure is the key indicator of compensatory shock. Volume replacement at this stage is usually associated with a good outcome. b. Early decompensatory The second stage of shock is called the middle or early decompensatory stage of shock. This stage occurs when uid losses continue. There is a reduction in the blood ow to the kidneys, gastrointestinal tract, skin, and muscles. There is an uneven distribution of blood ow. Oxygen consumption in the tissues becomes dependent on oxygen delivery and anaerobic metabolism, which causes lactic acidosis. Anaerobic metabolism is less efcient than aerobic metabolism. In anaerobic metabolism there is production of lactic acid and hydrogen ions, which lead to metabolic acidosis after exhaustion of the buffer systems. Acidosis leads to cellular swelling with loss of extracellular uid volume into the cells. As cell integrity is lost, with progression of lactic acidosis, there is development of multiple organ dysfunction. Clinical signs of early decompensatory shock in the bird include hypothermia, cool limbs and skin, tachycardia, decreased blood pressure, pale mucous membranes, prolonged capillary rell time, and mental depression. Aggressive uid therapy to support blood pressure and heart rate is required in this stage. c. Decompensatory or terminal shock The nal stage is called decompensatory or terminal shock in all animals. Irreversible hypovolemic shock occurs when the neuroendocrine compensatory responses fail to restore and maintain tissue perfusion. Once greater than 60% of the intravascular volume is lost, the neuroendocrine responses to hypovolemia become ineffective and irreversible organ failure begins.

378

At this stage, the intrinsic compensatory mechanisms no longer provide delivery of oxygen to the tissues, and the heart and brain begin to fail with the severe hypoxia. The pathophysiological prole of terminal shock is a continuum of that described for the early decompensatory stage, except that the damage has overwhelmed the bodys natural protective mechanisms and multiple organ failure has occurred. The clinical signs are bradycardia with low cardiac output, severe hypotension, pale or cyanotic mucous membranes, absent capillary rell time, weak or absent pulses, hypothermia, oliguria to anuric renal failure, pulmonary ooedema, and a stupor to comatose state. Cardiopulmonary arrest commonly occurs.

5 PERFUSION VERSUS HYDRATION

The transport of uid and oxygen through the blood vessels to the capillaries is called perfusion. Tissue perfusion is directly dependent on adequate intravascular volume and a normally functioning cardiovascular system. Perfusion should be evaluated rst followed by an evaluation of hydration. The effects of perfusion are demonstrated on physical examination by perfusion parameters, which include mucous membrane colour, capillary rell time, and resting heart rate as well as blood pressure. These parameters change in response to the various stages of shock as was discussed above. Interstitial hydration is the presence of uid in the interstitial space. Adequate hydration provides support for the cells and transport media for molecules. Clinical parameters evaluated during assessment of interstitial hydration include mucous membrane moisture, position of the ocular globe, and skin turgor. When dehydration is estimated to be greater than 8%, an intravascular volume decit is expected to occur from osmotic uid shifts. Interstitial volume decits are typically associated with a decrease in skin turgor and dry mucous membranes. Rehydration of the interstitial compartment is best accomplished using an isotonic replacement uid. The rate of uid administration depends primarily on the rate of uid losses and clinical status of the animal, as indicated by the physical exam and laboratory parameters. For animals with evidence of interstitial dehydration on physical examination but stable cardiovascular parameters, uid decits can be replaced over 12-24 hours. If the interstitial volume is rapidly lost, then the interstitial uid decit should be rapidly replaced (2-4 hours). Isotonic replacement uids are administered according to the patients estimated dehydration, maintenance needs, and anticipated ongoing losses.

6 CLINICAL TECHNIQUES FOR THE MEASUREMENT OF BLOOD PRESSURE

The gold standard for measuring blood pressure would be the direct method, which entails placing a catheter in an artery and connecting it to a pressure transducer. Unfortunately, arterial catheterization in birds is technically difcult and can be time-consuming, so this method is usually not warranted. Although there are several indirect

379

or non-invasive methods (i.e., oscillometric and Doppler) available, it is sometimes impossible to obtain a reading on the avian patient. The Doppler method is more versatile than the oscillometric method, and is the method used by the author for all exotic patients. The ultrasonic Doppler ow detector (Parks Medical Electronics Inc., Aloha, OR) uses ultrasonic waves to detect and make audible blood ow in an artery distal to the blood pressure cuff. In the birds, the cuff is placed on the distal humerus or femur. The Doppler probe is placed on the basilica and metatarsal arteries respectively. The cuff bladder is inated to a suprasystemic pressure with cut-off of the Doppler signal. The cuff is deated with the rst sound heard and marked as the systolic pressure. Trends of change in systolic pressures obtained using the Doppler ow detector has been found to correlate well with direct pressure measurements in anesthetized ducks (unpublished data done by the author). Normal systolic blood pressure for birds under isourane anaesthesia, measured at the authors clinic is between 90-150 mmHg.

7 RED BLOOD CELL REGENERATION AFTER ACUTE BLOOD LOSS

After acute blood loss, mammals are dependent on red blood cell (RBC) regeneration to maintain oxygen delivery to the tissues. In response to tissue hypoxia, erythropoietin stimulates RBC production by the bone marrow. Reticulocyte release from the bone marrow in mammals occurs rarely after 2-4 days and most commonly longer than 5 days after acute blood loss. Reticulocytosis, or polychromasia, is the hallmark of intensied erythropoiesis in mammals and birds, allowing classication of anaemias into regenerative or non-regenerative types. A previous study done by the author, on acute blood loss in the duck documented a early regenerative response shown by presence of polychromasia starting at 12 hours after blood loss. The relatively short live span of the red blood cell (28-45 days) and presence of a nucleated red blood cell (RBC) may account for a birds ability to mount a very early regenerative response. The use of early supportive care with uid therapy in avian shock may help bridge the gap for the rst 24 hours, after which birds can mount their own RBC regenerative response.

8 FLUID SELECTION
Individual characteristics of available uids inuence the dose, type, and volume of uid administered. Crystalloids solutions can be used together with colloids during the resuscitation phase. Crystalloids are the mainstay of the rehydration and maintenance phases of uid therapy. The three basic groups (i.e., crystalloids, synthetic colloids, and Haemoglobin-Based Oxygen Carriers) of uids will be discussed. a. Crystalloids: Crystalloids (also called replacement uids) are uids containing sodium chloride and other solutes that are capable of distributing to all body uid compartments. Replacement uids have electrolyte concentrations that resemble extracellular uid

380

whereas maintenance uids contain much less sodium (40-60 mEq/l) and more potassium (15-30 mEq/l). The most commonly used replacement uids are 0.9% saline (Baxter, Deereld, ILL), lactated Ringers solution (Abbott lab, North Chicago, Ill) and Normosol-R (CEVA Laboratories, Overland Park, KS) or Plasmalyte-A (Baxter, Deereld, ILL). Buffered solutions are usually indicated for resuscitating patients in shock since administration of a highly acidic solution may worsen a preexisting metabolic acidosis. Buffered solutions usually contain lactate, gluconate or acetate. The liver must metabolize lactate whereas many cells in the body metabolize acetate and gluconate. Therefore uids containing lactate may not be tolerated well by animals with end-stage liver disease. There is evidence to support the use of 0.9% sodium chloride, the highest sodium-containing isotonic crystalloid, to animals with head trauma, to avoid rapid changes in osmolality. Normal saline can also be used for treating conditions causing hyperkalemia (e.g., urinary obstruction and oliguric renal failure), hypercalcemia, and hypochloremic metabolic alkalosis. Since approximately 80% of extracellular uid is in the interstitial space, crystalloids will rapidly redistribute and after approximately 1 hour there will be only 20% of the administered volume remaining in the circulation. On a short-term basis crystalloids will expand the intravascular space, but this effect will be short-lived. Thus, crystalloids should be thought of as interstitial dehydrators, not intravascular volume expanders. This increase in interstitial uid can lead to tissue oedema (thus decreasing the ability of oxygen to diffuse to the cells). Interstitial oedema may be extremely detrimental in cases of cerebral oedema and pulmonary oedema. b. Colloids Colloids are uids containing large molecular weight substances that generally are not able to pass through capillary membranes. Colloids can be considered intravascular volume expanders. Since most patients in shock require sustained intravascular volume expansion, colloids are indicated frequently during uid resuscitation. Examples include synthetic colloids such as hetastarch (Braun Medical Inc, Irvine, CA), and biologic colloids such as whole blood, plasma, and albumin. Ideally blood should be used for resuscitation if the patient has lost whole blood. The lower the haematocrit becomes, the more important this is to ensure adequate oxygen delivery to the cells. If the patient has lost clotting factors, clotting factors should be replaced. The availability of blood products in sufcient quantities to meet the needs of the avian species is often the limiting factor in survival. Most hospitals do not have readily available donors for species specic donation and commercial blood banks do not carry exotic pet blood products. Hydroxyethyl starch is a molecule made from maize or sorghum and is primarily an amylopectin. This synthetic colloid uid contains large-molecular-weight particles that effectively increase the COP beyond what can be obtained with blood product infusion alone. They maintain intravascular osmotic pressure because their molecular size is too large to pass through the normal capillary pores. Their negative charge attracts sodium and water (Gibbs-Donnan effect), producing a nal intravascular volume greater than the volume infused. It expands the volume by about 1.4 times the volume infused.

381

Hetastarch has an average molecular weight of 450,000 Daltons and has a half-life of 25 hours. Synthetic colloids are administered with isotonic crystalloids to reduce interstitial volume depletion. The dose of crystalloid administered is only 40 to 60% of what it would be if crystalloids were used alone during resuscitation. c. Haemoglobin-Based Oxygen Carriers Haemoglobin-based oxygen carriers (HBOC) are indicated during resuscitation when increased oxygen delivery to tissues is desired. Although HBOC are colloids, they have the added advantage of carrying oxygen to the tissues. Oxyglobin is a puried, polymerized bovine haemoglobin that is in a modied lactated Ringers solution that is approved for use in dogs. It is isoosmotic and has an average molecular weight of 200,000 making it a very effective colloid. It has a pH of 7.8 which means it will not contribute to the acidosis of the patient during infusion, as many crystalloid solutions may. It has a COP (20-25 mmHg), similar to that of plasma. Its oxygen afnity is dependent upon the chloride ion concentration not the concentration of 2, 3-diphosphoglycerate (2, 3-DPG). This provides a distinct advantage over blood that has been stored longer than 1 week that may have signicantly depleted 2, 3-DPG levels leading to increased oxygen binding and decreased oxygen delivery at the tissue level. In addition the normal oxygen afnity of Oxyglobin is lower than that of normal canine blood which enhances delivery of oxygen to the tissues. It has a lower viscosity than canine blood that may improve microvascular ow. Because it is a smaller molecule than red cells it is able to perfuse tissue beds that red cells may not be able to reach. Oxyglobin can be administered via standard intravenous administration sets and standard intravenous infusion pumps can be used for delivery. Because it contains no antigens cross matching is not required and there is no possibility of transfusion reactions. Filters are not required. It can be kept at room temperature and has a 3year shelf life, which makes it useful for hospitals that cannot keep blood products readily available. Once opened the bag must be discarded within 24 hours due to the production of methaemoglobin. Oxyglobin is now available in 60 ml containers. Oxyglobin is up to 10 times more effective than blood when given during uid resuscitation to animals in hemorrhagic shock. For this reason, low volumes of Oxyglobin can be used effectively to treat hemorrhagic shock. It has a short half-life (30 to 40 hours); however, the length of clinical benet is currently unknown Oxyglobin can be administered at any time to any species in need of a blood transfusion or during acute and critical situations in which an acute increase in blood volume and oxygen carrying capacity are needed. It is an excellent uid to use in animals suffering form acute blood loss secondary to trauma or surgical mishap and should be administered to patients that have become haemodiluted (PCV< 15-18%) from crystalloids. Oxyglobin can also be administered to patients suffering from chronic anaemia with PCV values less than 10%. Appropriately used, it offers the opportunity to decrease patient morbidity and mortality.

382

9 FLUID THERAPY PLAN

The administration of uids is almost always thought of as the single most important therapy during surgery and for the treatment of patients suffering from hypovolemic shock. The benecial effects of uid administration are most obvious in haemorrhaging or traumatized patients because of the absolute losses of blood and uids from the vascular and interstitial uid compartments. The uid therapy plan typically has a resuscitation (correction of perfusion decits), rehydration (correction of interstitial decits), and maintenance phase. Resuscitation implies an urgent need to restore tissue perfusion and oxygenation. The intravascular volume must be replaced initially. The type, quantity, and rate of uid administration required to reach the desired resuscitation endpoints are determined and depend on the type of shock and underlying disease process. Re-evaluation of the hydration status is necessary before planning the rehydration phase. The maintenance phase provides uids and electrolytes to replace ongoing losses, meet metabolic demands, and restore intracellular water balance. Recommendations for uid resuscitation subsequent to severe blood loss in birds have included the administration of whole blood. However, the use of avian blood products is limited by the lack of availability. A pilot study of acute hemorrhagic shock in cockatiels treated with HES and Oxyglobin found no adverse side effects associated with either uid. Rapid bolus administration (doses of 5 ml/kg over 1 minute) of Oxyglobin or HES with crystalloids (10 ml/kg) has been given to hypovolemic birds (birds with acute blood loss) in research and clinical trials without side effects. In a study on acute blood loss (loss of 60% of their total blood volume), in ducks, bolus infusion of Oxyglobin and crystalloid was effective in correcting heart rate and blood pressure within 30 to 120 seconds after infusion. Since uid resuscitation in critically ill birds is difcult, the importance of small volume resuscitation with one bolus of crystalloids with Oxyglobin to hypovolemic birds may have clinical relevance.

10 FLUID THERAPY-BIRDS
Any sick, debilitated bird presenting for emergency care, should immediately be placed in a warm incubator (Temperature at 85-90F [29.4-32.1C]) with oxygen supplementation for 2-4 hours. When active external haemorrhage is present, this must be stopped immediately. Most birds benet from the administration of warmed crystalloids at 3 ml/100gBW IV, IO or SQ. Birds should be offered food and water during this time. When the bird appears stable (alert, responsive) and can be safely anesthetised with mask isourane (Abbott Lab, North Chicago, ILL) or sevourane (Abbott Lab, North Chicago, ILL), diagnostics and treatment for hypovolemia and dehydration can be performed. Blood pressure monitoring using Doppler and an ECG can be used during these procedures.

383

The Doppler cuff can be placed on the distal humerus or femur and Doppler probe on the medial surface of the proximal ulna or tibiotarsus respectively. The blood pressure of various avian species under isourane or sevourane anaesthesia at the authors clinic is 90-140 mmHg systolic. When blood pressures are below 90 mmHg systolic, birds are treated for hypovolemia as given below. Bolus administration of crystalloids (10 ml/kg) and colloids (HES or Oxyglobin at 5 ml/kg) can be given IV or IO until blood pressure is greater than 90mmHg systolic. In the authors experience 1 or 2 bolus infusions are usually required. In severely dehydrated birds that are not eating IV or IO catheters are placed for replacement of dehydration losses with crystalloids. Estimation of the uid decit is based on estimated dehydration and body weight: Estimated Dehydration (%) x Body Weight (g) = Fluid Decit (ml). Daily maintenance uid requirements (2 ml/kg/hr) are added to the uid decit volume.

11 PAIN MANAGEMENT IN THE CRITICAL AVIAN PATIENT

Most seriously ill birds experience pain and anxiety during hospitalization. Administration of analgesics is strongly recommended in these birds. Butorphanol (0.5-1 mg/ kg) intramuscularly has been shown as a useful analgesic is birds. Non-steroidal antiinammatory drugs as meloxicam (0.5 mg/kg IM) can be used for pain relief, when perfusion parameters and kidney parameters are normal.

12 NUTRITION
Critically-ill avian patients suffering from anorexia, maldigestion, and weight loss need nutritional support. The importance of nutrition early on is so important and emphasizes the saying when the gut works, use it. Perform other procedures before tube feeding to prevent regurgitation and aspiration. Palpate the crop before feeding. Do not feed a bird with food still present in the crop. There are several enteric feeding formulas on the market which range from less than 1.0 to 2.0 Kcal/ml. Hand-feeding formulas for baby birds can also be used. Tube feedings should be warmed in hot water to body temperature to avoid crop stasis. The volume is estimated at 3ml per 100g of bird. Baby birds may be fed up to 5ml per 100g as they have a greater crop capacity. A curved metal feeding needle with a balled tip (Dosing Needles, Ejay International, Glendora, CA) is recommended. Red rubber feeding tubes (Sovereign Feeding Tubes, Sherwood Medical, St. Louis, MO) can be used, however, considerable care should be taken to ensure the bird does not bite the feeding tube and swallow the end. Hold the bird in an upright position with the neck extended. The tube or feeding needle is gently passed into the left side of the beak, over the tongue, and down the right side of the oesophagus into the crop. Palpate the crop and verify that the feeding needle or tube is in the crop. The formula is infused into the crop, the tube or feeding needle is carefully withdrawn, and the bird is released. If the bird reuxes into the mouth during the procedure, release the bird and allow the bird to clear the food from

384

its mouth. The suggested volumes of food to be administered in different species, is given below. The feedings should be done three to four times a day. Persistently regurgitating birds or birds with beak, oesophagus or crop injuries may need to have a feeding tube placed into the proventriculus. This tube is termed an oesophagostomy tube, and can be left in place until the bird starts eating on their own.

Suggested Volumes for Tube Feeding Volumes Species of bird (ml) Budgerigar 1-3 Lovebird 2-3 Cockatiel 3-6 Conure 4-12 Parrot 15-35 Cockatoo 20-40 Large Macaw 35-60 13 CARDIOPULMONARY-CEREBRAL RESUSCITATION
CPCR is a comprehensive term used to describe both the basic principles of cardiopulmonary resuscitation (CPR) as well as advanced life support and post-resuscitation care. The word cerebral resuscitation was added to cardiopulmonary resuscitation to identify the importance of being alive with complete neurological function. The most recent International Heart Association guidelines for CPCR and emergency cardiac care in humans were published in 2000. The primary focus was use of evidence-based medicine. These guidelines were used to extrapolate the basic principles for mammals. I have taken this information and applied the same principles to the avian species. The prognosis for respiratory arrest especially when caused by isourane anaesthesia overdose is good. Cardiac arrest carries a poor prognosis, because direct compression of the heart is not possible because of the overlying sternum. Because birds do not have a diaphragm, closed-chest compressions in birds cannot utilize the thoracic pump mechanism to increase overall negative intrathoracic pressure. Always monitor birds with a Doppler blood pressure and electrocardiogram when placing under gas anaesthesia. The goal is to use these monitoring devices to identify cardiovascular instability early. Early recognition of cardiovascular instability is the key to success in avian medicine. If the bird arrests on anaesthesia, stop the anaesthesia administration. Place an endotracheal tube and start positive-pressure ventilation with 100% oxygen. An alternative method for positive pressure ventilation is placement of an air-sac breathing tube. The bird is given an injection of naloxone (0.2 ml for a large bird and 0.1 ml for a small bird) IM (intramuscularly) to stimulate the respiratory centres. Epinephrine (0.01 mg/kg) and atropine (0.04 mg/kg) can be given IV (intravenous), IO (intraosseous), or via the endotracheal route (using a tom cat catheter inserted down the endotracheal tube and doubling the dose used for IV). Intracardiac injections should be avoided, because of risk for laceration of the coronary vessels. An electrocardiogram, Doppler blood pressure, and end-tidal CO2 monitor can be used to evaluate the effectiveness of CPR resuscitation.

385

14 ACUTE RESPIRATORY DISTRESS (ARD)

Differential diagnosis a. Causes of large airway disease include: obstruction by foreign body (i.e., seed in cockatiels), mass, Aspergillus granuloma or oropharyngeal granulomas. b. Causes of parenchymal disease include: cardiogenic pulmonary oedema; smoke inhalation; pneumonia; Aspergillus, Chlamydophila, Gram-negative bacteria, poxvirus, Pachecos disease and reovirus. c. Causes of coelom space disease include: heart disease; hypoalbuminemia; liver disease; egg-related peritonitis; organomegaly. d. Small airway disease includes inhalant respiratory irritants as smoke, Teon toxicity, aerosols and candles. Treatment Resuscitative intervention is required most often prior to aggressive diagnostics to minimize stress. Oxygen supplementation by mask or cage oxygen administered during resuscitative procedures. a. Upper airway obstruction requires rapid anaesthetic induction with sevaurane/isourane anaesthesia, rapid intubation and ventilation with 100% oxygen. If there is a total upper airway obstruction, emergency air sac tube placement is performed. b. Parenchymal disease may benet from use of parental or nebulisation of bronchodilators and antibiotics. An air sac tube may partially improve respiration in birds with primary lung and air sac disease. i. Careful auscultation for a heart murmur or gallop or persistent arrhythmia suggestive of cardiogenic oedema. If heart failure is suspected, administration of furosemide (2-4 mg/kg IV) and nitroglycerine ointment on the back is administered and the bird is allowed to stabilize prior to further diagnostics. ii. Birds with a history of regurgitation or vomiting may benet from broadspectrum antibiotics (e.g., enrooxacin, cephalosporins, trimethoprim sulfadiazine) and bronchodilators when aspiration pneumonia is suspected. iii.Birds suspected of aspergillosis (leukocytosis and pulmonary nodules) should be treated with terbinane hydrochloride (Lamisil, Norvartis Pharmaceuticals, East Hanover, NJ) at 10-15 mg/kg BID for 4 weeks. iv. Birds suspected of psittacosis (leukocytosis , clinical signs varying from chronic unthriftiness to acute anorexia, diarrhoea and respiratory distress, and lime green urates) should be treated with drugs inhibiting the growth of Chlamydophila (e.g., tetracyclines, macrolides, chloramphenicol, and the uoroquinolones). There is no gold standard test that can be used to unequivocally determine if a particular test is accurate. The author recommends the use of two tests run simultaneously (e.g., serology antibody test and a PCR test) to lessen chances of falsepositive and false-positve results.

386

c. Small airway disease will require: Bronchodilators (terbutaline at 0.01mg/kg IM) can be given parenterally or terbutaline can be given by nebulisation. d. Coelomic distension will require immediate coelomocentesis when ascites is present. i. Coelomocentesis is done on the right lateral coelom just cranial to the cloaca. Use a 25-22 gauge needle and 12 ml syringe. ii. Often ultrasound can help guide thoracentesis if the uid is loculated and difcult to aspirate. iii. Save any uid recovered for culture/sensitivity and cytology. iv. Organomegaly, egg binding or a mass lesion is suspected when the coelom is rm on palpation. Radiology or ultrasonography is useful when differentiation is not obvious.

AUTHORS ADDRESS
The Animal Emergency Center, Glendale, WI United States of America Email: AECMARLA@AOL.COM

387

Westgate Pet and Bird Hospital1, Austin, Texas, US and Perrysburg Animal Care1, Perrysburg, Ohio; College of Veterinary Medicine2, The University of Tennessee, Knoxville, Tennessee, USA, and the Lafeber Company2, Cornell, Illinois, United States of America

A MASTER CLASS OF THE URINARY SYSTEM : FROM ANATOMY TO TREATMENT OPTIONS

M.S. Echols1 DVM, Dipl ABVP (Avian) and S.E. Orosz 2 PhD, DVM, Dipl ABVP (Avian), Dipl ECAMS

KEYWORDS
Urinary system Kidneys - Renal disease - Calcium levels - Protein levels - Water excretion Urinalysis Casts Electrophoresis Urography - Glomerular ltration Diuresis - Renal brosis Amyloidosis Hyperuricemia - Gout

ABSTRACT
The kidneys of birds are retroperitoneal and rmly attached to the synsacrum. The avian kidney contains 2 basic types of nephronscortical or reptilian-type nephrons and the medullary or mammalian-type nephrons that contain medullary loops of Henle. The urinary concentrating ability generally varies inversely with body mass. Concentration of urine may occur by retroperistalsis. The unique anatomic and physiologic features of avian kidneys lead to many differences in the types, diagnosis and treatment of renal disease when compared with mammals. Important consideration should be given to the birds diet and exposure to toxins and infectious agents in addition to a variety of diagnostic tests. Given anatomical constraints, avian renal surgery is often restricted and therapy is frequently limited to supportive care. However, renal biopsy and histologic evaluation is often the only means of a denitive diagnosis and can better guide the clinician to more accurately treat kidney disorders in birds.

1 GROSS ANATOMICAL CONCEPTS

The kidneys of birds are rmly attached to the synsacrum and sit within the renal fossae. Their kidneys are retroperitoneal in location, and extend from the lungs cranially to the distal end of the synsacrum caudally. Extensions of the abdominal air sacs from the pelvis can be found between the synsacrum and the kidneys. Birds without salt glands have 0.8% of their body mass as kidney tissue, while those with salt glands have 1.4%.

388

The kidneys of birds do not have lobes as in mammals but have anatomical divisions. Each kidney has a cranial, middle, and caudal division. From a clinical perspective, it is important to note that the spinal nerves from the lumbar and sacral plexuses pass through the substance of the kidney. Nerve impairment to the pelvic limb from swelling or pressure as with neoplasia can result in neuropraxis or proprioceptive decits to the limb.

2 HISTOLOGIC STRUCTURE
In general, the basic unit of the avian kidney is the lobule. Renal lobes may be recognized in some species, but they are often obscured. The lobules are oriented with their long axis perpendicular to the long axis of the kidney as it sits in the renal fossae. They tend to be pear-shaped and wedged between the interlobular veins of the renal portal system. The widest portion of the lobule sits near the surface of the kidney, while the smaller end is where the collecting tubules converge to form branches of the ureter. This stalk-like region of the narrowed end of the lobule is the medullary region or the medullary cone. This area contains collecting tubules and the nephronal loops of Henle from the medullary type nephrons. The wide, supercial portion represents the cortical region of the lobule and contains nephrons of both the cortical and medullary type nephrons.

Figure 1. Parts of a cortical (reptilian) nephron type (top) and a medullary (mammalian) nephron type (bottom).

389

As suggested, the avian kidney contains 2 basic types of nephronscortical or reptilian-type nephrons and the medullary or mammalian-type nephrons that contain medullary loops of Henle. The cortical type of nephron is found in the cortical region only and does not have loops of Henle. They form the majority of the nephrons in the kidneys of birds. This is the type of nephron found in reptiles and the nephrons secrete primarily uric acid. The medullary type has a loop of Henle, as occurs in mammals that has a potential to produce and, to a certain extent, concentrate urine. However, a recent study suggests a gradient of nephrons with the outermost representing cortical nephrons and the innermost nephrons (with the largest loops of Henle) representing the mammalian types. Both types of nephrons have a renal corpuscle at their proximal ends. The renal corpuscles lie about midway between the inter- and intralobular veins. All of the nephrons have a proximal and distal portion to their nephrons, but only the mammalian type have the loop of Henle. The cortical nephrons have their proximal segment in the outer portion of the lobule and form approximately half the length of this nephron type. The middle segment of this nephron is poorly dened but is short and convoluted. The distal segment of the reptilian nephron has compact convolutions near the intralobular veins. The medullary nephrons have, as their intermediate segment, the loop of Henle, which is anatomically similar to the short mammalian nephron type with the proximal descending loop enlarging prior to the bend. An inner ring is formed by the ascending limbs of the loops of Henle. The outer ring of collecting tubules of each lobule consists of perilobular collecting tubules, while those that are part of the medullary region are termed medullary collecting tubules. Commonly, these medullary tubules combine to form a single larger vessel, a collecting duct. When several of these collecting ducts combine, they form a secondary branch of the ureter. Birds, like mammals, have a juxtaglomerular apparatus, which consists of a macula densa, an epithelial thickening at the beginning of the distal convoluted tubule. Amyloidosis Amyloidosis is occasionally noted in association with avian renal disease. Amyloid deposits are often related to chronic inammatory disease and usually found systemically, but can affect specic tissues.

3 GENERAL MECHANISMS AND CONSEQUENCES OF RENAL INJURY

Initiation of renal disease Proposed mechanisms of the process of initiation of renal injury and perpetuation of disease are complex, but the mammalian model may shed some light on avian renal disease. For this reason, some of the inammatory cascade that occurs with renal disease is described. The products resulting from the arachidonic acid cascade have effects throughout the body.

390

The renal medulla and papilla are a rich source of an enzymatic group known as prostaglandin synthetases. The action of prostaglandin synthetase, cyclo-oxygenase upon arachidonic acid results in the formation of numerous prostaglandins (PE2, PGF2, and PGD2) and thromboxanes (thromboxane A2 [TXA] and thromboxane B2) all of which have varying actions on cells. In response to renal ischemia and vasoconstriction, prostaglandin and thromboxane production is altered (primarily increased). These alterations subsequently result in varying effects on the body and kidney including changes in renal vascular resistance, blood ow, recruitment of inammatory cells, and other physiologic effects. Non-steroidal anti-inammatory drugs act to inhibit prostaglandin synthetase and represent another method to alter these arachidonic acid by-products and their subsequent actions. TXA production is considered the main cause of renal vasoconstriction associated with acute renal failure and is believed to play a pathogenic role in many forms of kidney disease. Thromboxane A2 is produced by glomerular epithelial and mesangial cells, renal medulla tubular cells, and especially platelets. In mammals, TXA causes mesangial cell contraction and is a potent vasoconstrictor. Both of these actions can result in decreased glomerular ltration rate (GFR) resulting in renal damage. Thromboxane A2 also promotes platelet aggregation and may be partially responsible for haemostatic abnormalities noted with renal disease. Nephrosis: Nephrosis is a non-specic histopathologic change characterised as any degenerative, non-inammatory lesion of the kidney, from cloudy swelling to necrosis, whatever the cause. Nephritis: Nephritis is simply inammation of the kidney and may involve the interstitium, tubules, and/or the glomerulus (although glomerulonephritis is typically reserved for glomerular lesions). Post-mortem renal change Renal post-mortem changes are noted in chickens as soon as 22 minutes following death at 37oC (98.6oF). Early renal post-mortem changes occur in the proximal tubular epithelium, followed by collecting tubule epithelium and glomerular nuclei. Haemostatic abnormalities: Abnormalities of haemostasis can be noted. Platelet aggregation and activation occur secondary to complement activated antigenantibody interactions and renal endothelial damage. Selected toxic and nutritional diseases Vitamin D intoxication: As a result of excessive calcium uptake, visceral calcinosis, nephrocalcinosis, visceral gout, and urate nephrosis are considered frequent complications of vitamin D intoxication in birds. Symptoms of hypervitaminosis D include hypercalcemia, anorexia, nausea, polyuria, polydipsia, demineralization of bones, disorientation, painful joints, and muscle weakness. Hypervitaminosis D may occur when feeding chicks vitamin D containing supplements.

391

Hypercalcinosis: High calcium intake has also been directly correlated with renal disease in birds. Dietary supplementation with calcium, as well as limestone sand substrate, has been correlated with renal disease. In a study involving young and adult budgerigars (Melopsittacus undulatus), increasing dietary calcium levels were shown to be more renal toxic than was excess vitamin D3. Parent birds were fed diets containing 0.3%, 0.7%, and 1.5% dietary calcium with a range of 500, 1000, 1500, and 3000 IU/kg/feed of vitamin D3. The adults subsequently fed the young the same diet. When fed a diet containing 3000 IU/kg/feed of vitamin D3 there was a questionably increased mortality rate only in the birds receiving 1.5% dietary calcium. However, there was a clear correlation with mild and severe metastatic (renal) mineralization in birds fed 0.7% and 1.5% calcium, respectively. The young birds fed 0.7% and 1.5 % calcium died by 24-32 days old and never edged (32-35 days). Growth rate and hatchability were poor only in the groups fed 1.5% calcium. While only a few adults died by 5 months on diets containing 1.5% calcium, most had metastatic renal mineralization when fed 0.7% calcium. Birds fed 0.3% calcium had no evidence of metastatic mineralization and had good hatchability and growth rates. This study suggests that some species, such as budgerigars, may be very sensitive to dietary calcium levels and that supplementation should be used cautiously. Hypovitaminosis A: Hypovitaminosis A may also lead to renal disease in avian patients. In birds with hypovitaminosis A, the ureters and renal collecting ducts may undergo metaplasia. These epithelial changes can result in decreased mucin production and excessive keratin leading to plug formation and ureteral obstruction. High protein diets: High protein diets have been associated with renal disease in birds, but only under specic conditions. Extraordinarily high protein levels in the diet of genetically predisposed chickens have been shown to cause gout, but a direct relationship with renal disease has not been established. Diets high in urea have also been linked to nephritis outbreaks in poultry. Fish-meal adulterated with urea was linked to high (6-8%) mortality in 2 separate farms. Diet-induced renal disease of colour variety psittacine birds: There may be form of renal disease induced by feeding predominately pelletised diets to colour mutations in psittacine birds. Lesions have been limited to non-specic tubular nephrosis and were reversible after feeding a non-pelletised diet for 1-3 months. Mycotoxic nephropathy: Mycotoxic nephropathy, primarily due to ochratoxin A, has been reported in chickens and ducks. Ochratoxin A is produced by several species of Aspergillus and Penicillium. Ochratoxicosis induces hypertrophy of renal proximal convoluted tubules. Because of the multiple sources of toxin, multiple avian species can be exposed to ochratoxin resulting in kidney damage. Lead nephropathy: Lead toxicity is the most common cause of metal poisoning in waterfowl and affects a wide variety of birds. Renal lesions may include proximal tubular necrosis and degeneration (nephrosis), visceral gout, and in some birds, acidfast intranuclear inclusion bodies.

392

4 THE VENOUS SUPPLY TO THE KIDNEYS

Blood from the area around the loops of Henle are drained by the venulae recta into the intralobular veins. The intralobular veins drain blood into the efferent renal veins or branches. Depending on the division of the kidney, the blood then ows into renal veins of its appropriate division.

Figure 2. Ventral view of kidneys of the domestic chicken illustrating the renal portal system. The renal portal system is involved in the secretion of urates in the blood so that it can be excreted by the kidneys in birds. It has been suggested that the renal portal system provides about two-thirds of the blood supply to the kidneys that bypasses the glomeruli. This has important ramications for the removal of urates along with other components including drugs. The renal portal system forms a venous ring with both kidneys. There are numerous small venous branches that take origin from this ring to penetrate the parenchyma of the kidney. Renal portal valves control blood ow to the kidney and systematically. These valves are found within the lumen of the common iliac veins between the renal and the portal veins. Each valve is innervated by adrengeric and acetylcholine receptors. If

393

the valves are open, then blood will ow directly into the caudal vena cava and not the kidney. Valve closure is inhibited by norepinephrine and epinephrine so that with ight there is available venous return to the heart. When the valve is closed, blood ows into the parenchyma of the kidney. Acetylcholine stimulates its closure. Clinically, these connections with the renal portal system help explain the spread of neoplasia and infectious agents to other parts of the body. Blood from the renal portal system can ow through the portal valve into the vena cava, into the caudal mesenteric vein to the liver, and /or into the internal vertebral venous plexus within the vertebral canal. This shunt can result in the bypass of the kidney completely but often is only partially activated so that blood may go a variety of directions. A potential consequence of the venous supply: Gastrointestinal ulcerations are reported in some animals with uraemia and advanced renal disease but are rarely mentioned concurrently in clinical reports of birds with kidney disorders. In chickens, gizzard erosions have been associated with naturally-occurring urolithiasis. Intestinal inammation may lead to renal disease. In humans, inammatory bowel disease (IBD) can be related to renal disorders. The avian caudal mesenteric vein drains the mesentery of the hindgut into the hepatic portal and/or the renal portal vein. Therefore, colitis may serve as a source of infectious agents, toxins, and inammatory products to the avian kidney. Gout Renal disease may lead to numerous other conditions, including gout, which can further damage the kidneys or other organ systems. Gout reportedly may be caused by reduced excretion of urates or by increased dietary protein Dehydration, obstructed ureters, and general kidney damage can decrease uric acid elimination. As blood uric acid rises and exceeds its solubility (hyperuricemia), urate crystals precipitate. Gout may be only a nutritional disease in birds with hypovitaminosis A. Visceral gout results secondarily from elevated plasma uric acid levels and its resultant deposition on visceral organs. Experimentally, visceral gout has been induced in chickens fed excessive dietary calcium and a diet decient in vitamin A, administered various nephrotoxic agents, and following ureteral ligation and urolithiasis. Articular gout results from accumulation of urates in the synovial capsules and tendon sheaths of the joints. Diffuse urate deposits on visceral surfaces do not occur in articular gout. However, visceral and articular gout can be present in the same bird. Spontaneous articular gout in birds without underlying renal pathology is relatively uncommon and appears to have a hereditary basis, at least in chickens. Continuing damage Once renal damage occurs, persistent and progressive kidney damage is likely to occur, even if the initial insult is treated and cured. Although no refereed literature describes the post-treatment progression of renal lesions in living avian patients, the author has observed from repeated biopsies that some mild renal lesions persist even if the patient is clinically normal or improved.

394

Infectious diseases Bacterial: Certain patterns may be expected with bacterial nephritis. Chickens experimentally infected with E. coli [E. coli 01K67(B12)], Staphylococcus aureus and Actinomyces pyogenes, developed a fairly consistent pattern and progression of renal disease. Birds inoculated subcutaneously developed more severe renal lesions and these lesions were noted earlier than those exposed to bacteria Per Os. Gross renal changes included congestion, enlargement, and hemorrhagic foci. Lesions progressed to nephrotoxic nephritis and included tubular epithelial cell degeneration and necrosis with the formation of hyaline casts and eosinophilic material. The typical lesions suggestive of bacterial nephritis include tubular dilatation and impaction with inammatory cells. As nephritis becomes chronic, tubular necrosis, cyst formation, distortion, and interstitial brosis with mononuclear cell inltration become evident. Using sterile collection and culture methods, bacterial nephritis is denitively diagnosed by recovering bacterial organisms from affected kidneys. Light microscopic identication of bacteria within renal tissue may be difcult as has been noted in dogs and swine with renal disease. As is likely true of most viral and fungal renal diseases, bacterial nephritis is often a component of systemic infection and multiple organs may be involved. In summary, any septicaemia can potentially result in kidney infection and inammation. Viral: Viruses perhaps have the most varied effect on avian kidneys. Numerous viruses may infect and affect avian kidneys. Histologic patterns are highly variable as some viruses directly affect the kidneys, such as pheasant coronavirus-associated nephritis, while others like psittacine herpesvirus and polyomavirus damage renal tissue as a part of a more systemic process. Some viral infections, such as the West Nile virus, are best identied in the kidney and provide an additional reason to save extra renal tissue (frozen and/or formalinised) for later testing. Parasitic: Renal coccidian Primary and secondary renal parasites have been noted throughout the avian literature and some contribute to signicant morbidity and mortality. Renal coccidiosis, found predominately in some waterfowl and marine species, is the most frequently reported avian renal parasite in those species and has been clearly associated with disease in some cases. Renal Eimeria sp. oocysts are passed in faeces via the ureter and sporulate rapidly in the environment. Crowding likely favours transmission. The prepatent period appears to range between species and has included 5 to 21 days. Although transmission between different avian species is not clear, one study suggested that renal coccidia of geese do not infect ducks. Most clinically affected species are young birds, many of which are wild with concomitant intestinal parasites. Affected birds are typically emaciated and may have diarrhoea with or without blood. Grossly, the kidneys are often enlarged with white to yellowish nodules containing urates and/or oocysts. The tubules are often distended with endogenous developmental stages (micro- and macrogamonts, macrogametes) and maturing Emeria sp. oocysts.

395

Sarcocystis Canaries (Serinus canaria) experimentally infected with Sarcocystis falcatula developed mild multifocal interstitial renal inltrates and glomerular hypertrophy with mesangial hyperplasia that modestly progressed with duration of infection. Sarcocystis organisms have also been noted histologically in the renal parenchyma of cockatiels, but again the signicance is unclear. Microsporidia Microsporidia (Encephalitozoon sp.) have been reported in numerous avian species with variable effects on the kidney including a PBFD positive Eclectus parrot, 3 species of lovebirds, budgerigars, and a double-yellow headed Amazon parrot. Cryptosporidia Although renal cryptosporidiosis is infrequently reported, it has been directly associated with kidney lesions in a 4-month-old black-throated nch (Peophila cincta), an 8-week-old Sonnarats jungle fowl (Gallus sonneratii), 4-month old pullets, and adult laying hens. Four-day-old chickens co-infected with Mareks disease virus have also been studied. Clinical signs ranged from acute death (nch and jungle fowl) to thinning, depression, leg weakness and respiratory distress (4-month-old pullets and 4-day-old chicks) to slightly increased morbidity and mortality (adult chickens). Pulmonary cryptosporidiosis was also a common feature of the pullets. Cryptosporidium sp. is associated with visceral gout most likely from partial ureteral obstruction. Several authors have hypothesized that urinary tract Cryptosporidium infection originates in the cloaca and retrogrades into the kidneys via the ureters. Although relatively uncommon, urinary tract cryptosporidiosis and associated disease seem to primarily be a concern in chickens, especially those with concurrent immunosuppressive illness. Fungal: Fungal nephritis is uncommonly reported in birds. Given the close association between the air sacs and kidneys, direct extension from the respiratory system (rather than primary renal invasion) is likely the cause of the necrotic fungal lesions in the kidneys. Neoplasia The avian kidney, just as with other animal tissue, is susceptible to neoplastic conditions. Nephroblastomas are the most commonly reported avian renal tumours. Nephroblastomas and renal adenocarcinomas comprise the majority of kidney tumours in budgerigars. Renal carcinomas are the most frequently reported tumours of the urinary system in non-domestic free-ranging and captive birds. There are likely many causes of renal tumours in birds but there is little information regarding denitive aetiologies. Avian leucosis virus (ALV) can induce renal tumours in chickens. While ALV has been found in budgerigars with renal tumours, a denitive

396

association has not been made. A common presentation with renal cancer is unilateral to bilateral leg weakness or paralysis and slight ataxia. Other clinical signs may vary but often include diarrhoea, dyspnoea, abdominal distension, and weight loss. The lumbar plexus lies dorsal to the cranial renal division while the sacral plexus runs through the middle division parenchyma. Because of this close association, any parenchymal inammation or pressure on, or from within, the kidney can potentially result in nerve dysfunction and resultant lameness. Additional neoplastic extension to the overlying spinal column may also result in nerve dysfunction. Peripheral neural compression should result in peripheral neuropathy with eventual loss of the withdrawal reex, not seen with most spinal cord lesions. In addition to lameness and muscle atrophy, ipsilateral osteopenia was noted in a cockatiel (Nymphicus hollandicus) with a renal adenocarcinoma.

5 REGULATION OF WATER EXCRETION

The kidney lters a large volume of uid daily with up to 11 times the total body water for a 100-g bird. Most (approximately 95%) of this volume is reabsorbed by tubular reabsorption. This likely result from a change in the rate of ltration and/or the rate of reabsorption. These changes can result from the increased levels of the circulating antidiuretic hormone, arginine vasotocin (AVT). AVT is an 8-amino-acid peptide hormone that is released by the neurohypophysis. The most common reason for AVT release is a rise in extracellular uid osmolality. In addition, a decrease in the extracellular uid volume using a haemorrhage model results in the release of AVT. Dehydration also results in an increase in circulating levels of AVT. Associated with the rise in AVT is a reduction of the glomerular ltration rate (GFR), which leads to reduced urine ow because of reduced ltration and also, indirectly, enhanced tubular reabsorption. The reduction of GFR may result from constriction of afferent arterioles that causes a reduction or even a cessation of ltration in some or all nephrons. Reptilian or cortical nephrons are the most sensitive to AVT. This is important as these nephrons lack a loop of Henle and therefore the ability to concentrate urine. The other effect of water overload and/or an increased extracellular uid expansion results in an increased GFR to compensate. However, the mechanism is not known. Birds also have the ability to concentrate their urine by tubular reabsorption, with a range of less than 70% to more than 99% of the ltered volume. For this reason, the urine can range from dilute, at approximately 40mmol/kg, to hyperosmotic at 23 times the osmolality of plasma. Studies using isolated nephrons or those using micropuncture techniques suggest that the proximal tubules are capable of reabsorbing 70% of the uid volume, as occurs in mammals. This reabsorption is dependant on active sodium reabsorption, but not bicarbonate. As in mammals, reabsorption depends on a counter current multiplier system of the loop of Henle that is produced by an osmolality gradient in the medullary region.

397

The thin descending limb (TDL) is permeable to Na+ and Cl- but has low water permeability. However, the thick ascending limb (TAL) allows for the transport of Na+ and Cl- against a concentration gradient while having a low permeability to water and net water transport. This results in reabsorption of ions in the ltrate as it moves through this TAL portion of the nephron. This produces the highest osmolality at the tip of the loop and the development of a gradient outward from there. Water permeability of the collecting ducts, as they run through this osmolality gradient, produces the concentration of urine over plasma. Exactly how AVT alters this system remains unknown, but it may involve tubular reabsorption and/or reduction of ltration by cortical nephrons. This latter action would result in an increased osmolality gradient in the medullary region. The net effect is that birds are able to concentrate their urine often at 23 times that of plasma. The urinary concentrating ability generally varies inversely with body mass so that small birds (1025 g) typically concentrate to 1000 mmol/kg, while birds greater than 500 g concentrate to 600700 mmol/kg. Further concentration of the urine may occur by retroperistalsis up into the coprodeum and the large intestine, which are able to reabsorb water.

6 DIAGNOSTIC TESTS
Urinalysis Biochemical and cytological sediment analysis of avian urine has been advocated as potentially useful in diagnosing avian renal disease. In birds, hematuria may be noted with renal disease but should be carefully differentiated from bleeding originating from the gastrointestinal and reproductive tracts. Hemoglobinuria, as noted in Amazona spp. parrots with lead intoxication and in other species with differing disorders, may or may not be related to renal disease. Toxic, neoplastic, bacterial, and viral nephropathies may be more frequently seen associated with hematuria in birds. White blood cells were seen in 45% of urine sediment from pigeons with paratyphus, many of which had interstial nephritis. Sediment analysis may be complicated by anatomical factors. Urine may be mixed with faeces when voiding. Ureteral urine may be reuxed orad into the colon up to the ceca where water, and sometimes electrolyte, reabsorption takes place. Additionally, diseases of the lower intestine (eg, gastrointestinal bleeding, inammation) may alter urine production and composition. In short, the urine present in a dropping is not the same urine produced from the kidneys. Collection: True urine can be collected in birds with some difculty. Once emptied of faeces, specially designed cannulae can be inserted into the cloaca for collection of ureteral urine. A foley catheter was used in chickens.. Casts: Urinary casts represent cellular and/or acellular material sloughed from the inner lining of various renal tubules. Protein and cellular casts, albumenous casts in renal tubules, hyaline casts in kidney sections or renal tubules, eosinophilic granular

398

casts in renal tubules, eosinophilic tubular casts, eosinophilic casts in renal collecting tubules, and epithelial casts in urine samples have been identied in various species. Urinary casts may be a sign of renal disease but there is little information correlating them with any type of renal disease in birds. Urine chemistries and electrolytes: Standard mammalian dipsticks may be used but not all components are applicable to avian urine. Chicken urine reportedly contains non-uric acid chromagen. Non-protein chromogens are known to interfere with refractometric and chemical measurement of plasma proteins and may also apply to avian urine sampling. Few studies mention test strips for use in avian urinalyses. One study evaluated commercial urine dipsticks (Combur-9 stix [Boehringer Mannheim]) on normal urine of 35 ostriches (Struthio camelus). Because ostriches can eliminate urinary waste separate from faeces, these values may not apply to most other birds. In the study, 31/35 (89%) and 35/35 (100%) of the urine samples were positive for nitrite and protein, respectively. The urine chemistry strips were negative for glucose, urobilinogen, bilirubin, and ketones in all ostriches. No association with renal disease was made. Because of inconsistent results for other analytes in other species, limited critical studies, difculty in obtaining ureteral urine, and clinical experience, the author (M. S. E.) believes that chemistry strips currently available have limited value for avian urinalyses. Urine electrolytes and chemistries can be collected, but there is limited information on their interpretation. LUMEIJ suggested that because renal intracellular enzymes are likely voided in the urine, urinary chemistries might be useful in detecting kidney damage. Urine sodium and potassium were measured, and insignicantly changed, in house sparrows undergoing trials with the antidiuretic arginine vasotocin. One study noted that in normal and dehydrated starlings (Sturnus vulgaris), cloacal urine contained signicantly higher concentrations of magnesium, phosphate, potassium, and total osmolality than found in ureteral samples. Osmolality and specic gravity: Avian urine is typically isosmotic with its osmolality maximally increased to 2.0-2.5 times that of plasma. This is minimal compared to mammals that can concentrate urine osmolaltiy 25 to 30 times that of plasma. There is limited information on urine specic gravity or osmolality in avian health or disease. GAVAERT et al. noted consistent polyuria and hyposthenuria (60% had specic gravity below 1.007) in Salmonella typhimurium-infected pigeons, many of which had interstitial nephritis. In a separate evaluation, urine osmolality signicantly increased up to 3 times control levels in post-ight and dehydrated pigeons. Urine pH: Urine pH is highly variable in birds. The urine pH may be acid (down to 4.7) in egg laying female birds during calcium deposition. Once the egg is laid or calcium is no longer being deposited, urinary pH may climb to 8.0. Male birds have an approximate urine pH of 6.4. Hypoxia, as noted in diving ducks, may drop urine pH to 4.7. Normal ostriches have a urine pH range of 6.1 to 9.1, with a mean of 7.6.

399

Electrophoresis Plasma protein electrophoresis: Properly determined hypoalbuminemia (via plasma electrophoresis) is not reported in conrmed active cases of avian renal disease. However, it is possible that birds may develop low albumin/protein with some kidney disorders. Biochemically determined hypoalbuminemia has been noted in some active avian renal disease cases. Urinary protein electrophresis: Avian urine normally contains a large amount of protein (average of 5 mg/ml up to 15 mg/ml), especially when compared to that of mammals (< 0.09 mg/ml in dogs and humans). Proteinuria is likely necessary to maintain the excreted uric acid-containing spheres in a colloidal suspension, preventing aggregation and renal tubular blockage. The reux of urine into the cloaca may be a mechanism to recover some of the urinary protein as cloacally voided uid contains very little protein compared to ureteral samples. Serum albumin, among other proteins, is found in both the liquid urine and uric acid spheres in chickens. Imaging Radiography: Plain and contrast radiography, nuclear scintigraphy, ultrasound, magnetic resonance imaging, and computed tomography can be used to image the avian kidneys. Because the avian kidney is surrounded by bone dorsally and air sacs ventrally, imaging of the avian renal system may be difcult. Indirect methods such as positive contrast radiography of the alimentary tract may be helpful in outlining renal masses. A lateral view is the best method to view the kidneys radiographically. As viewed with a lateral radiograph, the absence of the normal dorsal diverticulum of the abdominal air sac (dorsal to the kidney and ventral to the synsacrum) may indicate renal enlargement. Improper positioning can artifactually change the appearance of this air-lled diverticulum. Because the renal silhouettes are superimposed on a lateral view of the abdomen, an oblique view may also be used to distinguish each kidney. Renal density and gross size changes may indicate renal disease. Radiographically visible renomegally was noted in a salmon-crested cockatoo (Cacatua moluccensis) with chronic interstitial nephritis and calcication as the result of hypervitaminosis D3. Nephrocalcinosis was detected radiographically in ostriches and appeared as multiple radio-opacities throughout the renal parenchyma. Ultrasound: Ultrasonographic imaging of normal avian kidneys is difcult because of the surrounding air sacs (ventrally) and bone (dorsally and laterally). In one study of 386 mixed bird species that underwent ultrasonographic evaluation of the urogenital tract, abnormalities, such as renal cysts (6), cancer (12), and inammatory nephromegaly (11), were identied in only 29 patients. Sonographic imaging of the normal kidney was deemed not possible. Some disease conditions that either obliterate the air sacs or result in uid accumulation in the coelomic cavity may actually improve renal ultrasonographic imaging. In these abnormal situations, ultrasonography can serve as a non-invasive and safe means to evaluate coelomic structures such as the kidneys.

400

Intravenous excretory urography: Intravenous excretory urography has been described in birds as a method to gain information on kidney size, shape, and function. LUMEIJ reports using organic iodine compounds (Urographine 76) given IV at 2 mg/kg in the basilic vein. The organic iodine can be visualized radiographically in the heart and pulmonary artery within 10 seconds and outlining the kidneys and ureters 20 to 50 seconds later. After 2 to 5 minutes, the cloaca will be outlined. This technique should not be used in birds with severe renal compromise. Intravenous excretory urography may have some limited uses in a clinical setting. DENNIS and BENNETT successfully used a water-soluble iodinated contrast agent (400 mg/kg IV in right medial metatarsal vein; Renogran-76, Squib Diagnostics, Princeton, NJ, USA) to evaluate the ureters post-ureterotomy in a double-yellowheaded Amazon parrot (Amazona ochrocephala). Radiographic images taken at 1, 2, 7, and 10 minutes post-injection were used to evaluate ureter peristaltic movement and size.

7 WATER DEPRIVATION TESTING

Water deprivation testing is considered when attempting to rule out unknown causes of polyuria/polydipsia (PU/PD) including central and nephrogenic diabetes insipidus, and psychogenic polydipsia. Some of the many causes of PU/PD in birds to consider include organic (liver, kidney, intestine, cardiac, etc), endocrine (diabetes mellitus), and metabolic (hypercalcemia) diseases. The avian patient is caged with no food or water for the duration of the test. Evaluate both blood and urine parameters every 3 to 24 hours for 12 to 48 hours, depending on the species and physical condition of the bird. As a normal response, some birds such as European starlings may become distressed within 24 hours. Smaller birds should be evaluated at more frequent intervals. A presumptive diagnosis is based on whether birds can concentrate their urine. Birds with diabetes insipidus become dehydrated (as supported by plasma variables) but maintain dilute urine (low specic gravity and osmolality). Hereditary diabetes insipidus has been described in a strain of chickens that produce low osmolaltiy urine and maintain high circulating level of AVT. Identifying uric acid crystals Gout results when uric acid precipitates out as a solid, chalky substance in joints (articular) or on tissue surfaces (visceral). Articular gout material may be recovered using ne needle aspiration. Uric acid crystals are easily conrmed using microscopy or the murexide test. Cytologically, gouty material typically presents as uric acid crystals surrounded by a pyogranulomatous inltrate, usually without organisms. The needle-shaped crystals are easy to identify on direct and stained smears. Due to their water-soluble nature, urates will dissolve in formalin and therefore the crystalline form will not be seen on conventionally xed tissue. However, urates can be seen in alcohol xed tissue using Gomoris methenamine silver impregnation technique.

401

Biopsy When history, physical examination, and/or laboratory abnormalities support the presence of renal disease, consider biopsy. Currently, the only way to denitively diagnose avian renal disease and specic pathologic patterns is with a kidney biopsy and histopathologic evaluation. A renal biopsy is most frequently performed during endoscopic examination of the coelomic cavity and specically, kidneys. Before a renal biopsy is performed, the cost: benet of the surgical procedure versus conservative therapy must be considered as many birds have compromised health, especially if they have kidney disease. Several methods of renal biopsy, primarily via endoscopy, and detailed accounts of avian kidney anatomy and physiology have been previously discussed. For the most part, renal tissues can be stored in 10% formalin for light microscopy. If available, additional tissue may be stored in glutaraldehyde (electron microscopy), culture media (organism recovery), and alcohol (visualizing uric acid crystals) or frozen (PCR studies). Renal histologic lesions are rarely pathognomonic for a specic disease process. Many different diseases cause similar renal lesions and different pathologists may make differing morphologic diagnoses on the same renal tissue. It is often useful to combine the pathologists interpretation with the veterinarians rst-hand knowledge.

8 AVIAN RENAL DISEASE TREATMENT

Therapeutic considerations Treatment options for renal disorders in birds depend upon the cause and type of kidney disease and secondary complications present. Most renal disease patients are medically managed, as kidney surgery is difcult and often not needed. Because of the location within the renal fossae, avian kidneys are difcult to remove surgically. The close associations with the lumbar and sacral plexuses and extensive vascular network surrounding the kidneys lead to the high probability of signicant haemorrhage expected during surgery and possible neurologic damage. However, therapeutic surgery (including endoscopic biopsy) for supercial renal lesions and the ureters may sometimes be useful. Given the concern of serious haemorrhage, most surgical renal disease cases are managed medically. A few accounts of therapeutic renal surgery exist. Cloacaliths and other masses within the cloaca may be easily removed, relieving a potential ureteral obstruction. Renal stones were successfully removed via extracorporeal shock wave lithotripsy in a Magellanic penguin (Spheniscus megallanicus) and ureteral stones successfully removed from a 21-year-old male double-yellow-headed Amazon parrot. The author (M. S. E.) has also used minor surgery in articular gout cases. Small incisions are made over the gouty lesions and the thick material expressed. Anaesthesia is recommended as this can be quite painful. Bandaging is often required to stem bleeding and prevent secondary infection.

402

Diuresis and uid therapy As in other animals with renal disease, maintaining hydration is important in birds with most kidney disorders. Acid/base and electrolyte disorders may likely be present in birds with renal disease. Anuric and oliguric patients should be diuresed. Although manitol and lasix have been recommended to induce diuresis in birds, these drugs are poorly studied in avian species. Clinically, providing parenteral uids often induces diuresis in birds, even with most forms of renal disease. Until acid/base and electrolyte disorders are better evaluated in birds with renal disease, balanced electrolyte solutions, such as Lactated Ringers Solution (LRS), should be used to maintain hydration, replace uid losses, and/or induce diuresis as needed. The estimated daily uid requirement for most birds is 40-60 ml/kg/day. LUMEIJ recommends that 10% of the birds body weight should be given in uids when the patient is in renal failure. Warmed uids are given either with food (tube/ syringe fed), SC, IV, or intraosseously (IO). The IV and IO routes are most appropriate for critically ill patients. Subcutaneous uids are not adequate to rehydrate patients with severe dehydration, shock, or hypothermia. Oral uids are reserved for stable patients with mild dehydration that have normal gastrointestinal function and are often contraindicated in critically ill birds. The author (M. S. E.) typically diureses ill and severely hyperuricemic renal disease patients. Severe hyperuricemia is identied when one or more of the following conditions are met in clinically ill non-carnivorous and appropriately fasted carnivorous birds: uric acid levels exceed 30 mg/dl uric acid levels are elevated (> 10 mg/dl for most species) and rising over a period of several days (even if below 30 mg/dl) there is evidence of rapidly progressive articular or visceral gout Depending on the patients condition, the author will typically give 50-100 ml/kg of uid q12h via the SC, IV, IO, or combination routes. Fluid therapy (combined with other medications if needed) is generally continued until the blood uric acid level drops to either normal or mildly elevated levels (10-20 mg/dl) and the bird shows signs of improvement (eating, more active, etc). Antibiotics Antibiotics are indicated in patients with known or suspected bacterial nephritis. Bacterial renal infections in birds may result from an ascending ureteritis, extension from local tissues (peritonitis, oophoritis, salpingitis, etc), and haematogenously. Because of the renal portal system and possible shunting of blood from the intestines directly to the kidneys, alimentary tract organisms may contribute to kidney disease and should be considered when using antimicrobial therapy. Drug choices are based on an isolated renal organism (ie, identied during kidney biopsy sampling) or a suspected infectious agent (blood, ovarian, salpinx, or cloacal/faecal cultures, and/or supportive histopathology). Clinical consideration regarding potential antimicrobialinduced toxicities is important.

403

The distribution, elimination, and toxicities of many antimicrobials are poorly dened in most bird species. Although mammalian literature warns of potential nephrotoxicity with amphotericin B, cephalosporin, ouroquinolone, trimethoprim/sulfonamide, and tetracycline use, only aminoglycosides have been consistently and denitively associated with renal disease in birds. Those drugs with known potential nephrotoxicity should be used cautiously in birds with renal impairment. Antimicrobials that reach high concentrations in the renal tissue and urine without inducing toxicity should be chosen preferentially and used cautiously in kidney disease patients. Managing hyperuricemia, renal brosis and amyloidosis Allopurinol: Allopurinols main action is to decrease uric acid production. Specically, allopurinol inhibits xanthine oxidase, which is required to convert hypoxanthine to xanthine and subsequently to uric acid. Allopurinol has been specically shown to prevent renal synthesis of urates and allow the excretion of unchanged xanthine. Clinical and experimental data show decreased plasma/serum and/or urinary uric acid levels in birds treated with allopurinol. Allopurinol has been shown to be toxic to red-tailed hawks (Buteo jamaicensis) at 50 mg/kg PO q24h or at higher doses. Toxicity was attributed to oxypurinol, the active metabolite of allopurinol. Allopurinol administered at 25 mg/kg PO q24h was shown to be safe, but had no signicant effect on plasma uric acid concentrations. With the exception of red-tailed hawks, allopurinol use is reported to be non-toxic in birds (in studied chickens). Although the long-term effects are not clear, allopurinol given to chickens increases oxidative activity by lowering plasma uric acid, an important avian antioxidant. The author (M. S. E.) uses allopurinol as a rst-line drug to lower uric acid when uid therapy and diet modication alone are not sufcient or when hyperuricemia is severe. Colchicine: Theoretically, colchicine can reduce serum uric acid levels in birds and be used to control hyperuricemia. In chicken livers, colchicine reversibly inhibits xanthine dehydrogenase (compared to a pseudo-reversal with allopurinol). Colchicine prevents the progression of renal disease in humans with familial Mediterranean fever, a disease of recurring fever often complicated by amyloidosis. In humans, colchicine is best known for its anti-gout activity. In small animals, colchicine blocks the synthesis and secretion of serum amyloid A and decreases the formation and increases the breakdown of collagen. For these reasons, colchicine has been used to treat amyloidosis and hepatic brosis, respectively. Clinical use of colchicine suggests possible benet in reducing hyperuricemia in birds with renal disease. The author (M. S. E.) has also used colchicine successfully to reduce renal (and hepatic) brosis. The author uses colchicine as a second-line drug to reduce hyperuricemia and a primary medication for histologically conrmed tissue brosis. Allopurinol and colchicine are generally well tolerated when given together. If diagnosed pre-mortem, colchicine may be used in birds with amyloidosis.

404

Urate oxidase: Urate oxidase has also recently been discussed as an alternative method to manage hyperuricemia in birds. At least in humans, urate oxidase is reported to degrade the excess of uric acid to allantoin, which the kidneys can more easily clear than uric acid. A study with pigeons and red-tailed hawks concluded that urate oxidase is much more effective compared with allopurinol but further evaluation is needed. Dietary modication As a general note, birds should be fed diets appropriate for their species. Renal disease patients should be monitored regularly for weight loss. Protein: The question of dietary protein restriction in the face of renal disease remains controversial. Current human and veterinary literature both supports and refutes protein restriction in patients with renal disease. Careful supervision and adequate calories are urged in the human literature. Management of hypoproteinemia may also be important in birds with renal disease, but the identication of hypoproteinemia and its association with renal disease is unclear. Avian kidney disease patients should be fed a well-balanced diet appropriate to the species. Birds fed protein-restricted diets should be carefully monitored. A safe recommendation is that birds with hyperuricemia and/or gout should not consume diets with protein levels greater than is normal for the species. Nutritional supplementation Treatment: omega-3 fatty acids: Omega-3 fatty acids (N-3 FA) have gained popularity for their anti-inammatory, lipid-stabilizing and anti-neoplastic effects, renal protective properties, and other potential qualities. N-3 FA are those rich in eicosapentaenoic (EPA), docosohexaenoic (DHA), and/or linolenic acid. Flax seed and menhaden (cold water plankton-feeding sh) oils contain predominately linolenic acid and EPA and DHA, respectively and therefore have different N-3 FA compositions. Studies evaluating N-3 FA in mammals serve as the basis for potential treatment value in birds with selected renal disease. At this time, only anecdotal information exists regarding use of N-3 FA in birds with renal disease. In mammals, N-3 FA can signicantly reduce thromboxane A2 (TXA) synthesis in platelets and glomerular cells and increase production of vasodilatory prostaglandins. N-3 FA partially substitutes EPA and DHA acid for arachidonic acid in membrane phospholipid. This pathway decreases the release of arachidonic acid and, subsequently, the cyclooxygenase-mediated synthesis of TXA. In contrast, most animals readily convert omega-6 fatty acids (N-6 FA) to arachidonic acid and subsequently eicosanoids (prostaglandins, TXA). As with arachidonic acid, EPA also serves as a substrate for the formation of vasodilatory prostaglandin/cyclins (PGI/ PGE) and their respective products (PGI2 /PGE2 and PGI3 /PGE3), all of which have similar biologic potency. These vasodilatory prostaglandin/cyclins increase renal blood ow and single nephron GFR.

405

Specic toxicities associated with N-3 FA supplementation are poorly described but some potential adverse effects may occur. Supplementing the diet with N-3 FA increases the requirements for dietary vitamin E. Supplementation with 160 mg/kg of vitamin E (dl--tocopheryl acetate) was shown to prevent loss of -tocopherol in tissues and normalize or increase resistance to lipid peroxidation in chickens fed a commercial diet supplemented with 3% tuna oil (N-3 FA). Although specic doses have not been established, some believe that the appropriate N-6 to N-3 FA ratio is more important to inhibiting detrimental eicosanoid synthesis from arachidonic acid than is the absolute amount of N-3 FA. A dietary N-6 FA: N-3 FA ranging from 5:1 to 15:1 has been proposed as desirable for dogs and cats with renal disease. The author has successfully used supplements containing N-6 FA: N-3 FA of 4-5:1 to 1:3 (0.22 cc/kg body weight, PO, SID) combined with low-dose aspirin (0.51.0 mg/kg PO q 12 h) together to manage histologically conrmed glomerulopathies in avian patients. Vitamin A: Hypovitaminosis A is a reported cause of renal failure and results from metaplasia of the ureters leading to hyperkeratinization, decreased mucin production, and impaction. In birds with suspected hypovitaminosis A and renal disease, appropriate diet modication and short-term parenteral vitamin A is a logical therapeutic component. In such situations, the author gives a single IM vitamin A injection at the beginning of the therapy and recommends correcting the patients diet to improve long-term nutritional status. Non-steroidal anti-inammatories Non-steroidal anti-inammatory drugs (NSAIDS) are frequently discussed for use in human and animal renal disease patients. In general, NSAIDS such as aspirin and ibuprofen are non-specic cyclo-oxygenase inhibitors. Low doses of aspirin may actually inhibit platelet cyclo-oxygenase production but allow benecial (vasodilatory) prostacyclin formation and may be safe. Consequently, low dose aspirin therapy has been suggested to reduce platelet aggregation and subsequent thromboembolism and to minimize glomerular inammation for mammalian patients with some glomerulopathies. More specic NSAIDS, such as thromboxane synthetase inhibitors, have been shown to attenuate renal dysfunction/damage. Unfortunately, the benecial effects of low dose, or specic, NSAID therapy have not been studied in birds with renal disease. Although there are limited avian studies, most NSAIDS are eliminated by renal clearance and should be used with caution, as they have been associated with a variety of renal lesions in birds and mammals. Flunixin meglumine (Banamine) induced glomerular lesions in bobwhite quail (Colinus virginianus) in proportion with the dose. Aspirin has been associated with signicant inhibition of prostaglandin synthesis in Japanese quail (Coturnix japonica) and with temporary diuresis in Pekin ducks (Aix galericulata) when administered IV. Diclofenac-treated livestock caused several Gyps spp. vultures to die of renal failure and gout, while therapeutic administration of diclofenac has been implicated in the decline of the Oriental whitebacked vulture (Gyps bengalensis).

406

Even with the noted toxicities and lack of therapeutic studies in birds, low dose aspirin, and possibly other NSAIDS, can be benecial in treating avian kidney disease patients. In the authors experience (M. S. E.), low dose-aspirin (0.5-1.0 mg/ kg PO q12h) combined with N-3 FA supplementation is safe and may be effective at reducing the severity of some forms of avian renal disease, especially glomerular disorders. Aspirin (and N-3 FA) therapy can be used chronically. Discontinue use when evidence of renal disease is gone or the disorder is satisfactorily managed. Treatment summary Treatments of avian renal disease should be individualized according to the patients needs, accurate renal histologic diagnosis (if available), concurrent disorders, and client considerations. Treatment of bacterial nephritis with appropriate antibiotics should be based, in part, on culture and sensitivity results when available. Otherwise, suspected bacterialinduced nephritis should be treated with broad-spectrum, bactericidal antibiotics that reach high kidney concentrations and are non-nephrotoxic. Provide antibiotics for a minimum of 6 weeks. Antibacterials should also be considered when concurrent colitis is present. Nephrosis may best be managed by removing known nephrotoxins and addressing secondary complications. Such secondary complication of any renal disease may include dehydration, hyperuricemia, brosis, infectious diseases, and anorexia. Parenteral vit A may also be provided. Consider N3-FA supplementation. Dietary-induced renal diseases can be managed with diet change or supplementation depending on the aetiology. For colour variety psittacine birds, discontinue pellets and change diet over to whole grains, seeds, fruits, and vegetables as is appropriate for the species. If after 3-6 months all signs of renal disease are gone, pellets (< 50% of total diet) can be cautiously added to diet. Anti-neoplastic treatment of certain avian renal tumourss may be indicated and should be considered. Specically identifying and managing underlying diseases that may be concurrently present may best control glomerulopathies. Conrmed glomerular disorders in birds without an obvious underlying disease may be managed in some cases with low-dose aspirin and N-3 FA supplementation. If identiable, remove/control any source of infection/inammation. N3-FA can be given chronically if needed. Nutritional management such as weight loss, providing a balanced diet, and vitamin A supplementation may also be indicated. For renal brosis, use colchicine until histologic brosis resolves. Otherwise use colchicine for 6-12 months, or until laboratory abnormalities normalize. N3-FA may also be benecial. Articular gout can be treated with colchicine and allopurinol together until all signs of gout and hyperuricemia have resolved. Consider diagnosing the cause of probable underlying renal disease and manage appropriately. Give vitamin A if hypovitaminosis

407

A is suspected. Articular gout lesions may also be surgically opened and expressed to speed removal of uric acid crystal accumulation. Again, N3-FA may be benecial. Use aggressive uid therapy if articular or visceral gout is accumulating rapidly. Secondary infections, dehydration, unacceptable weight loss, etc should be managed as needed. Combination therapy should be considered when two or more histologic renal lesions are present. Prognosis There are limited studies that estimate the outcome of selected avian renal disorders. Based on the authors experience, several forms of renal disease can be successfully managed, and some resolved, giving a good prognosis for long-term health to the individual patient.

AUTHORS ADDRESS
MS Echols DVM, Dipl ABVP (Avian) Westgate Pet and Bird Hospital, Austin, Texas, USA Email: Spotdvm@aol.com

408

Department of Pathobiology, Pet Avian, Exotic Animal and Wildlife Section Utrecht University, The Netherlands

PASSERINE AND SOFTBILL MEDICINE AND SURGERY

G. M. Dorrestein, DVM, PhD

KEYWORDS
Passerines - Husbandry - Diseases - Treatment

ABSTRACT
In a short overview basic information is presented of housing, feeding, management and diseases of the small passerines (canaries and tropical nches). Special attention is paid to the diagnostic possibilities and infectious and parasitic diseases. In tables basic haematology, blood chemistry, and therapeutics will be summarized. In 2 diagnostic tables the most common problems in small passerines will be covered including suggestions for additional diagnostic tests.

1 INTRODUCTION
Many of the veterinarians will be relatively unfamiliar with the passerines. The aviculture, diagnostic procedures and common diseases and their treatment will be discussed based on own experience and recent publications. Passerines (songbirds) are regularly presented for veterinary care as aviculturists recognize that a visit to an avian veterinarian can result in a successful medical treatment, even in these oftentiny patients. The order Passeriformes contains over 5700 species, with body weights ranging from 4.8 to 1350 grams. Toucans and mynahs are often grouped together, but are from different families. Diseases in these birds are very much inuenced by nutrition, housing and stress. For an optimal approach of the veterinary problems, including diagnosis and treatment, it is a must to become familiar with aviculture, housing and husbandry of these species. Supportive care and measures to minimize stress are often needed to maintain the hosts defence mechanisms.

409

2 BIOLOGY AND HUSBANDRY

2.1 Species The most common representatives of the passerines in captivity are canaries and nches. The canary (Serinus canaria) are bred and kept for their song (e.g. the Harzer and the Spanish timbrado), their colours, or their build and shape (e.g. the border fancy and the Norwich). Their weight is 15 - 25 g. The sexes are alike and their lifespan is 6-16 years. There are several hundred species of nches and they have a worldwide distribution. The more domesticated species are bred in captivity since many decades. There is a fair amount of size disparity between the common nch pets (smallest: Gold-breasted waxbill 7 g, largest: Java rice sparrow 20 g.). Most common nches belonging to the family Fringillidae and Estrildidae, eg., zebra nch (Poephila guttata), Lady Gouldian (Chloebia gouldiae) and parrot nch (Erythrura psittacea) are kept for breeding but also as ornamental birds. Bengalese (or society nch (Lonchura striata domestica) and zebra nches are used as fosters for breeding Australian nches. This gives special problems because they can be carriers of diseases that can kill the foster edglings. i.e. Cochlosomosis and Campylobacter infections. 2.1 Housing The small Passeriformes are kept in captivity as individual pet birds and as ocks in aviaries. Two types of aviaries can be distinguished: mixed ornamental aviaries and breeding aviaries. The former are usually located outside and different species are kept in it together, mostly for ornamental purposes. In the latter, large numbers of the same species are kept, mostly indoors, for breeding and selecting. These fanciers often go to shows and competitions and there is exchange of birds (and possibly pathogens). In mixed aviaries, the population, consisting of individuals or couples of different species, is less dense and species specic diseases are restricted to only a few of the occupants of the aviary. The birds are in the aviary all year with a shed for shelter and a y pen outside. Planted aviaries are popular for these passerines because the vegetation provides observers a more natural view of a birds behaviour. Disease control in planted aviaries can give difculties related to controlling microorganisms and in medicating individual birds. These plants, however, are often necessary to get breeding results. In breeding aviaries located in a garden house or in a loft, the housing depends on the season. Nowadays canaries are mostly bred indoors. Normally canaries will start breeding when the following conditions are met: maturity and good health, an accepted partner, a minimum length of the day, presence of nest and nesting material, enough water and food, a minimum temperature and photoperiodic stimulation. In the breeding-season the birds are mostly kept in couples in small box type cages (50x40x40 cm). Normally the fancier breeds two to three rounds. The weanlings are

410

housed in communal ights with or without outside quarters. In the winter season (resting season) the males and females are housed as separate groups in pens. Singing canaries are housed individually in small sing-cages (21x20x15 cm) for more then 5 months to be trained and to enter the singing competitions. The breeding aviaries are relatively easy to clean. 2.2 Diet and Husbandry Dietary and husbandry requirements are diverse. Most common passerine species are primarily seed eating or granivorous and have been domesticated for centuries, while others species are nectivorous, frugivorous, insectivorous, omnivorous or, carnivorous. The majority of commercially available passerine diets are seed mixes and therefore multi-decient. The composition of the basic diet will be affected by the species of bird in question. Some species adapt readily to commercially available diets, while others may require live food. Common nutrient deciencies of seeds include lysine, calcium, available phosphorus, sodium, manganese, zinc, iron, iodine, selenium, vitamins A, D3, E and K, riboavin, pantothenic acid, available niacin, vitamin B12 and choline. The inadequacies of these seeds for growth apply generally for reproduction and, to a lesser extent, for maintenance of adults.

3 DIAGNOSTIC PROCEDURES
Diagnostic and treatment options in passerines may be limited by owners nancial constraints and difculties in collecting samples from small birds. Veterinary care in these species is frequently directed toward appropriate preventive husbandry measures and approaching medical problems from a ock perspective. The main clinical diagnostic procedures for these small birds are history, examination of the cage, physical exam and limited clinical procedures. In many cases, especially in ocks, these procedures should be followed by a diagnostic necropsy. Larger passerines like mynahs are more expensive. The basic approach like in parrot-medicine will lead to proper diagnosis and treatment, but are beyond this presentation. 3.1 Clinical diagnostics The history should include information on the species, age, symptoms, diet and housing. A careful history will provide much information needed to arrive at a diagnosis. Examination of the cage or aviary can provide a great deal of useful information. Attention should be paid to the droppings, the feed dishes, and the oor. Most breeders of passerines bring in their birds in transport boxes or cages. Birds should be put in a birdcage as soon after arrival as possible, even before the history is taken. The birds can acclimate then to their new surroundings and fresh stool for examination is likely to be produced. Transport in their own cage is recommended whenever possible. Light out/perches out catching techniques are almost mandatory, and strong lighting in combination with a magnication device will greatly facilitate any examination of the tiny birds. When handling the birds, keep the windows closed!

411

The physical examination and clinical procedures are limited in the smaller passerines, but nevertheless very important. Most gram scales can provide an accurate weight if the nch is contained in a paper box or bag, but the container must be weighed or tarred. The usual physical examination is performed as for any other bird. Listen for respiratory sounds. Do not interfere with the movements of the sternum, which could kill the patient. Special attention should be paid to the state of moult, the pectoral muscle mass (chronic or acute problem), the abdomen (blow the feathers apart and look for an enlarged liver and dilatation of the gastrointestinal tract), and the skin (search for pox lesions and parasites). Routine diagnostic procedures also include the following: Faecal examination. Helminthic infections are very rare indeed in small passerines, but are more often seen in wild-caught mynahs and insectivorous passerines. Coccidia, which are common in small passerines, are excreted mainly between 2 pm and darkness. Yeasts and protozoal cysts (e.g. Giardia spp) are found with direct wet preparations or after otation techniques. The diagnosis of cochlosomosis in Society nches or Australian nches can only be made in direct wet mounts of fresh and warm stool without dilution. Because passerines are not considered to have a normal gut-ora, no bacteria or other microorganisms should be found in stained faecal smears. Routine microbiologic aerobic cultures should be negative and microaerophylic strains (e.g. Campylobacter jejuni) can be found in stained faecal smears. Crop swabs. Crop swabs are essential for the diagnosis of trichomoniasis and crop candidiasis. Blood samples. For additional information in small individual passerine birds, blood can be collected in heparinised capillary tubes after puncturing the medial metatarsal vein. From lager birds, but also with some practice from small nches, blood can be collected from the right jugular vein. One drop is used for a blood smear, which can be examined for blood parasites and if, present, for the type of anaemia. The packed cell volume (PCV) normally ranges from 40% to 55%; any reading less than 35% indicates anaemia. Total protein (TP) determinations provide a very signicant diagnostic measure. Normal haematological and serum biochemical references are presented in table 1.

3.2 The diagnostic necropsy A necropsy should always be performed on birds that die from unknown causes so that aws in management can be rectied and a possible epidemic can be forestalled. The necropsy is also the ultimate method to conrm a diagnosis. The following procedures can provide much additional information during the necropsy: direct wet preparations of the gut contents and of the coating of the serosae; scrapings from the mucosa of the crop, proventriculus, duodenum, and rectum; and contact or impression smears from a freshly cut surface of liver, spleen, lungs, and any altered tissues. The smears are stained routinely with Romanowsky stains (e.g. Giemsa) or Quick stains (e.g. DiffQuick) and searched microscopically (cytology) with oil immersion. Bacteriologic, mycologic, virologic, serologic, and histopathologic examinations and immunodiagnostic techniques are special techniques to come to a diagnosis.

412

4 TREATMENTS
Compared to the other orders of the class Aves passerines, including mynahs, have an extremely high basal metabolic rate (K=129). The resulting effective blood levels after administration of drugs are often of a very short duration. For the different dosage rates see the different formularies. Medications can be easily administered by several methods. For parenteral administrations (intramuscular or subcutaneous) a 27-gauge needle is required, and even these can cause signicant haemorrhage if not used with caution. To minimize risk, the injection site should be located in the caudal third of the breast muscle. Application via the drinking water is a common route of drug administration in birds. In many disease cases and after adding drugs to the water the water intake will be reduced substantially. The normal water intake is already irregularly spread over the day. This results in very irregular or even a lack of effective blood levels, which can be the cause of disappointing therapeutic results and the development of resistance. Water medication can be successful against mild infections of the gastrointestinal tract in which the drug may have a local effect in the gut. Administration of medication in the drinking water is especially not recommended in soft bills and fruit-eating nches because they generally do not consume large quantities of water. They obtain most of their daily uid requirements from fruit. Oral administration in larger passerines can be given directly, by metal or rubber gavage tube, or by placement of a capsule or granules in the centre of a grape or raisin. In ock-treatment of small passerines, to achieve better therapeutic results, the drug should be administered at the same time in both drinking water and the soft or egg food, resulting in self-medication several times a day (Table 2). Because the elimination is so rapid, there will with oral administration through the food and water be no measurable blood levels during the night in most cases. It is possible to increase water and food intake during the night by changing to a 24-hour light regimen, which will result in a more even distributed blood levels over a period of 24 hours. This change of day-night rhythm, however, will disturb breeding and can induce moulting. Antibiotics must be administered for at least 7 days and bactericidal antibiotics are preferred above bacteriostatics. Treatment of skin or leg lesions with ophthalmic ointments often results in intoxication and the death of the patient. A few drops swallowed during cleaning its feathers are sufcient to kill the bird. The owner should be instructed to apply the ointment very sparingly or put a collar on the bird.

5 COMMON DISEASES AND THEIR TREATMENT

A quick algorithm for determining a possible diagnosis based on the information collected and the most common diseases in passerines is presented in Table 3 Additional diagnostic test and the differential diagnosis are listed in Table 4.

413

5.1 Problems in mixed aviaries Nutritional problems especially those resulting from an unbalanced diet, are often seen in mixed aviaries and individual pet nches. Even granivorous birds need a certain amount of soft bill food as an unbalanced diet enfeebles these birds and can lead to problems with Enterobacteriaceae (eg. E. coli, Klebsiella spp. and Enterobacter spp.) and yeast infections (esp. Candida albicans). The breeding results are also disappointing in birds with an unbalanced diet. The primary cause of many problems in Australian and other tropical nches is an unbalanced diet; therefore, when treating disease problems in these birds, improvement of the diet has to be the rst objective. A good starting point is a controlled feeding of three parts of a seed mix supplemented with one part soft food. It may be difcult, however, to make the birds eat the soft food. Management and hygiene-related problems. Many problems in aviaries, however, are management and hygiene-related problems due to location food and water utensils. Overcrowding leads to aggression, and insufcient nesting sites results in poor breeding results. The control of ecto- and endoparasites is a matter of constant attention. Trauma. Picking is a common problem in aviaries. This can range from a few feathers lost on the back of the head to cannibalism. Zebra nches are prone to indulging in this behaviour. It can also be the result of inappropriate sexual behaviour of one or more dominant male birds. Aggression is the main drive when aviaries are overcrowded or nesting sites or territory is involved. Sick birds may attract aggressive behaviour, therefore the attacked bird should be separated and the underlying problem addressed. Hemorrhagic enteritis. Often is diagnosed at necropsy, but it is not enteritis in the true sense. It should be considered as a hemorrhagic diathesis (blood leaking into the gut). It is seen in small birds that for some reason did not eat at all for over 24 hours, perhaps because they were too ill to eat (e.g. because of an infection or intoxication) or, more often, when they get the wrong food or no food at all (e.g. if the owner was away and someone else fed the birds). A typical sign is an empty crop. A similar interpretation should be given to swollen, white kidneys which are the result of uric acid precipitation in the collection tubules that occurs when birds do not drink. This condition is often falsely called renal gout, but it should not be interpreted as nephritis or gout. It should be differentiated from visceral gout based on impaired renal function or a high protein diet. Articular gout is a poorly understood chronic condition with no relation to renal function. Haemochromatosis or iron storage disease is the most common non-infectious disease in soft bills including mynahs. Clinically dyspnoea, abdominal distension (hydrops ascites), and weakness are seen with hepatic haemochromatosis. In general, fructivorous, insectivorous, and omnivorous birds accumulate more iron in their liver than carnivorous, piscivorous, and granivorous birds, even within the same order. Diets with 100 ppm iron or less have been recommended to reduce dietary

414

sources of iron, because most birds shown to have the disease had been consuming diets in excess of 100 ppm iron. Because in a practical situation diets with less than 100 ppm iron are difcult to formulate, this observation may prove to be more in line with what is available to feed birds than what is needed to prevent excessive iron storage. Even diets with 100 ppm iron are in excess of the requirements for growth of poultry, which generally require 60 to 80 ppm. In birds that are prone to develop iron storage in the liver, diets below 50 to 60 ppm can prevent induce an iron storage. New information shows good effects of adding tannins or phytines to the diet. Radiographs revealing enlarged liver and ascites make the diagnosis; a liver biopsy conrms haemochromatosis. One effective treatment, phlebotomy, is usually performed in conjunction with low iron diets. A less invasive treatment is documented using deferoxamine (100 mg/kg q24h, SC) combined with a low iron diet (65 ppm) for periods as long as 4 months until the iron content in the liver of a toucan normalized. 5.2 Infectious diseases Many infectious diseases are species specic, although and are exceptions and can infect any species. Although is often diagnosed in nches, most birds appear to have their own coccidian species. These coccidia are said to belong to the Isospora lacazei group. 5.2.1 Viral Diseases Avian pox. In passerine birds kept in captivity, avian pox as a septicaemic problem is seen almost exclusively in canaries and other Serinus sp. Typical ndings are skin-pox lesions, respiratory dyspnoea and high mortality. Preventive vaccination is possible by the wing web method preferable in early summer. In masked bull nches (Pyrrhula erythaca) a poxvirus has been demonstrated causing tumour-like lesions in the head-region and inside the beak. Papovavirus. Papovavirus and polyoma-like virus infections occur in nch aviaries across Australia and the United States and are probably more common than the number of cases actually diagnosed would indicate. The disease causes both young nestling mortality and a more chronic disease in which poor development and beak abnormalities predominate. The diagnosis is made by a specic uorescent antibody test on liver and spleen impression smears and electron microscopic examination of the intranuclear inclusions Paramyxovirus. PMV-3 infections are commonly seen in many nches (e.g. African silverbills [Lonchura malabarica cantans], zebra nches, and Gouldian nches) with torticollis. The diagnosis is based on the symptoms and can be conrmed by serology and virus isolation. The necropsy is a specic. A severe pancreatitis is found on histological examination. Herpesvirus and Cytomegalovirus. These viruses cause respiratory problems in Australian nches.

415

Other virus infections. Infections with Inuenza virus have been reported in nches as well as imported mynahs 5.2.2 Bacterial Infections E. coli (and other Enterobacteriaceae): These bacteria are isolated very often from the faeces or from the intestinal contents of diseased passerine birds, with and without diarrhoea. In general there is a malaise with some birds showing conjunctivitis and rhinitis; a few birds may die. These are secondary pathogens, however, and should be considered as a symptom of poor health or hygienic conditions. Possible causes are unbalanced diet, housing problems or management problems. Other primary diseases may be present (e.g. atoxoplasmosis or coccidiosis). Treatment with an antibiotic does improve this condition but only temporarily; the clinician must search for the real cause. Enterobacteriaceae are regularly cultured from cases with diarrhoea in passerine nestlings (sweating disease). The antibiotics of choice are neomycin or spectinomycin because they are effective and not absorbed from the gut. The drug is administered via the soft food. In edglings, extra water, like green food, will prevent dehydration. Yersiniosis (pseudotuberculosis): An infection with Yersinia pseudotuberculosis is regularly seen in the wintertime in Europe in canaries and wild nches. The clinical signs are not specic. At necropsy a dark, swollen congested liver and spleen with small, yellow, focal bacterial granulomas are found. The diagnosis is conrmed after culturing the microorganisms. Treatment of choice is amoxicillin via the drinking water and the soft food. Salmonellosis (paratyphoid.) Infection with Salmonella typhimurium is clinically, and at necropsy, identical to pseudotuberculosis, although a chronic course is seen more often in salmonellosis. Carriers are not known in canaries. The diagnosis is conrmed after culturing the microorganisms. The antibiotics mostly effective are trimethoprim (with or without sulpha), amoxicillin, or enrooxacin. The therapy needs to be combined with hygienic measurements. Campylobacter fetus subsp. jejuni is found very often in tropical nches, especially in Estrildidae. Society nches often are carriers without conspicuous clinical symptoms. Demonstrating the curved rods in stained smears from the droppings or gut contents, and cultivating the bacteria on special microaerophilic media conrm the diagnosis. There is no evidence for zoonotic importance of this infection. Cocci infection. Infections with Streptococcus spp. and Staphylococcus spp. are seen often. The clinical signs are abscesses, dermatitis, bumblefoot and conjunctivitis, as well as, less often, sinusitis, arthritis, pneumonia, and death. Cocci will be seen in the impression smears. Local and systemic treatment with ampicillin or amoxycillin is the therapy of choice.

416

Enterococcus fecalis. has been associated with chronic tracheitis, pneumonia and air sac infections in canaries. Clinically affected birds have harsh respiratory sounds, voice changes and dyspnoea. Pseudomonas infections. Inexpertly prepared sprouted or germinated seeds and dirty drinking vessels or baths can be the source of Pseudomonas spp. infection causing a foul smelling diarrhoea. A polluted ower-spraying syringe, used for spraying the birds, can cause a severe necropurulent pneumonia and aerosacculitis. Avian tuberculosis. The classic tuberculosis with tubercles in the organs is seldom seen in small passerines. Mostly it are infections with atypical Mycobacterium avium or Mycobacterium-avium-intracellulare-complex and M. genavense). Individual infections with acid-fast bacilli are seen relatively often. On histological examination, macrophages loaded with acid-fast bacilli can be found in many organs. No alterations are apparent at necropsy, except perhaps a dark, slightly swollen liver. Ornithosis. This is relatively uncommon problem in passerines. Mycoplasma spp have been isolated from canaries and many cases of conjunctivitis and upper respiratory disease in canaries respond to tylosin; however, there has been no conclusive work proving that Mycoplasma is associated with this syndrome. Other bacterial infections. The following bacteria are occasionally isolated from diseased passerines: Erysipelothrix rhusiopathia, Listeria monocytogenes and Pasteurella multocida (cat-bite?). 5.2.3 Mycotic Infections Mycotic infections are not a signicant problem in canaries, but are much more common in tropical nches and mynahs. Especially cases of candidiasis are seen. It is not uncommon to identify the yeast Candida albicans in cultures of the gastrointestinal tract of toucans and other soft bills. Chicks that have poor daily weight gain or poor feeding response should be examined for potential bacterial or yeast overgrowth. Cytology stain or cultures of the crop or cloaca should be performed. In canaries infection with the yeast Macrorhabdus ornithogaster (formerly called Megabacteria)of the proventriculus is very common, predominantly found on the mucosal surface and in the ducts of the glands. 5.2.4 Protozoal Infections The most important protozoal infections in canaries are atoxoplasmosis, coccidiosis, toxoplasmosis and trichomoniasis. Atoxoplasma like infections and cryptosporidiosis are found only occasionally in nches, but are restricted to individual birds. It is never seen as a ock problem. Coccidiosis, cochlosomosis and trichomoniasis are very common in nches.

417

Atoxoplasmosis is caused by Isospora serini in canaries. The clinical symptoms are huddling and rufing of the feathers, debilitation, diarrhoea, occasionally neurological signs (20%), and death. An enlarged liver can be seen as a blue spot at the right side of the abdomen caudal to the sternum, referred to by fanciers as thick liver disease. At necropsy an enlarged, sometimes spotted liver, in the acute phase with necrosis, a huge, dark red coloured spleen, and often an oedematous duodenum with vascularisation are seen. In the imprints of the liver, the spleen and the lungs the parasites are found in the cytoplasm of the monocytes. This infection is also a common problem in other European nches kept in captivity (e.g. goldnch, siskin, greennch and bullnch). Atoxoplasma-like infections are seen in tropical nches, mynahs and other Sturdidae. Coccidiosis can be a problem in passerines of all ages older then 2 months. The clinical symptoms are diarrhoea and emaciation. In wet preparations from the droppings large amounts of oocysts can be seen. Therapy consists of strict hygienic measurements and treatment with coccidiostatic drugs. Toxoplasmosis: In the acute phase of toxoplasmosis the birds (canaries and mynahs) may show severe respiratory signs. Canaries become blind many weeks after the infection. In cytology from the lungs trophozoites are easily demonstrated and in histology slides from the brains (pseudo)cysts can be found. Serology, immunouorescence on brain tissue slides, or infection of mice conrms the diagnosis. No effective treatment is known. Trichomoniasis. Infections with Trichomonas sp are sporadically seen in canaries, but common in Australian nches. The diagnosis can be made in a live bird with a crop swab. At necropsy trichomoniasis presents a thickened, opaque crop wall. Cochlosomosis. The agellate, Cochlosoma sp., living in the intestinal tract of Society nches, can cause many deaths among Australian nches fostered by these carriers. The diagnosis is based on demonstrating the agellates in fresh and body-warm faeces from the nches. Plasmodium and other blood parasite have incidentally been reported in passerines. 5.2.5 Helminthic parasitism Worms are of no signicance in small passerines. In outdoor aviaries an infection with gapeworms (Syngamus trachea) is incidentally seen. Acuaria skrjabini infections of the gizzard with mucosal necrosis were reported in adult nches in Australia Tapeworminfestations in insectivorous nches (particularly parrot nches and diamond retails) are common and are regularly seen in Sturdidae.

418

5.2.6 Arthropods Ectoparasites, including blood-sucking mites (Dermanyssus gallinae and Ornithonyssus sylviarum), skin mites (eg. Backericheyla sp. and Neocheyletiella media) and feather mites (eg. Epidermotidae, Dermation spp) in the calamus of the feathers. Meal mites (Tyroglyphus farinae) can cause unrest and irritation. Air sac mites. Sternostoma tracheacolum are found occasionally in canaries, they are seen mostly Australian nches. Knemidocoptes pilae is occasionally seen on the beak base of nches. It is easily found and recognised in scrapings from the altered beak. Local treatment with any oil or 0.1% ivermectin applied locally will cure the birds. This infestation should not be confused with the so-called tassel-foot in the European goldnch (Carduelis carduelis), which is caused by a papillomavirus. Table 1. Some selected normal haematologic and serum biochemical values in passerines (adapted from CARPENTER JW, MASHIMA TY, RUPIPER DJ: Exotic Animal Formulary, Manhattan, KS, Greystone Publications, 1996, pp 156 and ALTMAN RB, CLUBB SL, DORRESTEIN GM and QUESENBERRY K, Saunders Co, 1997, p 1021) Measurement Haematology: PCV (%) RBC (10 /l)
6

Canary 45-60 2.5-4.5 4-9 20-50 40-75 0-1 0-1 0-5 45-345 120-350 5.1-13.4 1.6-5.6 200-400 2.0-4.5

Finch 45-62 2.5-4.6 3-8 20-65 20-65 0-1 0-1 0-5 150-350 200-450 3-5

Mynah 44-55 2.4-4.0 6-11 25-65 20-60 0-3 0-3 0-5 130-350 (600-1000) 9-13 190-350 2.3-4.5

WBC (106/l) Heterophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%) Chemistries: AST (IU/L) LDH (IU/L) Ca (mg/dl) P (mg/dl) Glucose (mg/dl) TP (g/dl)

419

Table 2. Dosage regimens for chemotherapeutics and antibiotics for canaries and small passerines Drug Amoxycillin Ampicillin Chloramphenicol Chlortetracycline Dimetridazole Doxycycline1 Enrooxacin2 Erythromycin Furazolidone Ivermectin3 Lincospectin Ketoconazole Metronidazole Neomycin Nystatin
4 1

Conc. in drinking water (mg/l) 200 - 400 1000 - 2000 100-150 1000 - 1500 100 250 200 125 100-200 800-10003 100-200 200 100 80-100 100 000 IU 50 000 IU 400 200-400 200-400 150-300 150 50-100 250-400

Conc. in soft food (mg/kg) 300 - 500 2000 - 3000 200 - 300 1500

1000 200 200 200

200 200 100 100 200 000 IU 50 000 IU 400 400 400 100 400

Polymyxin Ronidazol Spectinomycin Spiramycin Sulphachlorpyrazin Sulphadimidine Trim/sulpha Tylosin

5

-----------------------------------------------------------------------------------------------------1 In case of ornithosis, 30 days. 2 In case of ornithosis, 21 days. 3 Concentration in MICROGRAMS/L, alternative by topical application, one drop of 1% solution. 4 For the treatment of Candida albicans for 3-6 weeks. 5 This dosage is for the trimethoprim part alone.

420

Table 3. Diagnostic table for canaries and nches

Species: 2 12 7 3 4 5

- Canary - Australian nch - Mixed aviary Age: - Nestling - Juvenile, under one year of age - Any age

- Interior of the nests are yellow stained by diarrhoea of the nestlings, the feathers sticky, the E. COLI DIARRHEA youngsters stunt, and there is much mortality between 1 and 3 days of age.

-Very pale membranes visible by opening their beaks and weak in stretching their necks. Females B L O O D - S U C K I N G can be found death sitting on the eggs. MITES

421

-The youngsters show huddling and rufing of the feathers, debilitation, diarrhoea, sometimes ATOXOPLASMOSIS neurological signs (20%) and death. Mortality can be as high as 80%. 6 7

-Respiratory distress

-Respiratory symptoms not main sign

-Dyspnoea, debilitation with scabs and pox-lesions, esp. on eyelids, commissure of the beak and in feather follicles. Diphtheric lesions can be found in the mouth and larynx. Birds of all ages can be AVIAN POX affected and the mortality lays between 20 and 100%; the infection spreads quickly. TOXOPLASMOSIS BLOOD SUCKING MITES STERNOSTOMOSIS TRICHOMONIASIS

-Severe respiratory signs, general illness and central nervous symptoms and iridocyclitis, which often results in blind birds after 3 months due to a panophthalmia. -Minor to severe respiratory symptoms with anaemia and sometime a high mortality. The main complaint from the owner is usually a general depression in the bird. - Loss of voice, decline of physical condition, respiratory distress, wheezing, squeaking, coughing, sneezing, nasal discharge, head shaking and gasping. A low mortality. -Apathy, respiratory symptoms, regurgitation, blowing bubbles and emaciation, but seldom diarrhoea.

- Diarrhoea 9

- Diarrhoea not specic

-A general decline of the physical condition, huddling and rufing of the feathers, debilitation, COCCIDIOSIS diarrhoea and emaciation. The mortality is low.

-Several birds demonstrate a general malaise, with or without diarrhoea and some birds showing COLIBACILLOSIS conjunctivitis and rhinitis. Some birds may die. 10 11

- Obvious wasting - Sudden death of several birds

10

-Most infections are seen in winter. The clinical signs are apathy, decline in food and water intake, PSEUDOTUBERCULOSIS debilitation, emaciation, diarrhoea, respiratory symptoms, rufing of the feathers, and high mortality. SALMONELLOSIS

-Especially in outdoor aviaries, clinically indistinguishable from pseudotuberculosis, more often chronic

-Apathy, diarrhoea, debilitation, nasal exudates and conjunctivitis. The mortality is usually less then 10%.

422

- Birds grow thinner, but only low mortality

11

-Not specic. CNS-symptoms, often a obvious salivation and dyspnoea or diarrhoea in apathic birds

CHLAMYDIOSIS MACRORHABDUS ORNITHOGASTER TOXICOSIS

-Often after a weekend when some other than the owner fed the birds. Sometimes black-stained STARVATION droppings or diarrhoea. Weakness is often interpreted as a CNS-symptom. 13 16 14 15

12

- Age:

- Nestlings and edglings under the age of 3 months

- All ages affected

13

- Bengalese or Society nches as foster parents

- Natural breed or foster parents

14

-From the age of 10 days until 6 weeks, there are debilitation, shrivelling and yellow- staining of the edglings, difculties with moulting and parts of or whole seeds in the droppings. The foster parents COCHLOSOMOSIS show only watery droppings.

15

-High losses of nestlings, adult Estrildidae can show apathy and yellow diarrhoea or yellow solid CAMPYLOBACTER droppings due to large amounts of undigested amylum.

-In nestlings the crop is bloating, and a thickened crop wall is relatively common. In weanlings and CANDIDIASIS adult birds diarrhoea and moulting problems are more prominent. 17 18

16

- Respiratory distress

- Respiratory distress not the main symptom

17

-Respiratory distress, wheezing, squeaking, coughing, sneezing, nasal discharge, loss of voice, STERNOSTOMOSIS head shaking and gasping. The mortality is low.

-Apathy, respiratory symptoms, regurgitation, blowing bubbles and emaciation, sometimes TRICHOMONIASIS diarrhea. 19 7 PARAMYXOVIRUS 11

18

- CNS-symptoms

423

- CNS-symptoms not a main symptom

19

-Torticollis is the main symptom. As long as these birds can still eat, mortality is low.

- Sudden death of several birds

Table 4. Special hints for further diagnostics A necropsy and demonstration of the parasites in imprints Atoxoplasmosis of several organs
Avipox Campylobacter Candidiasis Chlamydiosis

Necropsy, histology and virus isolation In smears from the dropping the microorganisms can be demonstrated after staining with Diff-Quick. Cultivation only on special media. A direct wet preparation and/or a stained smear. Culture

Necropsy and demonstration of the agent by staining, IFT or Elisa. Parasitical examination of droppings collected between Coccidiosis 2 and 6 p.m. Flagellates in a wet mount of fresh and body-warm faces Cochlosomose from the nches. Analysis of the situation for other factors in combination Colibacillose with the isolation. Not important in small passerines. Helminthic infestation Syngamus very occasionally. Detailed case history. A direct conformation often Intoxication impossible, when the toxin is not known.
Mites Macrorhabdus sp Paramyxovirus Pseudotuberculose Salmonellosis Starvation Sweating Disease Sternostomose Trichomoniasis

Demonstration of mites in the nest or bird-room crevities Demonstration of the yeasts in a scraping of the mucosa of proventriculus Serological and virologic screening. In the histology a pancreatitis Necrotic foci at necropsy in liver and spleen and agent isolation. Necrotic foci at necropsy in liver and spleen and agent isolation. Haemorrhagic diathesis (bleeding into the gut) at necropsy Demonstration and isolation of bacteria in the faces. Diagnostic necropsy and demonstration of the parasite Demonstration of agellates in crop-swab. Necropsy Serology and demonstration of the parasite in brain-smears, organ-smears or histological

Toxoplasmosis

AUTHORS ADDRESS
G. M. Dorrestein, DVM, PhD Department of Pathobiology, Pet Avian, Exotic Animal and Wildlife Section, Utrecht University, The Netherlands Email: g.m.dorrestein@vet.uu.nl

424

Highbury Veterinary Clinic, Burwood, Victoria, Australia

AVIAN CLINICAL ANATOMY FROM AN EVOLUTIONARY PERSPECTIVE

P. Macwhirter B.V.Sc. (Hons), M.B.A., M.A., F.A.C.V.Sc. (Bird Medicine)

KEYWORDS
Bird clinical anatomy Evolution - Therapoda

ABSTRACT
Birds are ying dinosaurs that trace their ancestry to bipedal therapods of the Mesozoic Era. The Cretaceous-Tertiary boundary extinctions of c. 65 million years ago and the development of strong winds following the severing of the link between greater Australia and Antarctica c. 33 million years ago were key events in the emergence of modern volant families of birds. Avian anatomy and physiology, including musculo-skeletal structure, reproduction, nesting behaviour, respiration and renal function reect birds reptilian ancestry as well as constraints of powered ight. These have implications for the day to day medical and surgical care of birds and include, for example, presence of pneumatic bones and fusion and strengthening of bones throughout the body; unique jaw and beak structure; air sacs; lack of a diaphragm and use of air capillaries rather than alveoli; small clutch sizes compared with reptiles; development of the more heavily calcied avian egg and parent incubation.

1 INTRODUCTION
There are currently over 9,000 species of birds on the planet, more than any other group of vertebrates. It is not possible for veterinarians to learn detailed anatomy of each of these species and clinicians are often challenged by types of birds they have rarely or never previously encountered. An understanding of how birds evolved from reptilian ancestors is helpful in understanding the anatomy, physiology and diseases of present day avian patients. Fossil remains of Archaeopteryx, a primitive bird from the Upper Jurassic, about 150 million years ago (mya), in Europe have been known to science since the 1860s but discoveries of the late twentieth century, particularly that of Protoavis, recovered

425

from Triassic (225 mya) fossil beds in Texas and a trove of feathered dinosaurs from the Cretaceous (about 125 mya) from China have changed and expanded current knowledge of the evolution of birds. Interestingly, the much older bird, Protoavis had pectoral girdle suggesting it was capable of limited powered ight while the pectoral girdle structure of Archaeopteryx suggests that this bird was primarily a glider. The feathered dinosaurs of China most likely did not y but used their feathers for other purposes. * Examples showing clinical relevance of avian evolution are highlighted with this symbol in these notes.

2 THE AMNIOTE EGG

Since multicellular organisms rst emerged on earth some 4 billion years ago, periods of gradual diversication of life forms have been punctuated with periods of abrupt extinctions (DE DUVE 1995). In the Carboniferous Period, 350 mya, crocodile shaped amphibians called labyrinthodonts dominated a land vegetated with primitive psilophytes, lycopods and horsetails. The continent of Laurasia, which included Europe and parts of North America had emerged from the sea, moved south and coalesced with the southern continent of Gondwana to become a single land mass, Pangaea. Flora and fauna between the two landmasses intermingled, enabling a vastly increased pool of natural variation and selection pressures that favoured organisms able to reproduce independently of an aquatic environment. In this context the amniotes emerged. These were vertebrates that produced eggs containing specialised membranes that provided the developing embryo with a liquid environment, gave oxygen in exchange for carbon dioxide, stored food as yolk and isolated nitrogenous waste. The earliest amniotes were anapsids without any lateral openings on the side of the cranium posterior to the orbit. Tortoises and turtles have been traditionally classied as members of the Anapsida. Independently from primitive anapsids the synapsids (mammals) evolved with a single lower opening on the skull posterior to the orbit and the diapsids (reptiles) evolved with two lateral openings, one above the other. These openings allowed for larger, more powerful jaw muscles for chewing and capturing prey. Birds subsequently evolved avidiapsid crania in which the two lateral cranial openings merged into a single opening, allowing for streptostyly, a sliding action of the quadrate bone that enabled independent movement of the upper beak. * Clinical relevance: hatching difculties, retained yolk sacs, jaw function and injury repair.

When a wave of global extinctions occurred 245 mya, large amphibians over most of Pangaea were wiped out while descendants of the small reptiles survived and evolved to ll a wide range of ecological niches. These included the progenitors of turtles, lizards and snakes as well as the archosaurs which in the warm humid climate of the Mesozoic Era gave rise to the crocodile family, the pterosaurs, the dinosaurs and, later, birds.

426

3 WHEN AND WHERE DID FEATHERED REPTILES FIRST APPEAR?

It has been strongly argued that birds should be placed as a subgroup within Reptilia rather than given equal rank with Mammalia and Reptilia. Bird bones are particularly fragile and do not preserve well, so it is not surprising there are gaps in the fossil record. However, phylogenetic analysis and analysis of fossils with possible pro-avian features emerging from both Asia and North America have lent weight to the case that birds are embedded in the bipedal dinosaur class of therapoda at the highest node of dinosaur evolution. They are living members, indeed the only the only living members, of the dinosaur family (CHATTERJEE 1997, MARTIN 2002). Crocodiles are their closest living relatives. Feathers are the key difference between birds and (other) reptiles but the earliest birds did not y and the evolutionary steps between ectothermic reptiles and the wide diversity of endothermic, ighted and non-ighted avian species of today has been the subject of much speculation. Critical bio-geographical events over the 225 million year period since the rst appearance in the fossil record of possibly feathered reptiles have included the gradual break up of Pangaea and Gondwana due to tectonic plate movement, the expansion of winged insects and emergence of owering plants c. 120 mya the Cretaceous-Tertiary (K-T) Boundary Disaster that marked the end of the Mesozoic Era, possibly due to a meteor crash near present day Mexico c. 65 mya, la grande coupure - the separation of South America and then greater Australia from Antarctica with resultant circumpolar currents, global cooling and increased winds, c. 33 mya. ongoing sea level changes, uctuating climates and the emergence and global expansion of mammalian species, especially pinniped, felids, canids and humans.

4 HOW DID REPTILES EVOLVE INTO FLIGHTED BIRDS?

If a species is to survive and multiply, evolutionary change needs to confer immediate advantage for the next generation. The paucity of the avian fossil record, particularly from the Mesozoic Era, leaves many blanks in the sequence but key events in the transition from reptiles to birds must have included the development of feathers along with changes in thermoregulation, reproduction, nesting behaviour, respiration, renal function and musculo-skeletal structure. Efcient apping ight appears to have occurred later in the sequence. Bipedalism with feathers for thermoregulation, buoyancy or ight

427

In general, ectotherms are at an advantage in hot climates where food sources scarce but endotherms perform better when the climate is cooler and food sources abundant. The at foot structure of early fossil birds suggest they leaped, waded, walked or ran on two legs rather than perched on small branches as their more curved footed descendants came to do. They perhaps leaped or hang-glided, using a long neck and bill, still with reptilian teeth, to catch prey, their forelimbs free for balance and other purposes. In common with other therapods, most birds have the phalangeal formula of 2-3-4-5 with absence of the fth digit. Probably in response to a bipedal cursorial lifestyle proximal tarsal bones fused with the tibia to form the tibiotarsus and distal tarsal and metatarsal bones form a single tarsometatarsal bone. * Clinical relevance: tibiotarsus, tarsometatarsus and intertarsal joint structure and disease; leg fractures & repair.

Feathers do not appear to be modied scales but rather emerged in therapod dinosaurs as independent tubular structures that became progressively more complex. Insulation, balance and perhaps buoyancy, rather than ight, may have been the key initial benets that feathers provided (PRUM and BRUSH 2004). Winged insects pre-dated the emergence of birds. Such insects, along with aquatic creatures could have provided an abundant food source, around the shores of the Tethys Sea where Archeopteryx lived or in the Triassic swamps of Texas where Protoavis lived. Both species had feathers and were almost certainly endothermic but, in addition, fossil evidence suggests that Protoavis also had stereoscopic vision, a deep keel bone and a pectoral girdle structure with a rudimentary triosseous foramen suggesting the bird was capable of limited powered ight. Enanatiornithines, birds that rst appear in the fossil record around 120 mya showed features of a reduced tail, a pygostyle and fused carpal and metacarpal bones. Both features improving their manoeuvrability and capacity for powered ight. Later in the fossil record, Hesperornis, a toothed, loon-like Cretaceous marine bird from the northern hemisphere, couldnt y but feathers aided in buoyancy and it used its large feet for swimming. * Clinical relevance: problems associated with large avian eyes; signs of pituitary tumours; stress lines and feather formation; scaly and feather mites; pygostyle injuries in wing clipped birds, pododermatitis in webbed and other feet.

5 TERTIARY BIRDS, THE K-T BOUNDARY DISASTER

Given the global distribution of enantiornithines and more than a dozen other genera of land and sea based Mesozoic bird fossils, it would seem that a variety of primitive birds were widespread at the time of the great Yucatan meteor crash of 65 mya. It is likely that birds and bird-like reptiles living near the impact would have had little chance of surviving the initial explosion, res and the ensuing long dark winter. Those living at a distance, for example southern South America, Antarctica and Australia (which at that time were still conjoined as Gondwana) may have escaped the worst of the global disaster. Evolving near the Antarctic circle, these birds might have

428

been better adapted to the cool, dark conditions and could provide bird populations from which the northern hemisphere could be repopulated. The ability to y or swim would have aided dispersal. Fossil evidence and distribution of endemic bird species suggest that following major extinctions at the K-T boundary, Western Gondwana might have been the site of geographic origin of many bird families of today. Flightless land birds, penguin-like birds using their wings as ippers and loon-like marine birds using their feet as paddles were amongst primitive avifauna of the southern regions of Early Tertiary times. Bony-toothed pelicaniformes were found globally (WARHEIT and KENNETH 2001). * Clinical relevance: Ratite, penguin and pelican afnities and diseases.

Urogenital adaptation and nesting behaviour Birds reptilian ancestors had two ovaries and laid large clutches of eggs simultaneously which were incubated in nests on the ground in warm, moist conditions and gave rise to precocial young. Crocodiles, birds closest extant reptilian relations, still nest in this manner. For both birds and reptiles an excretory system based on the production of insoluble urates (rather than soluble urea) enabled waste material to be compartmentalised within the egg but away from the developing embryo in a nontoxic form and the capacity to adapt to dry conditions. * Clinical relevance: Megapod reproduction, renal function, cloacoliths, gout.

For pro-avians, using body heat to shorten the incubation time (and later developing specialised brood patches) would have been an advantage in reducing risk of predation and enhancing survival of their young, particularly in cool climates. It would have also favoured birds having small enough clutches to incubate against the parents bodies. Grebes, an ancient family that builds oating nests, might be a model for this type of incubation. Alternatively, feathered, partially endothermic avian ancestors may have nested in burrows as, for example, some penguins (another ancient avian family) do today (KAVANAU 1987). * Clinical relevance: commorant and cockatiel posture; incubation patterns in waterbirds, kiwis, penguins.

The ability of feathered pro-avians to use their own body heat to speed up the incubation process may have been advantageous in the short term in enabling birds to make use of nest sites in tree hollows, branches or bushes but this also required a change in egg structure to enable the embryos to survive in conditions with lower humidity. Laying small clutches of eggs individually rather than as a simultaneous clutch eliminated a need for two ovaries and pro-avian bodies could be streamlined to contain a single ovary and oviduct. Kidneys became compressed into fossa in the synsacrum. By producing a more heavily calcied, less porous egg shell in a shell gland, bird eggs became less prone to desiccation and birds could exploit nesting sites above the ground. The emergence of medullary bone lay down of calcium in egg laying females would have assisted this process. Flight and the avian urogenital package developed hand in hand.

429

Clinical relevance: medullary bone lay down, hernias, egg yolk related peritonitis, egg binding.

While primitive birds would likely have hatched precocial young, altricial young could be produced from smaller eggs relative to the size of the adult bird. This could have been an advantage for many ighted birds and might offset the greater parental care required to rear altricial young.

6 LA GRANDE COUPURE. C. 33 MYA

Following the K-T crisis, another critical biogeographical event for evolving avifauna was la grande coupure - the great cut, so coined by the Swiss palaeontologist Hans Stehlin. Beginning 60 mya Australia began to unzip itself from Antarctica as tectonic plate movement slowly drew the continent northwards. Fossils of giant penguin-like birds have been found near Adelaide, South Australia date to these times (VICKERSRICH and HEWITH-RICH 1996). By 40 mya, India had crashed into southern Asia and the Himalayan Mountains were rising. The Drake Passage between South America and Antarctica opened c. 35 mya and nally, around 33 mya, a long submarine rise that stretched from Australia to Antarctica severed allowing bottom water of the Antarctic circumpolar current to ood between the two continents for the rst time. This caused global cooling and strengthened coastal and trade winds. The effect was magnied as polar ice caps expanded, winds strengthened, temperatures dropped and global cold water currents carried rich marine food sources northwards (FLANNERY 2000). It was from this time that a dramatic expansion and diversication of ighted bird families occurred. Musculo-skeletal adaptations The strong circumpolar winds that followed la grande coupure may have been a key factor favouring birds with deep keel bones that were good at apping ight. Volant birds generally have dorsoventral rather than lateral attening of the body as the centre of gravity needs to be below the extended wing rather than above it (SIMKISS 1963). In avian ancestors rotation of the coracoid to the front of the chest and the scapula to the at of the back left the glenoid in a dorsolateral position. This positioning of the scapula facilitates climbing as well as the formation of the wing and it is also seen in mammalian climbers, including primates. However in the avian model the scapula is strap-like, narrow and xed compared with the broad triangular scapula of climbing animals. Flighted birds do not require strong muscles to raise their wings as lift performs this function, but they require strong muscles for the down beat a function performed by the supercial pectoral muscles. The two deep pectoral muscles (supracoracoids), whose tendons pass through the triosseous foramen and insert on the humeri, help to produce lift by altering the angle of attack and adjusting curvature of the dorsal aerofoil surface of the wing through their indirect effect on the patagial membranes.

430

Clinical relevance: distinguishing bones of the pectoral girdle on radiographs; effects of pectoral muscle injuries.

In addition to the change in the centre of gravity and reduction and xation of the scapula, skeletal renements useful for apping ight in birds include: pneumatic bones. replacement of teeth with lighter beaks fusion and strengthening of bones of the limbs, spine and synsacrum, swivel carpal joints for wing folding enlargement of the ulna to which secondary wing feathers attach, development of the alula from the second digit of the wing, development of uncinate processes on the ribs, shortening of the tail to become the pygostyle, fused clavicles forming the furcula, the enlargement of the coracoid, development of the keeled sternum, the triossesus foramen which enabled the tendon of the deep pectoral muscle to attach to the head of the humerus and control the angle of attack of the leading edge of the wing. Development of ight feathers with asymmetrical closed pennaceous vanes

The seeds of some of these changes could be seen in Archeopteryx, Protoavis and the Enantiornithines but the upsurge and explosion in diversity of birds capable of apping ight occurred after 30 mya when pectoral girdle and deep sternal conrmation typical of modern carinates was rened and became widespread. * Clinical relevance: patterns of bone fractures and coracoid and wing injuries; sternal injuries associated with wing clipping; spinal injuries at the exible joint T6-7.

Respiratory, digestive and immune system adaptations Respiratory systems of birds differ signicantly from reptiles in their extensive development of the air sacs, lack a diaphragm, presence of pneumatic bones and use of air capillaries rather than alveoli. These features generally make for lighter bodyweight and efcient respiration, both of which are cornerstones for leaping, running, swimming or ight. Ratites and penguins, both primitive avian families, have paleopulmonic parabronchi in which air ow is caudo-cranial, unidirectional and linked with air sacs. Storks, cormorants and cranes have this system as well as an additional network of parabronchi, the neopulmonic parabronchial net, in which air ow is bi-directional. Almost all other birds have well developed neopulmonic parabronchi. Birds rigid skeletal systems appear to have functioned as a bellowslike apparatus in breathing and aided in streamlining and lightening birds bodies for swimming or gliding before birds took to the air in apping ight.

431

Avian digestive systems reect diet diversity and weight constraints of powered ight. For example, while all present day birds lack teeth, large caeca may be present in herbivorous ground dwelling birds such as gallinaceous birds but are reduced or absent in passerines. Although not reected in the fossil record, the key avian immunoglobulins are IgM, IgA and IgY, as seen in reptiles rather than IgM, IgA, IgG and IgE as occur in mammals. * Clinical relevance: distribution of (e.g.) Aspergillosis lesions; use of abdominal breathing tubes for anaesthesia; disease patterns of Coturnix and Turnix quail; nature of allergic reactions in birds and response to intradermal skin testing.

7 CONCLUSION
Discoveries being made in the elds of palaeontology and molecular biology are giving greater insight into avian anatomy and physiology from an evolutionary perspective. Using an evolutionary framework is a useful aide in trying to understand and deliver quality veterinary care to our diverse range ying dinosaur patients.

8 ACKNOWLEDGEMENT
Illustrations from CHATTERJEE S. (1997). The Rise of Birds, 225 million Years of Evolution, will be used, with kind permission of the publisher John Hopkins University Press, in conducting this Master Class.

9 CITATION INDEX AND FURTHER READING

1. BAIRD R. Avian fossils from the quaternary of Australia. In: VICKERSRICH P, MONOGHAN JM, BAIRD RF and RICH TH (eds): Vertebrate palaeontology of Australasia. Melbourne: Monash University Publications 1996; 809 - 870. CHATTERJEE S. The rise of birds, 225 million years of evolution. Baltimore: John Hopkins University Press 1997; 1 - 224. DE DUVE C. Vital dust, the origin and evolution of life on Earth. New York: Basic Books 1995; 1 8. FLANNERY T. The eternal frontier. Melbourne: Text Publishing Company 2000. KAVANAU JL. Lovebirds, cockatiels and budgerigars, behaviour and evolution. Los Angeles: Science Software Systems, Inc 1987; 373 - 640. MARTIN L. An early archosaurian origin for birds. Beijing: IOC Proceedings 2002; 9. PRUM R and BRUSH A. Which came rst, the feather or the bird? Scientic American 2004. 72 - 82. SERENO P. Birds as dinosaurs. Beijing: IOC Proceedings 2002; 10. SIMKISS K. Bird ight. London: Hutchinson Educational Ltd, 1963; 13 - 46.

2. 3. 4. 5. 6. 7. 8. 9.

432

10. VICKERS-RICH P and HEWITT-RICH T. Sydney: Reed 1993: Wildlife of Gondwana. 11. VICKERS-RICH P. The Mesozoic and Tertiary history of birds on the Australian plate. In: VICKERS-RICH P, MONOGHAN JM, BAIRD RF and RICH TH (ed): Vertebrate palaeontology of Australasia. Melbourne: Monash Univ Publications 1996; 721 - 808. 12. WARHEIT and KENNETH. The seabird fossil record and the role of palaeontology in understanding seabird community structure. In: Schreiber E.A. and Burger J. Biology of Marine Birds. Boca Raton: CRC Press 2001; 17-56. 13. WILSON B. Birds. Scientic American 1979. 1 - 148.

AUTHORS ADDRESS
P. Macwhirter B.V.Sc. (Hons), M.B.A., M.A., F.A.C.V.Sc. (Bird Medicine) Highbury Veterinary Clinic, 128 Highbury Road, Burwood, Victoria, 3125 Australia Email: patvet@bigpond.com

433

Clinic for Birds and Reptiles, University of Leipzig Germany

IMAGING NON-INVASIVE DIAGNOSTIC TECHNIQUES OF THE UROGENITAL TRACT

M.E. Krautwald-Junghanns, Prof DrMedVet, Dipl ECAMS, M. Pees, DrMedVet

KEYWORDS
Urogenital system Kidney Testes - Ovary Oviduct Imaging techniques Radiology - Ultrasound

ABSTRACT
Radiology and ultrasound are important diagnostic tools in birds with alterations of the urogenital system. Both non-invasive techniques have its advantages and limitations. The combination of them gives information about the organs size and inner structure (radiodensity, echogenicity). Besides infectious diseases, alterations of traumatic, endocrine and neoplastic origin can be diagnosed.

1 KIDNEYS
1.1 Anatomy The paired kidneys are situated in the dorsocaudal part of the thoracoabdominal cavity. Each kidney is divided into three lobes all containing cortical and medullar tissue. Since a urinary bladder and a urethra are missing, the urine is conducted via the ureters directly into the urodaeum of the cloaca. 1.2 Indications for non-invasive diagnostic techniques Clinical signs indicating renal disease are generally non-specic and frequently superimposed by secondary changes caused by renal dysfunction. Dyspnoea, lethargy, diminished appetite up to emaciation, dehydration, defecation problems and changes in the urinary output are common signs of renal disease (LUMEIJ 1994). More specic are clinical ndings like non-traumatic unilateral or bilateral paresis of the legs, caused by compression or inammation of branches of the lumbosacral plexus, which pass through the kidneys. Subcutaneous urate tophi or urate accumulations in the joints are signs of hyperuricemia caused by renal disorder.

434

Alterations of blood chemical parameters like uric acid are further hints for renal disease. Nevertheless blood chemical values within the physiological range do not exclude a renal disease. Furthermore indications for sonographic examination of the urogenital tract consist of any disorder which is more or less connected with abdominal distension; and/or cases of radiologically diagnosed soft tissue masses in the region of the kidneys (HOFBAUER and KRAUTWALD-JUNGHANNS 1999). 1.3 Radiology Survey radiographs (laterolateral and ventrodorsal projection) provide information about the size and radiopacity of the kidneys. Enlargement of the renal shadow can be assessed in the laterolateral projection, since surrounding air sacs provide a good negative contrast. In the ventrodorsal projection, they are superimposed by the gastrointestinal tract, only high-grade alterations can be diagnosed. Besides the size, abnormalities like crystalline inclusions can also be detected. However in some severe cases only, urate tophi in the kidneys are visible as radiodense shadows. They are generally non-specic signs for renal insufciency of different aetiologies. Barium sulphate contrast of the gastrointestinal tract point out space-occupying lesions like renal tumours or cysts. Contrast studies using organic iodine compounds for visualisation of the kidneys/ the ureters are possible and may provide information about the functional status of the urinary system. However, due to anatomical peculiarities in comparison to mammals their use is limited in birds (KRAUTWALD et al. 1992). 1.3.1 Plain radiology Enlargement of the renal shadow is interpreted as a non-specic sign of many generalised infections (e.g. chlamydiosis). Non-infectious causes are vitamin A deciency, kidney cysts and neoplasia. The latter is one of the most frequent aetiologies of renal disease in the budgerigar (Melopsittacus undulatus). In cases of massive enlargement of the kidneys, the surrounding air sacs are displaced and it might be difcult to evaluate the cause of distension. In these cases, it may be necessary to have recourse to contrast radiography in order to dene the kidneys against the surrounding organs. This could involve GI contrast or renal contrast. Active gonads must not be confused with kidney tumours. In severe dehydration, gout and vitamin A deciency, there tends to be an increased density of the kidney shadow, but this is difcult to evaluate as the bony pelvis often obscures the image. The presence of radiodense, crystalline deposits in the kidney is readily demonstrated in both of the standard planes. Such crystals are evenly distributed throughout the organ and are due to the precipitation of uric acid and/ or renal calcinosis. They occur in chronic kidney infections indicating renal insufciency, often in association with a high blood uric acid level, but they are not specic for gout. They are also seen after prolonged uid deprivations.

435

1.3.2 Urography Renal contrast radiography is indicated in some instances to obtain information about functional disturbances of the urinary apparatus. A reduction or, indeed, complete absence of a contrast image in the kidney or ureter occurs sometimes when renal tumours or cysts are present. Elimination of the contrast medium is retarded in renal insufciency due to a variety of causes. Indications: Diseases of the kidneys and ureters that are associated with alteration in their size or shape, unless this has already been established in the plain radiograph (enlargement of the kidneys, for instance, in cases of acute nephritis). Dening the kidney against the surrounding organs, if this could not be achieved by the plain radiograph or GI-contrast study (for instance in ovarian cysts, gonadal tumours). Conditions of the kidneys and ureters accompanied by decreased function as, for instance, in renal neoplasia or cysts and in renal insufciency from whatever cause when excretion is retarded. In isolated cases: diseases of the other parenchymatous organs, where it is desirable to augment the demonstration of those organs. For urography the patient should be fasted for about 2 hours before the contrast medium is given. It may be necessary to aid emptying the gut by introducing liquid parafn into the cloaca. Organic iodine compounds are administrated intravenously, rarely orally (an example for oral use is Gastrogran), or by some other route, such as in sinography where it is injected directly into the sinus. For urography the organic iodine compounds is warmed to body temperature and injected slowly intravenously, usually into the ulnar vein. The dose is 2 ml per kg body weight, as a 70 % - 80 % solution of organic iodine compound or a solution containing 300 400 mg of iodine per ml (800 mg/kg intravenously (NEWELL 2000). The quality of the contrast obtained depends on the concentrating capacity of the kidneys, on the preparation used and on the iodine concentration of the medium. Aorta, heart and pulmonary arteries are demonstrated 10 seconds after the injection, kidneys and ureters are shown 30 60 seconds after injections, cloaca and terminal gut are visualised 2 5 minutes after the injection. 1.4 Ultrasonography Ultrasonography is of limited use for the assessment of the normal-sized kidney. In contrast, in birds with organ enlargements, abdominal swelling and especially in cases when ascites is present, the sonographical examination may provide more information than any other imaging technique. Kidney tissue is demonstrated in the area between the intestines and the total reexion of the spinal/pelvic bones. In cases of kidney enlargement, not only the size of kidney can be assessed, but also the density (echogenicity). Furthermore ultrasound is ideal for diagnosis of cystic alterations within kidney tissue (HOFBAUER and KRAUTWALD-JUNGHANNS 1999).

436

1.4.1 Approaches for ultrasonography of the kidneys There are three possible approaches to the organs of the avian urogenital tract (HOFBAUER 1996): The cranioventral approach, mid-abdominally directly behind the Margo caudalis of the sternum The caudoventral approach, situated between the pubic bones of the pelvis The lateral approach, in the ank directly behind the last rib Due to anatomic dissimilarities these approaches are not available in all species. On account of the complications caused by the caudal air sacs, particularly with the lateral approach, the gently caudally sloped scanner has to be angled about 70 to obtain a better image of the organs. Furthermore, it is advantageous to compress the air sacs, located underneath the body wall, by gentle pressure with the scanner onto the abdominal wall. This led to better imaging, not only in the lateral, but also in the ventral approaches. 1.4.2 Examination methods and evaluation criteria The sonographic demonstration of the normal kidney by transcutaneous ultrasonography is in most cases not possible. This is due to its position along the vertebral column, within the depressions of the pelvis and the surrounding abdominal air sacs, and also due to the rather small size along the ventrodorsal direction of the ultrasound waves. Using special sonographic equipment and transintestinal examination technique the demonstration of the normal kidney is possible. This has been described in birds with a bodyweight of more than 2 kg (HILDEBRANDT et al. 1995). However, at the present time, this technique is of little practice importance in pet birds, due to the high costs of equipment and the limited possibilities of application (small size of most avian patients). Kidney size: Whereas the ultrasonographic visualisation of the normal kidneys is difcult, in cases of organ enlargement, the examination is possible and both the size and the parenchyma can be assessed. This is possible not only in large birds but also in smaller ones (e.g. budgerigars) without problems. With a cross section (the beam plane perpendicular to the spinal column), the kidneys are presented lying in a W-shaped total reexion caused by the pelvic/spinal bones. With this view, both kidneys can be compared to each other. In this section view of the kidneys they are presented round to oval, and measurement of the size can be done. By turning the transducer about 90 degrees, the kidneys can be examined in their long axis (longitudinal to the spinal column). This should be done after identifying the renal tissue in the cross section. With this view, the major part of the kidney tissue can be examined for parenchymatous alterations (changes in echogenicity, see below). A direct comparison of both kidneys is not possible in this view. Kidney parenchyma: The echotexture of the enlarged inamed kidneys is homogenous and rather

437

anechoic with no recognizable inner structure. Neoplasia is visible as voluminous and often rounded single masses, frequently with diffuse inhomogenous echotexture. Sometimes, multiple poorly echoic foci mark necrotic areas within the irregular masses of the tumours. For identifying single-sided tumorous alterations, the vertical cross-section is preferable since the comparison of both kidneys is possible with this view. Also colour Doppler ultrasonography is possible for detection of the blood ow within the tumour, which is often signicant higher compared to the normal renal tissue surrounding the neoplasia. Besides neoplasia, also cysts of the kidneys are easy to detect by means of ultrasound. They appear sonographically as clearly dened, rounded anechoic structures, often with marked distal acoustic enhancement. In cases of renal cysts without renal enlargement, it might be difcult to differentiate between renal and ovarial cysts. In these cases, an ultrasound-guided puncture of the uid-lled cavities is easy to perform and the uid can be examined. Uric acid depositions and/ or calcications cause reexions (increased echogenicity), the renal tissue appears more inhomogeneous. However, diagnosis of renal gout by means of ultrasound is difcult; other techniques (radiology, endoscopy, blood chemistry) should be taken into account before making diagnosis.

2. GENITAL TRACT
2.1 Anatomy The gonads are situated in the dorsal part of the thoracoabdominal cavity, cranioventrally to the cranial lobes of the kidneys. The testes are paired, but the reproductive tract of the hen consists only of a left ovary and oviduct. The size of the gonads and the oviduct depends highly on sexual maturation and activity. 2.2 Indications for non-invasive diagnostic techniques Clinical signs in birds with disturbances of the genital system include abdominal swelling, depression/ anorexia, dyspnoea, tail wagging, changes in posture, defecation problems and rear limb paresis/ paralysis (JOYNER 1994). Behavioral changes of the patients are also frequently observed. If the hen has already laid eggs, the history often contains obvious indicators for a reproductive disorder (abnormal eggs, chronic egg layer). However, the patient history is only indicative in about half of the cases. In the other cases the patient owners report nonspecic symptoms which are also applicable for other diseases (for example respiratory disease). In cases of egg-binding, palpation proved to be a simple and reliable diagnostic technique for eggs with calcied shells in the caudal abdomen. However, laminated and thinshelled eggs, ectopic eggs, multiple growths as well as cases that are complicated by peritonitis cannot be denitively diagnosed by palpation (BOWLES 2001). In these cases, palpation can even lead to false negative ndings. Therefore the results of the clinical examination should be conned by means of radiology and/ or ultrasound. Indications for sonographic examination of the genital tract consist of any disorder which is more or less connected with abdominal distension; furthermore, cases of

438

radiologically diagnosed soft tissue masses in the urogenital region and/or formation of medullar bones. In males (particularly budgerigars), neoplasia of the testes may lead to emaciation, apathy, dyspnoea, anorexia and signs of feminization (in budgerigars: discoloration of the cere from bluish to brownish). Dyspnoea may be caused by distension of the air sacs, rear limb paresis due to compression of the nervus ischiadicus. 2.3 Radiography A denitive diagnosis of egg-binding is reached with the help of radiography in 30% of the cases. Radiography is helpful in diagnosing shell alterations, multiple and deep intraabdominal situated eggs and is therefore an indispensable tool in choosing a therapy. Unfortunately, it is not possible to differentiate radiographically between eggs without a calcied shell or with a very thin shell and other soft tissue masses (for example tumours) in the thoracoabdominal cavity. Radiographically diagnosed medullar bone and contrast radiographs of the gastro-intestinal tract can, however, be helpful (MC MILLAN 1994). By this means, common differential diagnoses in budgerigars, such as tumours of the liver and kidneys, can be fairly easily ruled out. Nevertheless, changes that occur in conjunction with an increased estrogen level, such as cystic changes of the ovaries and oviduct and gonadomas, cannot be ruled out. Laminated or thin-shelled eggs may be the cause of egg-binding in birds, which have radiographically diagnosed medullar bone. Also in birds with ovarial cysts and gonadal tumours, medullar bones are often seen in radiographic examination. Medullar bone is therefore a helpful nding in radiographs with otherwise unclear ndings. However, it is also found in other disease processes, and no denite conclusion on the existence of a pathological process can be made on this basis (KOSTKA 1992). Gonads In respect of the gonads one must differentiate between pathological enlargement and physiological activity but the clinical ndings give the necessary indications. Budgerigars are particularly prone to gonadal neoplasia which causes massive enlargement of the organ resulting in pressure on the air sacs and ventrocaudal displacement of the gastrointestinal tract (seen with barium-sulphate contrast studies). Alterations in the size and shape of the gonads, as they occur, for instance, in cystic ovaries, are often associated with other radiographic features such as polyostotic hyperostosis and abdominal hernia. Oviduct In some cases of salpingitis the oviducts shadow is enlarged and more dense than normal. In birds with egg retention the egg can often be palpated, provided it is calcied and not situated too deep in the thoracoabdominal cavity. A radiograph is not only necessary to provide information about the form, size, number and position of eggs, it can also help to decide between their surgical and non-surgical removal.

439

The eggs shell may be abnormal in salpingitis. On the radiograph the laminated eggs produce a distinct soft tissue shadow, but there may also be evidence of an abdominal hernia, displacement of the gastro-intestinal tract and an increased density of the long bones. 2.4 Ultrasonography Ultrasonographic examination proved to be a helpful tool in diagnosing genital tract physiology and abnormalities (HOFBAUER and KRAUTWALD-JUNGHANNS 1999). Its advantage is the direct diagnosis of soft tissue structures, as opposed to radiography, in which extensive contrast studies are necessary. It is, for example, possible to recognize developed follicles, laminated and thin-shelled (non-calcied) eggs and neoplasia as well as pathological processes in the oviduct (salpingitis). Laminated eggs are typied by their shape, varying echogenicity as well as, in most cases, a uid perimeter. For the evaluation of the shell quality of intact eggs, however, sonographic examination is only appropriate in combination with radiography, since not all changes are visible. As differential diagnoses neoplasia of other internal organs, especially of the liver and the kidneys must be considered. This differential diagnosis can be ruled out with the help of a contrast study of the gastro-intestinal tract with barium sulfate and/ or a sonographic examination. Cystic degeneration of various organs, especially kidneys and ovaries, should be considered in cases with nonspecic histories. Sonographic examination again allows a clear characterization of cysts. Radiographic and sonographic examination therefore compliments one another in cases of suspected genital disease. Both methods, however, also have their diagnostic limits. 2.4.1 Approaches for ultrasonography of the genital tract These are the same as described for the examination of the kidneys (see 1.4.1). 2.4.2 Examination methods and evaluation criteria General comments: The demonstration of the healthy gonads by transcutaneous sonography is only successful in cases of highly sexually active birds. By transcloacal sonography a sex determination in birds with more than 2 kg BW is possible (HILDEBRANDT et al. 1995) Gonads Testes: The sonographic demonstration of testes is only successful in the case of highly sexually active birds. The parenchyma of this organ shows a delicately granulated structure of average echogenicity. The closely tting testicular serosa occurs as a smooth, hyperechoic string surrounding the parenchyma, although testicular vessels normally cannot be identied. Neoplasia, inammatory processes and other alterations in conjunction with enlargement of the organ are principally sonographically observable. Tumours appear as voluminous and rounded single masses, often with diffuse inhomogenous

440

echotexture. Sometimes, multiple poorly echoic foci mark necrotic areas within these irregular masses. In no case were remnants of unchanged testicular tissue demonstrable. The sonographic complexity of the tumorous tissue allows often a clear demarcation from surrounding structures. But due to the extension, it is not possible to associate the masses denitely with the testes. Ultrasound-guided biopsy for histopathology is possible but difcult (risk of internal bleeding). Histopathologically most neoplasias of the testes are found to be sertoli cell tumours. Ovary: The sonographic demonstration of the ovary is successful in most of the active hens with a bodyweight of more than 70g. A clear differentiation of inactive ovaries, however, was not possible. Evidentially, the quality of sonographic images depends on the phase of development and, in connection with this, on the increase in size. The closer the ovaries were located to the body wall, the better the image of the organ was. Beyond that, in hens it is possible to differentiate follicles or eggs in the oviduct. In sonographic demonstration and interpretation both ovarial parenchyma and follicles of varying degrees of maturity were principally regarded as a morphological unit. The picture of active ovaries is characterised by the presence of follicles in different sizes representing various stages of development. Developing follicles are rst seen as round areas with indistinct, anechoic or hypoechoic inner structure. In advanced stages of development the follicles exhibit the more echoic content of yolk. Changes in the shape of the follicle were visible due to the movement of adjacent intestinal loops. The ovarial parenchyma, only visible to some extent between the follicles, occurred as an area of higher echogenicity. A number of rudimentary follicles within the ovary tissue results in an inhomogenous echotexture. More distally in the oviduct, in the magnum, the ova exhibit a distinct separation in echogenic yolk surrounded by poorly echoic perimeter of albumen. A decentrally positioned round area of minor echogenicity was often visible within the yolk, which gave it a characteristical appearance of a signetring. The hyperechoic shell, added in the uterus, is easily recognizable. However, its reexion and absorption of the sound waves prevents the examination of the inner structures of the completed egg. Ovarian neoplasias are distinctly demonstrable by means of sonography. Accompanied by massive enlargement of the affected organ, the well dened structures appeared as large rounded masses of mixed echogenicity, seen as marked focal or diffuse inhomogenous echotexture. In some cases multiple hypo- to anechoic foci within the masses, representing necrotic or cystic areas intensied the inhomogenous impression. Histopathologically the masses are often found to be adenocarcinomas or granulosa cell tumours. Because of their massive extension, the origin of the tumours cannot be determined. Furthermore, it is not possible to infer the character of the neoplasias. Ovarial cysts may occur as single large cysts or as polycystic changes of the ovary. Ovarial cysts appear sonographically as clearly dened rounded anechoic compartments, showing the phenomenon of distal acoustic enhancement. In polycystic abnormalities this is evident in the parenchyma.

441

Oviduct Irrespective of its functional state, the unchanged oviduct is often sonographically indistinguishable from the surrounding abdominal structures (intestines), essentially because of the similar echogenicity. Pathologic changes of the oviduct are commonly salpingitis. The inammation of the oviduct is frequently accompanied by an increased incidence of laminated eggs. In some cases salpingitis may be found after egg binding surgery and following hormonal therapy. The sonographic image of advanced salpingitis is, particularly in the sectional view, recognizable by increased thickness of the oviduct wall. Depending on the kind of effusion, the inammatory exudates may appear sonographically anechoic to hypoechoic. In pronounced suppurative processes the echogenicity of the content increases. In birds with high grade salpingitis it may be sonographically impossible to make a distinction between the laminated egg and the oviductal wall. Lowgrade oviductal inammation processes are sonographically indistinguishable. Furthermore, cysts of the rudimentary right oviduct may be detected. They show the same sonographic picture as ovarian cysts. Egg binding: Abnormal eggs are detected most frequently in suspected cases of egg binding. Egg binding in association with fully formed, correctly shelled eggs does occur quite often in birds. Because of their high echogenicity, roughness of the egg shells cannot be demonstrated in most cases. Furthermore, thin-shelled or non-calcied eggs, malformed eggs as well as eggs with destroyed shells are sonographically assessable. Laminated eggs are clearly demonstrated sonographically. These are seen as oval to round structures of varying echogenicity, due to different densities of the deposited material, which gives the laminated egg a sonographic appearance of onion layers. Moreover, they are sometimes surrounded by a margin of anechoic to hypoechoic uid.

3 CITATION INDEX
1. 2. 3. 4. 5. LUMEIJ JT. Nephrology. In: RITCHIE BW, HARRISON G and HARRISON L (eds): Avian Medicine Principles and Application. Lake Worth: Wingers Publishing 1994; 538 555. HOFBAUER H, KRAUTWALD-JUNGHANNS ME. Transcutaneous ultrasonography of the avian urogenital tract. Vet Rad Ultrasound 1999; 40: 58 64. MCMILLAN MC. Imaging Techniques. In: RITCHIE BW, HARRISON G and HARRISON L. (eds): Avian Medicine Principles and Application. Lake Worth, Florida: Wingers Publishing 1994; 246 - 326. HOFBAUER H. Beitrag zur transkutanen Ultraschalluntersuchung des aviren Urogenitaltraktes. Giessen: Doctoral thesis, 1996. HILDEBRANDT T, PITRA C, GRITZ F, et al. Transintestinal ultrasonographic sexing. Proc Euro Assoc Avian Vet, Jerusalem 1995: 37 - 41.

442

6.

KOSTKA VM. Rntgendiagnostik des aviren Skelettsystems. Gieen: Doctoral thesis, 1992. 7. KRAUTWALD ME, TELLHELM B, HUMMEL G, et al. Atlas of Radiographic Anatomy and Diagnosis in Cage Birds. Hamburg and Berlin: Paul Parey 1992. 8. NEWELL SM. Radiology of the avian patient Y2K and beyond. Proc Assoc. Avian Vet, Portland 2000: 421 - 424. 9. JOYNER KL. Theriogenology. In: RITCHIE BW. HARRISON G and HARRISON L. (eds): Avian Medicine Principles and Application. Lake Worth: Wingers Publishing 1994: 748-804. 10. BOWLES HL. Diagnosis and management of female avian reproductive diseases. Proc Assoc Avian Vet, Orlando 2001: 349-357.

AUTHORS ADDRESS
M. E. KRAUTWALD-JUNGHANNS, Prof DrMedVet, Dipl ECAMS Clinic for Birds and Reptiles, University of Leipzig, An den Tierkliniken 17, 04103 Leipzig, Germany Email: krautwald@vmf.uni-leipzig.de

443

European Association of Avian Veterinarians Conference POSTERS

Division of Zoo Animals and Exotic Pets, Vetsuisse Faculty, University of Zurich, Switzerland

INFLUENCE OF A SEED DIET VERSUS A FORMULATED DIET ON BODYWEIGHT AND INTESTINAL FLORA IN BUDGERIGARS (MELOPSITTACUS UNDULATUS) OVER A SIX MONTH PERIOD
I. Fischer, Dr. med. vet., P. Keller, Dr. med. vet., J.-M. Hatt, Prof. Dr. med. vet.

KEYWORDS
Diet Bodyweight Intestinal ora Budgerigar

ABSTRACT
Two groups (n=22) of budgerigars (Melopsittacus undulatus) were maintained under identical conditions with the exception of the diet for 6 months. One group was offered a commercial seed mixture plus carrots and a mineral supplement. The other group received a commercial formulated diet for budgerigars plus carrots. On day 0, 15, 20, 35, 90 and 180 days each animal was weighed and faeces were analysed microscopically with Gram stain. The bodyweight of the formulated-diet group dropped after diet-changing to a minimum average of 41.5g (16%) on day 15 compared to day 0. Following an increase of the formulated diet bodyweights increased. Average bodyweight of the seed-diet budgerigars remained constant during the trial. The evaluation of the faecal samples revealed no signicant differences between the both groups with the exception of Macrorhabdus ornithogaster which was found in 7.7% of samples in the seed-diet group versus 36.6% in the formulated-diet group. The results of this study suggest that transition of diet in budgerigars has to be carefully monitored and that the different diets showed no inuence on the intestinal ora with the exception of M. ornithogaster

1 INTRODUCTION
Clinical signs in birds are unspecic and diagnosis has to rely heavily on additional clinical investigative methods. Faecal examinations represent an important diagnostic tool in the clinical evaluation of diseased birds. In healthy granivorous psittacine birds gram negative bacteria, especially E. coli, do not belong to the normal intestinal ora (FIENNES 1959, HARRISON 2003), A recent study supported the assumption that E. coli is not part of the normal intestinal ora of granivorous birds, but showed that the composition of the food might have an inuence on the intestinal ora (GLUNDER 2002).

447

2 MATERIAL AND METHODS

Two groups including 22 randomly selected 2-year-old budgerigars (Melopsittacus undulatus) each were housed under identical conditions for 6 months with the diet being the only difference. One group was offered a commercial seed mixture ad libitum (90 Hammer, SWV English budgerigar-food, Jordi AG.Signau, Switzerland) plus carrots (0.5 g FM/bird/d) and a mineral supplement (Quikon, Firma Quiko, Bocholt, Germany). The other group was changed to a commercial formulated diet (Harrisons Bird Diet Adult Lifetime Fine Grind, Avifood, Grfelng, Germany) (4g FM/bird/d, manufacturer recommendation) plus carrots (0.5 g FM/bird/d). Changing to the formulated diet took place over a period of 14 days. The required amount of the formulated diet was offered with continuous decrease of the seed fraction. On day 0, 15, 20, 35, 90 and 180 days each animal was weighed and on day 90 and 180 faecal material was smeared in a thin layer onto a slide. The faecal smears were stained with Grams solution (Color Gram 2, BioMrieux, France) using the procedure described by the manufacturer. The slides were analysed under 1000x magnication in oil immersion by scanning several optical elds to evaluate the uniformity. In three to ve uniform optical elds the percentage of Gram positive rods, Gram positive cocci and Gram negative rods were recorded and the average was determined. Additionally, several elds were scanned for the presence of yeasts and their percentage of budding, and for Macrorhabdus ornithogaster. Each sample was evaluated by different persons. The formulated diet and the seed diet were analysed for nutrient contents (Tab.1). The contents for crude ash, crude protein, crude fat and crude bre were determined using standard procedures for Weender analysis. The phosphorus concentration was measured with an autoanalyzer (Cobas Mira, Roche, Basel, Switzerland) with a colorimetric method. The calcium concentration was measured by atomic absorption spectrometry (SpectrAA-20, Varian, Zug, Switzerland). Statistical analyses were performed with ANOVA.

3 RESULTS
The average bodyweight of the pellet-diet group dropped after diet-changing to a minimum average of 41.5g (i.e. a decrease of 16%) on day 15 compared to day 0. One bird died on day 16 due to emaciation as conrmed by necropsy. Following an increase of the formulated diet of 50% bodyweights increased by day 35 to 48.1g. At day 90 the average bodyweight (44.9g) dropped again (9.9%) compared to day 0 and subsequently increased again by day 180 (49.7g) without change to the diet. Average bodyweight of the seed-diet budgerigars remained constant during the trial. The evaluation of the faecal samples revealed no signicant differences between both groups, with the exception of Macrorhabdus ornithogaster which was found in 7.7% of samples of seed diet group versus 36.6% in the formulated diet group. Grampositive rods dominated (79.1%) over Gram-positive cocci (14.7%). Gram-negative rods were found in 25.6% of the samples. No yeasts were diagnosed.

448

Table 1. Analysis of the diets fed to budgerigars (Melopsittacus undulatus). Values are expressed on a fresh matter (FM) or dry matter (DM) basis. Unit Water Crude ash Crude protein Crude bre Crude fat Metabolizable Energy Calcium Phosphorus Sodium g/kg FM g/kg Dm g/kg Dm g/kg Dm g/kg Dm MJ/kg Dm g/kg Dm g/kg Dm g/kg Dm Pelleted diet 77.4 36.5 171 21.0 96 19.3 4.1 4.3 0.6 Seed diet (dehulled) 100.6 32.6 171 29.5 75 17.0 0.4 5.0 0.8

4 DISCUSSION
The present study shows that transition of seed fed budgerigars to a formulated diet is possible, but that bodyweights should be carefully monitored during transition and amounts fed to birds may have to be higher during transition than for subsequent maintenance. A possible explanation for the decrease in bodyweight at day 90 in the formulated diet group could not be found. The observation that the formulated diet did not have a signicant effect on intestinal ora differs from another study in 100 African grey parrots (Psittacus erithacus) (STANFORD 2003). Possible reasons may be that the durations of both studies were not comparable or that the different number of animals had an inuence on the interpretation. Additionally it may be that the diets and species used in the study of Stanford (2003) and this study may be too different to allow comparison. A possible reason for the increased occurrence of M. ornithogaster may be the stress of transition to the formulated diet. Furthermore it is not known if the vitamin and mineral-supplement had an effect.

5 CITATION INDEX
1. 2. 3. 4. FIENNES T. Report of the Societys Pathologist for the Year 1957. Proc Zool Soc London 1959; 132: 129 - 146. STANFORD M. Effects of Dietary Change on Fecal Grams Stains in the African Grey Parrot. Exotic DVM, 2003; 4.6: 12 - 13. GLUNDER G. Inuence of diet on the occurence of some bacteria in the Intestinal ora of wild and pet birds. Dtsch Tieraerztl Wochensch 2002; 109: 266 - 270. HARRISON G. Preliminary Field Study of Fecal Grams Stain Results in Two Free-Ranging Australian Parrot Species. Exotic DVM, 2003; 4.6: 10 - 11.

449

AUTHORS ADDRESS
Isabelle Fischer, Dr. med. vet. Division of Zoo Animals and Exotic Pets, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland, Email: ischer@vetclinics.unizh.ch

450

C.E.P.E-CNRS Strasbourg, France

EFFECTS OF FLIPPER BANDS ON FREE-LIVING PENGUINS

M. Gauthier-Clerc, DVM, PhD, J-P. Gendner, C. Le Bohec, Y. Le Maho

KEYWORDS
Penguins - Ticks - Borrelia burgdorfer - Reproduction

ABSTRACT
Changes in seabird populations, and particularly of penguins, offer a unique opportunity for investigating the impact of sheries and climatic variations on marine resources. This often implies large-scale banding to identify individual birds, but the signicance of the data relies on the assumption that there is no bias introduced in this type of long-term monitoring. After ve years using an automated system of identication of king penguins implanted with electronic tags, we can report that banding results in later arrival at the colony for courtship in some years, lower breeding probability and lower chick production. We also found that the survival rate after 2-3 years of unbanded king penguin chicks with tags is about twice as large as that reported in the literature for banded chicks at different colonies or years.

AUTHORS ADDRESS
M. Gauthier-Clerc, DVM, PhD Station Biologique de la Tour du Valat, Le Sambuc, F 13200 Arles, France Email: gauthier-clerc@tourduvalat.org

451

Station Biologique de la Tour du Valat Arles, France

INFESTATION BY TICKS IXODES URIAE IN PENGUIN COLONIES

M. Gauthier-Clerc, DVM, PhD

KEYWORDS
Penguins - Ticks - Borrelia burgdorferi - Reproduction

ABSTRACT
High densities of penguins in their colonies and the continuous use of these sites over consecutive reproductive periods increase the risk of development of tick populations. We have studied the effects of tick parasitism by Ixodes uriae in a colony of king penguins (Aptenodytes patagonicus) at Possession Island during three breeding seasons. We investigated the prevalence and periods of tick infestation during the one-year breeding cycle of penguins. The effects of tick parasitism on penguin breeding performance were assessed from photographs of the colony and with an automatic penguin identication system. We compared two groups of penguins carrying individual subcutaneous electronic tags, one group breeding in an infested area and the other in a non-infested area. Tick feeding activity coincided with the periods when adult penguins stayed ashore for six days or more, i.e. during the incubating period. This duration corresponds to the duration of a tick meal on the host. The level of infestation varied between years. Penguins showed a lower incubating success in infested areas during a year of high infestation. In an infested area, individuals seen with ticks had a lower breeding success in rearing a oneyear old chick than those seen without ticks. We compared the body mass, the haematocrit level and the behaviour during incubation according to tick infestation. Tick infestation had no effect on the body mass, the haematocrit or the behaviour of incubating penguins. Contrary to what might have been expected, birds breeding in the infested area did not show a lower delity to that area the next breeding season in comparison to birds breeding in the non-infested area. The survival was not different over the 32 following months between both groups. Borrelia burgdorferi antibodies were detected in 14% of tick-infested adults and in 6% of chicks sera.

AUTHORS ADDRESS

M. Gauthier-Clerc, DVM, PhD Station Biologique de la Tour du Valat, Le Sambuc, F 13200 Arles, France Email: gauthier-clerc@tourduvalat.org.

452

Scanelis,Toulouse, France

REAL-TIME PCR FOR DIAGNOSIS OF THREE COMMON INFECTIOUS DISEASES IN CAGED BIRDS : CHLAMYDIOPHILOSIS, BEAK AND FEATHER DISEASE AND AVIAN POLYOMAVIROSIS
S. Lafon, MS

KEYWORDS
Real-time PCR - Infectious diseases - Caged birds - Pathogen quantication

ABSTRACT
Chlamydophila psittaci, beak and feather disease virus (BFDV) and avian polyomavirus (APV) are common pathogens in psittacine birds. Diagnosis testing can be used to conrm or inrm a clinical suspicion and it makes possible to the veterinarian to prescribe a suitable treatment to the bird. Furthermore, as these diseases are very contagious, asymptomatic carriers have to be identied to prevent dissemination and economic losses in facilities and to reduce the zoonotic risk with Chlamydophila. The Polymerase Chain Reaction (PCR) is an effective method for the detection of these three different pathogens. Whereas these assays can be sensitive and specic, the provided qualitative result is often inconclusive: what is the meaning of the presence of the detected agent? For example, is it an acute or a subclinical infection? Real-Time PCR is an advanced PCR-based technology which enables to assess the quantity of one specic agent in various biological samples. Here, we describe this technique and, through some clinical examples, the interest to perform Real-Time PCR analyzes in birds veterinary medicine is shown. Direct diagnosis with a quantitative approach produces a conclusive result when others assays may sometimes not provide any solution for avian practitioners.

1 INTRODUCTION
Although clinical signs in affected birds can lead to a presumptive diagnosis, denitive diagnosis often involves demonstration of virus presence. Nucleic acid detection of a pathogen, as PCR, provides helpful results either for the approach of a clinical case or for the detection of asymptomatic carriers. A quantitative tool can make interpretation easier. Some real-time PCR assays for caged-birds infections and their interest are described bellow.

453

2 MATERIAL AND METHODS

DNA extraction: DNA was extracted from each eld sample with High Pure PCR Template Purication Kit (Roche). EDTA blood samples and feather calamus have been screened for BFDV and APV and cloacal swabs for C. psittaci. PCR Internal Control: a synthetic DNA was added in each sample at the rst step of the DNA extraction. The extraction yield and the absence of PCR inhibitors were checked with a specic real-time PCR. BFDV conventional PCR: a specic assay was used to amplify a fragment of BFDV genome. PCR products run in 1.5 % agarose gel. Realtime PCR: the TaqMan system, an improved PCR-based technology, was used on an ABI7900 SDS (Applied Biosystems). Conditions: 2X UDG Platinum Quantitative PCR (Invitrogen), a specic probe (100 nM) and primers (600 nM) for 60 cycles. The limit of detection of PCR was assessed on quantied templates. Specicity was checked on various pathogens.

3 RESULTS
3. 1 Whereas conventional PCR can not provide quantitative data, real-time PCR does! Real-time PCR in TaqMan system is a PCR with internal probes for the quantication of PCR products. The Ct (Threshold cycle) value is the number of cycles necessary to reach a dened uorescence threshold. The more the initial amount is high, the lower the Ct value is. Amplication curves show that very different initial pathogen loads lead to equivalent amounts of PCR products after 60 PCR cycles. The electrophoresis result conrms this nding: end-point quantication is not accurate.

3. 2 Interest of sensitivity and pathogen load in veterinary medicine: examples 3. 2. 1 A higher sensitivity for a best detection of APV healthy carriers As only 10 copies of APV genome lead to a positive result with our assay, the detection of asymptomatic but infected birds is improved. Also any trace of virus can be detected, allowing prevention of virus dissemination in the breeding. 3. 2. 2 A tool to interpret chlamydophilosis testing results The bacterial load can be helpful to assess the role of the detected Chlamydophila in the clinical signs observed. 3. 2. 3 Viral load monitoring for BFDV: transient viraemia or disease progress? For one bird, the relative quantication of the viral load at two different times provides very interesting information about disease evolution and/or the efciency of any treatment.

454

4 DISCUSSION
There are many applications of the real-time PCR in the veterinary eld. Compared to conventional PCR, this technology increases sensitivity and allows quantication, both improving the quality of the result. The increase of specicity and the end of carry-over problems are two other major sides which bring false-positive results with conventional PCR to an end.

AUTHORS ADRESS
S. Lafon, MS, Research and Development Scanelis - National Veterinary School of Toulouse - 23, chemin des Capelles, 31076 Toulouse Cedex 3, France Email: soph.lafon@scanelis.com

455

Wildlife Centre, Ecole Nationale Vtrinaire Nantes, France

VANADIUM CONTENT OF OILED BIRDS : IS IT A GOOD MARKER FOR OIL EXPOSURE ?

S. Le Dran-Quenechdu. DMV, PhD, M. Kammerer. DMV, PhD, H. Pouliquen DMV, PhD, ECPVT Diplomate, M. Larhantec

KEYWORDS
Oil spill - Ecotoxicology - Seabirds

ABSTRACT
Vanadium is a trace element, contaminating oil products. It also has a specic toxicity (cellular and DNA toxicity). In this poster, we present a study about the vanadium content of organs of oiled birds since the Erika oil spill in France. The aim is to study the differences between species and between the types of oil (with different vanadium value). Vanadium was measured by atomic absorption spectrophotometry in liver and kidney of several oiled seabird species, stranded on French Atlantic and Channel coasts that had died in wildlife care Centres, during the winters of 19992003. The concentrations were not higher in oiled birds than in dead birds found on the beaches without visible traces of petroleum. However, we showed that there were differences between species, probably in relation to their diet. Moreover, there appears to be differences between origins of birds (Atlantic vrs Channel) but not between types of oil. We suggest that vanadium may be not a good marker of acute oiling but may be better to mark a chronic toxicity.

1 INTRODUCTION
Seabirds that have been victims of accidental release or operational discharges from ships suffer primarily from the physical action of petroleum. The toxicity of petroleum is related to its composition, not only hydrocarbons but also metals such as nickel, aluminium or vanadium. A certain amount of data is beginning to be accumulated concerning its presence in marine organisms (MIRAMAND and FOWLER 1998), but its metabolism in other animal species is not well known. The aims of this study are to compare the vanadium content of oiled birds form different species (with different diet) and form different origin (different hydrocarbons).

456

2 MATERIAL AND METHODS

The analysed birds died naturally in wildlife centres during the Erika oil spill, 20002001, 2001-2002 (chronic pollution), the Prestige and Tricolor oil spills. Whole kidney and liver, without gall-bladder, were taken and separately stored at 20C in polyethylene asks. Moreover, liver and kidney were also collected on seven other Allcidae found dead without visible traces of petroleum, in shing-nets in the Channel, during summer 2001. These birds were used as controls. Vanadium concentrations were determined by using an atomic absorption spectrophotometer 1 100 B (Perkin-Elmer, Norwalk, CT, USA). The procedure has been described with details in KAMMERER et al. (2004).

3 RESULTS
The vanadium content of livers according to species varied between 26ng/g and 131ng/g. There were signicant differences between species (t guillemot vrs kittiwake = -3,092, p<0.001). There were also signicant differences between types of hydrocarbons (F=4.247, p=0.006). The vanadium contents were signicantly higher for birds that died during winter 2002-2003 than during previous winter. However, the variability of data was also higher. Moreover, the birds coming from Channel showed higher vanadium content than birds coming from Atlantic sea, even control birds that were not oiled. The relation between vanadium content of oil sampled on feather and of livers was not signicant but the sample is low (r2=0.1374, p>0.05, n= 8).

4 DISCUSSION
The values for concentrations of vanadium in the liver of birds found in the literature vary from 18 to 38 ng.g-1 (fresh weight) for poultry receiving a food with a vanadium content of 0.1 in 3 ppm (PULS 1994) and from 100 to 900 ng.g-1 for waterbirds living in polluted zones. Our results are widely superior to the values for not exposed poultry but the lack of reference value makes difcult the interpretation. Furthermore control birds showed vanadium contents higher than oiled birds (except Tricolor birds). The hypothesis we made was that the birds wintering in Channels wintered in polluted zones . The vanadium resumption, and thus the possible physiological and toxicological consequences seem however limited enough for oiled seabirds. The data showed an important individual variability, within the same sort. However, it is possible that this variability comes from the variability of exposure of birds. Our study does not allow the assertion that the vanadium represents a good biomarker of the pollution by oil to the birds. It would be interesting to complete it by other analyses for various types of hydrocarbon with content vanadium known and by dosages, in parallel with some hydrocarbons of feathers, in the skeleton. Indeed the bone could be a better marker of the exposure.

457

5 CITATION INDEX
1. 2. MASTAIN O, LE DRAN-QUENECHDU S, POULIQUEN H and LARHANTEC M. Liver and kidney concentrations of vanadium in oiled seabirds. The Sci Total Environ 2004; 333: 295 - 301. MIRAMAND P and FOWLER SW. Bioaccumulation and transfer of vanadium in marine organisms. In: NRIAGU (ed): Vanadium in the environment, Part 1: Chemistry and biochemistry. New York: John Wiley and Sons Inc, 1998; 167 - 197. PULS R. Mineral levels in animal health 2nd ed, Sherpa International, Clearbook 1994, Canada.

3.

AUTHORS ADRESS
Le Dran-Quenechdu S, DVM, PhD Wildlife Centre, Ecole Nationale Vtrinaire, Atlanple-Chantrerie, BP 40706, 44307 Nantes cedex 03, France Email: sldq@club-internet.fr

458

Wildlife Centre, Ecole Nationale Vtrinaire Atlanple-Chantrerie,BP 40706, 44307 Nantes cedex 03, France

CAUSE OF MORTALITY OF WILD BIRDS COLLECTED IN A WILDLIFE CENTER IN FRANCE : EXAMPLE OF NANTES WILDLIFE CENTER
Le Dran-Quenechdu S. DVM, PhD, Risi E. DVM, Lambert O.

KEYWORDS
Wild birds - Raptors - Seabirds - Toxicology - Infectious disease - Trauma

ABSTRACT
More than 1000 wild birds are collected each year in the wildlife center of the National Veterinary School of Nantes (France). Most of them are raptor and sea birds. The causes of the collect will be analyzed according to season and to species. The release rate will be also shown.

1 INTRODUCTION
The increasing human activity leads numerous modications of the environment and assaults the balance of the ecosystems. Every day, numerous animals of the wild fauna are victims of poisoning, accidents (road, hunting, electrocution, plate glass windows) and of diseyrie in breeding period. Since 1985, the wildlife centre of the National Veterinarian School of Nantes welcomes these injured animals, which alone could not survive. All these animals are examined and looked after with for objective their insertion or reintegration in their environment, in the respect for charter of the French Union of Wildlife Centres (UFCS). In Nantes, the animals are mainly birds (94% in 2003).

2 CAUSES OF CARE
Hundred and twenty sorts were so taken in among which 106 sorts of birds. The most frequent sorts are the sea birds (guillemots, gulls, mallards), birds of prey (common buzzard, common kestrel, barn owl). The causes of entrance vary according to the sorts. The collisions with vehicles or electric lines are the most frequent causes of entrance for birds of prey (58 barn-owls entry for collision with a car in 2003). The sea birds as the guillemots are welcomed because of oil. The waterbirds are

459

essentially welcomed because of illness (botulism). The causes of entrance also vary according to the season: oiling is more frequent in winter (because reach the wintering populations); in spring, the centre welcomes mainly young birds fallen from the nest.

3 RELEASE RATE
According to the state of the animal, several decisions can be taken. If the animal cannot be taken care on the centre (for example seal), it will be transferred in another centre, adapted to its species. If the animal presents too grave wounds after which we cannot look, it is euthanised. If we think that in spite of the care, the animal cannot be released in its natural environment (example of a bird of prey which we should amputate of a wing or which would have become blind), it is also euthanised: This obligation to look after animals savage in the objective to release them in the wild is registered in the rule and in the charter of the French Union of the Wildlife Centres. If the veterinarian thinks that the animal has chances to be able to be released in the wild after the care, it is taken care: he can then benet from medical care and / or surgical. It is hospitalised in rooms adapted to its species. Then it benets from a phase of rehabilitation / rehabilitation (for example the sea birds are put in swimming pool to re-learn to live on the water, to dive). Finally, it is released after having been ringed.

In 2003, 13% of the animals were euthanised, 46% died in care and 41% were released. The release rate is very variable according to the species and according to the causes of entrance. For example, in 2003, more than 85% of the little owls were released. On the other hand, only 12% of the barn owls were released.

AUTHORS ADRESS
Le Dran-Quenechdu S, DVM, PhD Wildlife Centre, Ecole Nationale Vtrinaire, Atlanple-Chantrerie, BP 40706, 44307 Nantes cedex 03 France Email: sldq@club-internet.fr

460

Department of Pathology, Bacteriology and Avian Diseases1, Department of Medical Imaging2, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

COMPUTED TOMOGRAPHIC ANATOMY OF THE PIGEON (Columba livia)

A. Martel1, A. Van Caelenberg2, I. Gielen2, L. De Rycke2, F. Pasmans1, H. van Bree2

KEYWORDS
Pigeon Anatomy Computed tomography

ABSTRACT
Routine radiography is still the most frequently performed imaging modality in avian patients, but the use of computed tomography (CT) technology is increasing, as this technique avoids superposition of overlying structures and provides a better soft tissue differentiation than classical radiology. The present study describes the CT anatomy of the head, thorax and abdomen of the pigeon, compared to anatomical sections of frozen pigeons.

1 INTRODUCTION
Routine radiography is still the most frequently performed imaging modality, but the use of computed tomography (CT) technology is increasing, as this technique avoids superposition of overlying structures. Several reports have already described the use of CT in psittacines, raptors and anseriformes (BARTELS et al. 2000, GUMPENBERGER and HENNIGER 2001, KRAUTWALD-JUNGHANNS et al. 1993, 1998a, 1998b; OROSZ and TOAL 1992). No references of tomographic anatomy for pigeons are currently available. The present study describes the CT anatomy of the head, thorax and abdomen of the pigeon (Columba livia), compared with anatomical sections of frozen pigeons.

2 MATERIALS AND METHODS

Two adult male and female pigeons were used in this study. The birds appeared healthy at clinical examination on the day of the study. To avoid movement and stress during CT examination, the birds were anaesthetized. General anaesthesia was

461

induced and maintained with isourane and oxygen by face mask. CT examinations were done using a CT Pace scanner (General Electrics) with the birds positioned in ventral recumbency. Transverse and sagittal sections were made. Twenty-six contiguous 2 mm thick transverse CT-slices from the head and heart region, and 33 contiguous 5 mm thick transverse scans beginning from the thoracic inlet and proceeding to the cloaca, were obtained without overlap. Eighteen 5 mm thick sagittal slices were taken. A 3-second scan time was used. CT images were made using 100 mA and 120 kV. The image acquisition time was 15 minutes. Afterwards, the euthanised pigeons were frozen at -20C and sectioned into approximately 10 mm thick slab sections using an electric planner. Anatomical sections were photographed and compared to the corresponding CT images.

3 RESULTS AND DISCUSSION

The results of the study will be shown in sequential matched photographs of the anatomical sections with their corresponding CT images (see poster). CT images provided good anatomical detail of the internal structures. Transversal CT images may be unable to differentiate between soft tissue organs in close proximity, such as the gonads and the cranial pole of the kidneys. However, this differentiation is visible on the sagittal scans. Effects of breathing motion are seen on occasional transverse images, but this is not a signicant problem. Contrast enhancement of the soft tissues during total body CT would help to identify and differentiate various organs. For example to distinguish the intestinal tract from other viscera, barium sulphate can be used. Despite these small limitations, CT is a useful tool in the study of avian anatomy.

4 CITATION INDEX
1. BARTELS T, KRAUTWALD-JUNGHANNS ME, PORTMANN S, et al. The use of conventional radiography and computer-assisted tomography as instruments for demonstration of gross pathological lesions in the cranium and cerebrum in the crested breed of the domestic duck (Anas platyrhynchos forma domestica). Avian Pathol 2000; 29: 101 - 108. GUMPENBERGER M. and HENNINGER W. The use of computed tomography in avian and reptile medicine. Sem Avian Exotic Pet Med 2001; 10: 174 - 180. HATCHCOCK JT and STICKLE RL. Principles and concepts of computed tomography. Veterinary Clinics of North America: Small Animal Practice 1993; 23: 399 - 414. KRAUTWALD-JUNHANNS ME, SCHUHMACHER F and TELLEHLM B. Evaluation of the lower respiratory tract in psittacines using radiology and computed tomography. Vet Radiol Ultrasound 1993; 34: 382 - 390. KRAUTWALD-JUNGHANNS ME, KOSTKA VM and DRSCH B. Comparative studies on the diagnostic value of conventional radiography and computed tomography in evaluating the heads of psittacine and raptorial birds. J Avian Med Surg 1998a; 12: 149 - 157.

2. 3. 4. 5.

462

6.

7.

KRAUTWALD-JUNGHANNS ME, SCHUHMACHER F and SOHN HG. Untersuchungen am unteren respirationstrakt von Psittacinae- und Amasonae- Arten mit hilfe der rekonstruktiven rntgentransmissionstomogr aphie. Tierrztl Prax 1998b; 26: 61 - 70. OROSZ SE and TOAL RL. Tomographic anatomy of the golden eagle (Aquila chrysaetos). J Zoo Wildlife Med 1992; 23: 39 - 46.

AUTHORS ADDRESS
A. Martel, DVM, PhD Clinic for Avian and Exotic Animal Diseases, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium Email: An.Martel@ugent.be

463

Wildlife Center National Veterinary School of Nantes, France

DETERMINATION OF THE POSSIBLE EXPOSURE OF RAPTORS AND WATER BIRDS TO FIVE ANTICOAGULANT RODENTICIDES IN LOIRE ATLANTIQUE (FRANCE 44)
H. Pouliquen, M. Larhantec, O. Lambert, C. Thorin, M. LHostis

KEYWORDS
Raptors Water birds - Exposure Anticoagulant rodenticides France

ABSTRACT
An epidemiological survey was performed in Loire Atlantique (France) on raptors and water birds which did not present with clinical signs or lesions of poisoning. The aim of this study was to evaluate possible exposure of these non-target wildlife species to anticoagulant rodenticides used against rodents. The exposure of raptors was large and signicantly greater than the exposure of water birds, principally due to their feeding habits. Among the 5 anticoagulants tested, bromadiolone was the most frequent detected or quantied in the liver of birds, reecting its wide use in Loire Atlantique against coypus.

1 INTRODUCTION
Many anticoagulant rodenticides are used in eld-treatments in France. Despite the fact that they are used against rodents, they might have a negative impact on non-target species, either directly by eating contaminated baits, carrots, apples or cereals, or secondary by eating contaminated preys (SHORE et al. 1996; BERNY et al. 1997). The aim of this study was to evaluate the exposure of on target species, such as raptors and water birds, to ve anticoagulant rodenticides (brodifacoum, bromadiolone, coumafen, coumatetralyl and difenacoum).

2 MATERIALS AND METHODS

Thirty raptors and 28 water birds from Loire Atlantique (France) collected in the wildlife centre of the national veterinary school of Nantes were included in this study : 15 diurnal raptors [4 kestrel falcons (Falco tinnunculus), 11 common buzzards (Buteo

464

buteo)], 15 nocturnal raptors [10 barn-owls (Tyto alba), 5 tawny owls (Strix aluco)] and 28 water birds [15 mallards (Anas platyrhynchos), 13 black coots (Fulica atra) and 1 common moorhen (Gallinula chloropus)]. The liver of each dead bird was sampled and kept at -18C until analyses. Identication and quantication of brodifacoum, bromadiolone, coumafen, coumatetralyl and difenacoum in liver were completed at the Toxicology Laboratory of the National Veterinary School in Nantes.

3 RESULTS
Anticoagulant rodenticides were detected or quantied in 22 raptors (73%) and 4 water birds (14%). For all birds, the concentration in liver was low (<1 g/g) except for one common buzzard (coumafene liver concentration = 2 g/g) and one black coot (coumafene liver concentration = 23.52 g/g). Bromadiolone was detected or quantied in 15 birds, difenacoum in 8 birds, coumafene in 5 birds, brodifacoum in 4 birds and coumatetralyl in only one bird.

4 DISCUSSION
Our results show that the raptors exposure to anticoagulants rodenticides is large and similar ndings were reported by STONE et al. (1999, 2003). Furthermore, exposure of raptors is signicantly greater than exposure of water birds, probably because of their feeding habits (MERSON et al 1984). The anticoagulant most frequently detected or quantied in liver was bromadiolone, due to its regular and common used in wetlands from Loire Atlantique in the form of contaminated carrots or apples baits against coypus (Myocastor coypus).

5 CITATION INDEX
1. BERNY P, BURONFOSSE T, BURONFOSSE F, LAMARQUE F and LORGUE G. Field evidence of secondary poisoning of foxes (Vulpes vulpes) and buzzards (Buteo buteo) by bromadiolone, a 4-year survey. Chemosphere 1997; 35: 1817 - 1828. MERSON MH, BYERS RE and KAUKEINEN DE. Residues of the rodenticide brodifacoum in voles and raptors after orchard treatment. J Wildl Manage 1984; 48: 212 - 216. SHORE RF, BIRKS JDS, FREESTONE P and KITCHENER AC. Secondgeneration rodenticides and polecats (Mustela putorius) in Britain. Envir Pollu 1996; 91: 279 - 282. STONE WB, OKONIEWSKI JC and STEDELIN JR. Poisoning of wildlife with anticoagulant rodenticides in New York. J Wild Dis 1999; 187 - 193. STONE WB, OKONIEWSKI JC and STEDELIN JR. Anticoagulant rodenticides and raptors: recent ndings from New York, 1998-2001. Bull Environ Contam Toxicol 2003; 70: 34-40.

2. 3. 4. 5.

465

AUTHORS ADDRESS
H. Pouliquen Laboratoire de Diagnostic Toxicologique, Ecole Nationale Vtrinaire de Nantes, Atlanpole - La Chantrerie, 44307 Nantes Cedex 03, France Email: emmanuel.risi@wanadoo.fr

466

Aspen Wing Bird and Animal Hospital, Loveland, Colorado, United States of America

USE OF A COMPOSITE SILICONE DENTAL IMPRESSION MATERIAL TO CREATE A FORM-FITTING, FLEXIBLE, SUPPORT CUSHION TO FACILITATE WOUND HEALING IN BUMBLEFOOT
J. D. Remple, DVM, Dipl ECAMS C. Nurse

KEYWORDS
Birds Falcons Bumblefoot Metatarsal pad Digital pads - Pododermatitis Foot cast Cushion Silicone impression material

INTRODUCTION
Bumblefoot (pododermatitis) is a chronic, degenerative, granulomatous disease of the avian foot. Large, heavy, non-perching birds such as falcons, penguins and swans are particularly susceptible (HUMPHREYS 1996). Bumblefoot has been recognized in falcons for centuries, and most information on the disease emanates from that avian group. Bumblefoot occurs nearly exclusively in birds held in captivity, and lack of exercise, poor perching surfaces and increased body weight are factors in development (HARCOURT-BROWN 1996). The disease usually begins by repeated trauma and excessive pressure to plantar pedal skin resulting in devitalization of the epithelial barrier to infection with microbial entry into underlying tissues (REMPLE 1993). A foot cast is designed to relieve pressure (a major factor in the initiation of bumblefoot and an obstacle to healing) on prominent weight-bearing pads and redistribute body weight and pressure onto other plantar pedal surfaces. Owing to the pressure relief on lesion areas, foot casting has shown to assist healing and minimize recurrence of bumblefoot following surgical debridement (REMPLE and REMPLE 1987, REMPLE 1993, RIDDLE and HOOLIHAN 1993, REIDARSON ET AL 1999). Foot casting has also proven to be of value in preventing bumblefoot from developing in a healthy foot as a result of prolonged weight-shifting due to injury or disease in the contralateral limb (HARCOURT-BROWN 1996). This report describes the construction and application of a soft, exible, rubber foot cast for birds, made from a composite silicone dental impression material that has been modied for veterinary use as an equine sole support for acute laminitis (K B Cushion SupportR, Kentucky Blacksmith Farrier and Veterinary Supply, Inc., Indianapolis, IN).

467

MATERIALS AND METHODS

Step 1: Upon completion of the surgical procedure, the incision is overlaid with a thin, non-adhering dressing (TelfaR, Colgate-Palmolive). The metatarsus is lightly bandaged with a non-adhesive elastic wrap (VetrapR, 3M Animal Care Products), and compressed to the contours of the foot and proximal digits. A small piece of adherent padding (Dr Scholls MolefoamR, Schering-Plough) measuring the length of the incision X 5 mm wide is laid over the incision area and pressed onto the outside of the vetrap bandage. The padding, which is later removed from the cast, acts to create a dead space between incision and form-tting cast. Step 2: An assistant holds the bird and places the bandaged foot, plantar side down, in a normal standing position on top of a sheet of paper, and a rough outline of the foot is traced onto the paper. The tracing extends to the ends of each digit. Step 3: Equal parts (red and white) of the silicone composite impression material (KB Cushion SupportR) are quickly mixed in the palm of the hand to a uniform pink colour. The mixed composite is formed into a cylindrical shape, approximately the diameter of a piece of chalk, and placed onto the foot tracing. With the bird sedated, the assistant again holds the leg in a normal standing position, and presses the foot and toes into the silicone composite. At normal room temperature the composite has a moulding time of about 2 minutes and a setting time of 4 minutes. The foot is held motionless, while the cast maker works the composite into every nook and cranny along the sides of each toe and the metatarsus. When the composite has set (~4 minutes), the bird is lifted out of the foot cast, and the padding incision spacer is removed from the foot or cast (whatever it has adhered to) and discarded. Step 4: The ends of the cast toes are trimmed off just distal to the middle digital pads of digits 3 and 4 and approximately 2/3rds the distance along digits 1 and 2. Trimming at these locations allows for unhampered toe exion while the cast is being worn. Next, the exterior of the cast is trimmed with a #11 BP scalpel blade to form a neatly tting half-shoe. The cast is afxed to the foot with vetrap that encircles the toes and cast.

RESULTS AND DISCUSSION

The casting method described above is an updated version of the one previously developed at the Dubai Falcon Hospital (DFH); there silicone composite was placed into preformed moulds of falcons feet. Hundreds of silicone foot casts have been used at DFH over the past decade with excellent results. Casts were well tolerated by birds because they were comfortable and they allowed full use of feet while being worn. On several occasions, birds were prematurely released to their owners for hunting purposes with foot casts still on. These falcons all returned for cast removal weeks later after successful hunting with the foot casts still intact! Previous reports have described various rigid casts (REMPLE and REMPLE 1987, REMPLE 1993, RIDDLE and HOOLIHAN 1993). Although rigid casts relieve pressure on the metatarsal pad, they can cause discomfort, impede pedal function and/or transfer

468

foot pressure to hard cast areas with new bumblefoot lesions ensuing as a result. The silicone foot cast described above is the latest advancement in an evolution of foot casting, and it addresses all the concerns of rigid foot casts. The nished halfshoe forms a exible, mould of the foot that cushions and supports the entire foot while selectively relieving pressure from lesion areas.

CITATION INDEX
1. 3. 5. 7. 9. HARCOURT-BROWN NH. Foot and leg problems. In: BEYNON PH, FORBES NA and HARCOURT-BROWN NH (eds): Manual of Raptors, Pigeons and Waterfowl. Glouchestershire: BSAVA 1996; 163 - 167.2. HUMPHREYS PN. Wing and leg problems. In: BEYNON PH, FORBES NA and HARCOURT-BROWN NH (eds): Manual of Raptors, Pigeons and Waterfowl. Glouchestershire: BSAVA 1996; 313 - 314.4. REIDARSON TH, MCBAIN J, and BURCH L. A novel approach to the treatment of bumblefoot in penguins. J Avian Med Surg 1999; 13(2): 124 - 127.6. REMPLE JD and REMPLE CJ. Foot casting as adjunctive therapy to surgical management of bumblefoot in raptorial species. J Amer An Hospital Assoc 1987; 23: 633 - 639.8. REMPLE JD. Raptor bumblefoot: a new treatment technique. In: REDIG PT, COOPER JE, REMPLE JD and HUNTER DB (eds): Raptor Biomedicine. Minneapolis: University of Minnesota Press 1993; 154 160.10. RIDDLE KE and HOOLIHAN J. A form-tting, composite-casting method for avian appendages. In: REDIG PT, COOPER JE, REMPLE JD and HUNTER DB (eds): Raptor Biomedicine. Minneapolis: University of Minnesota Press 1993; 161 - 164.

11.

AUTHORS ADDRESS
J D Remple DVM, Dip ECAMS Aspen Wing Bird and Animal Hospital, Loveland, Colorado, United States of America Email: jdremple@earthlink.net

469

Wildlife Centre National Veterinary School of Nantes, France

CONGENITAL ABNORMALITIES ON A WILD YOUNG LONG-EARED OWL (Asio otus)

Risi E, DVM, Costiou P, DVM, PhD, LHostis M DVM, PhD

KEYWORDS
Long-eared owl - Asio otus Malformation Abnormalities - Congenital

ABSTRACT
A wild young long-eared Owl (Asio otus) was brought to the wildlife centre of the National Veterinary School of Nantes. The bird was a few weeks old, in a very poor condition, showed ataxia and died during the rst night. Several anatomical malformations were observed including 6 digits on each foot, 2 radius on the left wing, 2 radial carpal bones, 2 alular digists on the wings and 2 incomplete supernumerary tarsometatarsi on each leg. The poster presents the clinical aspect of the bird, radiographs and anatomical preparations to preserve the bones and muscles.

1 INTRODUCTION
In June 2002, a long-eared owl chick (Asio otus) was presented to the wildlife centre of the National Veterinary School of Nantes (France). The bird had been found in the wild, at the bottom of a tree, probably just under its nest.

2 CASE REPORT
The young owl was probably 2 to 4 weeks old, it was ataxic and unable to walk. It could only move by leaning its wings on the oor. The bird was in a poor condition and very weak. Physical examination showed skeletal malformations. The bird had 6 toes on each foot, and each wing bore 2 alular digits. Three bones were palpated on the left wing. A radiograph showed the presence of 2 radius on the left wing associated with a marked bowing of the radius and ulna on the left side and a slight bowing of the same bones on the right side, 2 radial carpal bones on both wings, 2 alular digits on both wings, 2 supernumerary metatarsal bones and 2 supernumerary toes

470

around the hind toe (digit I) of both feet. At necropsy, no internal malformations were observed. The bird died on the rst night after admission and the body was preserved in alcohol for anatomical research.

3 DISCUSSION
Anatomical abnormalities are not rare in farm breeding birds (poultry). Few cases have been documented in raptors and pet exotic birds. Wild birds with anatomical malformations are very rarely observed, because they certainly died in the wild before being found. The survival of this long-eared owl had probably been allowed by the parents raising up to the point of leaving the nest. Polydactylism is the most common malformation described in birds of prey and reports describe generally digit I duplications (COOPER 1984, CROSTA et al. 2002, BARREIRO et al. 2003) and less frequently an additional digit distant from the foot (FOX 1989). Up to 9 digits (in a hawk) have been described in raptors (CROSTA et al. 2002). Extra alular digits are a less frequent anomaly. One case has been reported in a goshawk (Accipiter gentilis) and a peregrine falcon (Falco peregrinus) (COOPER 1984) with a shortened carpometacarpus and an extra alula on one side only and one case in a common snipe (Gallinago gallinago) with bilateral supernumerary halluces (CROSTA et al. 2002). Absence of alular digits has been described in a wild tawny owl chick (Strix aluco) (BARREIRO et al. 2003). The case described in this poster seems to be the rst description of duplicated radius on a bird. On the left wing, 2 bowed radius could be observed and one of them was clearly enlarged. Radius anomalies are less frequently reported than those of the digits. One case of congenital dislocation of radius and carpometacarpus was described in a wild tawny owl chick (BARREIRO et al. 2003). A peregrine falcon (COOPER 1984) was found with bowing of one radius associated with carpometacarpal anomalies. In these 2 cases, the number of radial bones was normal. One case of incomplete duplicated radius was reported in a black vulture (Coragyps atratus) with an ectopic wing hanging from its cervical region (OSOFSKY et al 1990). The antebrachium of this third wing consisted of 2 bones, a radius and ulna, each of which was duplicated beginning at mid shaft (OSOFSKY 1990). Chemical and physical factors are known to be teratogenic in vertebrates including 6-amino nicotinic acidamide, retinoids (CROSTA et al. 2002), polychlorinated biphenyls (FERNIE et al. 2003), insulin (COOPER 1984), elevated temperature during early embryonic development (CROSTA et al. 2002). Unfortunately, no toxicological investigations were performed in our case. The aetiology of these anomalies remains unclear but hazardous material related genetic mutation can not be excluded.

4 CONCLUSIONS
This case is of interest for several reasons: - The large number of skeletal anomalies described (4 alular digits, 4 radial carpal bones, 3 radius, 2 bowed ulna, 6 metatarsal bones, 12 toes). - To our knowledge, this is the rst documented report in a long-eared owl. - To our knowledge, this case is the rst documented report of radius duplication. - The bird was found in the wild and did not come from a breeding centre (rarely found, low level of inbreeding).

471

5 CITATION INDEX
BARREIRO A et al. Congenital skeletal abnormalities in a tawny owl chick (Strix aluco). Avian Dis 2003; 47: 774 - 776. COOPER JE. Developmental abnormalities in two British falcons (Falco spp). Avian Path 1984; 13: 639 - 645. CROSTA L et al. Unilateral pentadactylism in a yellow-shouldered Amazon (Amazona barbadensis). J Avian Med Surg 2002; 16(1): 26 - 30. FERNIE K et al. Reproductive abnormalities, teratogenicity, and developmental problems in American kestrels (Falco sparverius) exposed to polychlorinates biphenyls. J Toxicol Environ Health A 2003; 66(22): 2089 - 2103. FOX NC. A unilateral extra digit in a wild commom buzzard (Buteo buteo). Avian Path 1989; 18: 193 - 196. OSOFSKY SA et al. An ectopic wing in a wild black vulture (Coragyps atratus). Avian Dis 1990; 34: 765 - 769.

AUTHORS ADRESS
Emmanuel Risi Centre Vtrinaire de la Faune Sauvage, Ecole Nationale Vtrinaire de Nantes, Atlanpole, La Chantrerie, BP 40706, 44307 Nantes cedex 03, France Email: emmanuel.risi@wanadoo.fr

472

Wildlife Centre National Veterinary School of Nantes, FRANCE

SUCCESSFUL TREATMENT OF A CERVICAL RHABDOMYOSARCOMA ON AN INDIAN FANTAIL PIGEON (COLUMBA LIVIA) BY SURGICAL RESECTION
Risi E, NGuyen F, Albaric O, Abadie J

KEYWORDS
Bird - Pigeon, - Rhabdomyosarcoma - Surgery, - Neoplasm

ABSTRACT
A domestic Indian fantail pigeon (Columba livia) was presented with a large ulcerated mass on the cervical region. After clinical, endoscopical and radiological examinations, it was decided to treat this mass surgically. The neoplasm was diagnosed as a rhabdomyosarcoma by immunohistochemistry. This poster presents the clinical aspect of the tumour and the successful surgical treatment. The pigeon was still alive two years after surgery. This case is the second published case of rhabdomyosarcoma on a pigeon and the rst published case of a successful treatment.

1 INTRODUCTION
Rhabdomyosarcoma is a rare malignant neoplasm in birds, previously described in hens, budgerigars, a pigeon and a vulture. All previous cases were observed at postmortem exmination, with metastasis in the lung, spleen, liver and/or kidneys. Here we describe a case of rhabdomyosarcoma in an Indian fantail pigeon (Columba livia), which was successfully treated by surgical resection.

2 MATERIAL AND METHODS

A 1-year-old Indian fantail pigeon was presented with a cervical mass of 4 weeks duration. Since 3 days, bleeding and head tilt were observed by the owner. The bird was not able to eat any more, because of the weight of the mass and the abnormal position of the head. The mass was located in the cervical region, near the crop. It was mobile, red, rm, round, 3 cm2, partially plucked and ulcerated. No other abnormality

473

was observed. By endoscopy, the internal wall of the crop was normal and seeds were present inside. Radiographs of the body in a lateral and a dorsal recumbency did not reveal any lesion, in particular any metastasis. A surgical resection of the mass was performed. The bird was anaesthetised with isourane by face mask and then maintained after intubation (isourane 5%, 02 1,5 L/min, then 1,5%, 02 1 L/min). The area was gently plucked and disinfected with iodine povydone (Vetedine). A sterile gauze was used as drape. Dissection was performed around the mass and along the crop wall. The crop wall was very thin and seeds were seen through. Two veins and one artery were ligated before removing the mass. After checking the absence of crop lesion, the skin was sutured with a interrupted suture pattern. Antibiotics were given for 5 days (amoxycillin-clavulanic acid SYNULOX 125 mg/kg bid PO). The biopsy was xed in formalin 10%, embedded in parafn, cut and stained with haematoxylineosin (HE). Serial sections were used for immunohistochemistry using the biotinavidin-peroxidase technique, with anti-vimentin (1:1000, Dako M0725), anti-desmin (1:200, Dako M0724), anti-smooth muscle actin (1:1000, Dako M0851) monoclonal antibodies, and anti-protein S-100 (1:4000, Dako Z0311) polyclonal antibody.

2 RESULTS
Arising from the striated muscle, there was an un-encapsulated, inltrative, densely cellular neoplasm composed of thick interlacing bundles. The neoplastic cells were spindle-shaped, 25 micrometers in diameter, or giant and polygonal, up to 100 micrometers in diameter, with abundant eosinophilic cytoplasm and one or two hypochromatic nuclei with prominent nucleoli. Abnormal mitotic gures were present. The neoplastic cells stained positive for vimentin, desmin, and smooth muscle actin, but negative for Protein S-100. The neoplasm was diagnosed as a cervical rhabdomyosarcoma, without evidence of vascular emboli. At the following presentation, 15 days after, the healing was good, the head was in a normal position and the bird was healthy. Two years after surgery, no recurrence was observed.

3 DISCUSSION
Reports of neoplasms in birds are numerous, but muscular neoplasms are not frequent (LATIMER 1994, SCHMIDT 1997). Smooth muscle tumours are reported twice as frequently as striated ones (LATIMER 1994). Rhabdomyosarcoma are generally described as irregular, elevated, lobulated and rm subcutaneous swelling of the wing or shoulder (LATIMER 1994). They are frequently immobile and rmly attached to the bone (LATIMER 1994, FERNANDEZ-BELLON et al 2003). In our case, the location and the mobility of the tumour may be considered as infrequent. Most cases are described on dead birds. In hens, rhabdomyosarcomas were reported in the anterior uvea, orbit (DUKES and PETIT 1983), heart, in skeletal muscles (FUJIMOTO and OKADA 1970), breast muscle (with metastasis in liver, lungs and kidney) (ERER and KIRAN 1989) and the biceps femoralis (with spleen metastasis) (FUJIMOTO and OKADA 1970). In a vulture, rhabdomyosarcoma within the myocardium was reported (DUNCAN and FITZGERALD 1997). In a racing pigeon (Columba livia), a

474

rhabdomyosarcoma was described on the ventral aspect of the wing (FERNANDEZBELLON et al 2003). The bird was euthanised and immunohistochemistry was positive for muscle actin and myoglobin and negative for desmin, neuron-specic enolase and S-100 protein (undifferentiated rhabdomyosarcoma) (FERNANDEZBELLON et al 2003). In a budgerigar, a surgical treatment of a rhabdomyosarcoma was not successful. Only 90% of the neoplasm was removed and the bird died 3 hours after surgery (RAPHAEL and NGUYEN 1980). On necropsy, liver metastases were found. Few cases of successful surgical treatments of neoplasms are described. Most of the time, benign tumours are successfully treated (lipoma) (SCHMIDT 1997). Intraabdominal tumours are known to be difcult to remove. In our case, the success of the surgical treatment may be related to the mobility and the location of the mass, and the absence of metastases. The large surgical resection was sufcient. To our knowledge, no successful treatments of rhabdomyosarcoma had been previously reported.

5 CITATION INDEX
DUKES TW and PETIT JR. Avian ocular neoplasia-a description of spontaneously occurring cases. Canadian J Comp Medi 1983; 47: 33 - 36. DUNCAN AE and FITZGERALD SD.. Multiple primary rhabdomyosarcomas within the myocardium of a vulture. Clinical challenge 1997; 28(4): 501 - 503. ERER H and KIRAN MM. A case of an undifferentiated rhabdomyosarcoma in a hen. Berl Munch Tierarztl Wochenschr 1989; 102(11):382 - 385. FERNANDEZ-BELLON H et al. Rhabdomyosarcoma in a racing pigeon (Columba livia). Avian Pathol 2003; 32(6): 613 - 616. FUJIMOTO Y, OKADA K. Rhabdomyosarcoma in the chicken. Jap J Vet Res 1970; 18: 109 - 115. LATIMER KS. Oncology. In RITCHIE BW et al, Avian medicine: Principles and Application, 1st ed., Lake worth, FL: Wingers publishing 1994: 640 - 672. RAPHAEL BL and NGUYEN HT. Metastasizing rhabdomyosarcoma in a Budgerigar. J Am Vet Med Assoc 1980; 177(9): 925-926. SCHMIDT RE. Neoplastic diseases. In ALTMAN RB et al. Avian Medicine and Surgery, 1st ed., Philadelphia, PA: WB Saunders 1997:590 - 603.

AUTHORS ADDRESS
Emmanuel Risi, Centre Vtrinaire de la Faune Sauvage, Ecole Nationale Vtrinaire de Nantes, Atlanpole La Chantrerie, BP 40706, 44307 Nantes cedex 03, France, Email: emmanuel.risi@wanadoo.fr

475

Central Veterinary Research Laboratory Dubai, United Arab Emirates

ENDO-PARASITES OF THE ASIAN HOUBARA BUSTARD (Chlamydotis undulata macqueenii)

R. K. Schuster, U. Wernery, J. Kinne

KEYWORDS
Chlamydotis undulata macqueenii - Acanthocephalans - Cestodes Nematodes - U.A.E.

ABSTRACT
In January 2004 thirty-one carcasses of houbara bustards (Chlamydotis undulata macqueenii) were sent for necropsy to the Central Veterinary Research Laboratory. All birds were heavily infested with endo-parasites. Two species of acanthocephalans (Empodius taeniatus, Sphaerirostris embae, three species of cestodes (Otiotaenia conoideis, Ideogenes otidis, Hispaniolepis falsata) and one nematode species (Harteria rotundata) were identied. In addition, one unidentied Eimeria species was found in a single bird.

1 INTRODUCTION
The houbara bustard (Chlamydotis undulata) occurs in 3 subspecies in Asia (C. u. macqueenii), Northern Africa (C. u. undulata) and on the Canary Islands (C. u. fuertaventurae), respectively. The Asian representative is a migratory bird with breeding sites in Central Asia. During the winter these birds migrate south and some of them enter the Arabian Peninsula. C. u. macqueeni is the favourite prey for hunting falcons and an unknown number of these birds are caught in countries north of the Arabian Gulf and smuggled into the U.A.E (BAILEY 2004). The knowledge on parasites of this bustard species is based on a small number of examined birds (WERNERY et al. 2001). In a larger survey in 2003 we examined 104 carcasses of originally wild caught houbara bustards kept at different places in Dubai (SCHUSTER and KINNE 2003). Most of these birds got however, anthelminthic treatment on arrival but drugs used were not indicated.

476

2 MATERIAL AND METHODS

In January 2004, thirty-one carcasses of conscated wild caught houbara bustards were sent to the Central Veterinary Research Laboratory for necropsy. Parasitological examination concentrated on endo-parasites. For this, the intestines were gently opened and helminths were isolated and washed in normal saline. For identication, nematodes and acanthocephalans were cleared up in lactic acid while cestodes were stained in lactic carmine. In addition, a faecal sample taken from the large intestine of each bird was examined using the otation method.

3 RESULTS
All birds were infested with endo-parasites. The helminth spectrum consisted of two acanthocephalans (Empodius taeniatus, Sphaerirostris embae, three cestodes (Otiotaenia conoideis, Ideogenes otidis, Hispaniolepis falsata) and one nematode (Harteria rotundata). Prevalence and burdens of these helminths are given in Tab. 1. The number of birds harbouring one, two, three, four and ve worm species was 5, 9, 10, 6 and one, respectively. In addition, a low number of unsporulated oocysts of an unidentied Eimeria species were detected in the faeces of a single bird. Table 1: Prevalence and number of helminths in houbara bustards (n = 31)
Parameter species Empodius Sphaerirostris Otiotaenia taeniatus embae conoideis 67.74 5.9 1 39 48.39 53.2 5 315 45.16 14.1 2 125 Ideogenes otidis 19.35 141.3 32 1200 Hispaniolepis Harteria falsata rotundata 32.26 40.5 3 100 58.06 11.8 1 156

prevalence% av. burden min. burden max. burden

4 DISCUSSION
All helminths isolated are considered to be bustard specic but some of them might be found in other birds sharing the same environment and having similar food preferences. All isolated worms have a heteroxenic life cycle with arthropods as intermediate hosts. In comparison to the previous investigation (SCHUSTER and KINNE 2003) the current study reects the helminthological situation in wild houbara bustards more realistically since no anthelminthic treatment was given. All birds harboured at least one parasite species and the prevalence of cestodes and H. rotundata was double high as in the former investigation where most of the birds got an anthelminthic treatment with one of the following compounds: praziquantel, fenbendazole, ivermectin and doramectin. While praziquantel is highly effective against cestodes, the macrocyclic lactones ivermectin and doramectin are administered for nematode infections, and fenbendazole can be used for the control of both cestodes and nematodes. None of the available anthelminthics are acting against acanthocephalans and for this reason the gures for prevalence and abundance of E. taeniatus and S. embae were comparable in both treated and untreated groups.

477

5 CITATION INDEX
1. 2. BAILEY T. Health status of free-living and rehabilitated houbara bustards. Proceedings 3rd Annual Wildlife Disease Association - Africa & Middle East Section, Abu Dhabi, in press. SCHUSTER RK and KINNE J. Acanthocephalans and other helminths of the Asian houbara bustard (Chlamydotis undulata macqueenii). 9th Internat Helminthol Symp, Programme and Abstracts, Stara Lesna, High Tatras 2003: 37. WERNERY U, MOLNAR L and HUNT K. Disease status of wild houbara bustards (Chlamydotis undulata macqueenii). Proc Euro Assoc Avian Vet, Munich 2001: 268 - 269.

3.

AUTHORS ADRESS
Prof. Dr. R. K. Schuster Central Veterinary Research Laboratory, PO Box 597, Dubai, United Arab Emirates Email: moniezia@zedat.fu-berlin.de

478

Dubai Falcon Hospital, Dubai, United Arab Emirates

ANTIFUNGAL SUSCEPTIBILITY TESTING OF FUNGI ISOLATED FROM THE RESPIRATORY TRACT OF FALCONS
C. Silvanose, BSc, DMLT; T. Bailey, MSc, MRCVS, PhD, Dip ECAMS and A. Di Somma, DVM

KEYWORDS
Aspergillus sp - Minimum inhibitory concentration - Amphotericin B - Itraconazole - Voriconazole

ABSTRACT
As new anti-fungal agents are introduced for the treatment of fungal infections in birds, establishing minimum inhibitory concentrations (MICs) of fungal isolates from birds is necessary. Biopsy samples were collected during endoscopy from the air sacs of forty-ve falcons diagnosed with fungal diseases of the lower respiratory tract. Our data indicated that antifungal tests before and during treatment may be helpful in guiding appropriate antifungal therapy in avian species.

1 INTRODUCTION
Aspergillosis is the most common avian mycosis, especially in captive waterfowl, wading birds, penguins, raptors, ostriches, pheasants and passerines (DEEM 2003, POLLOCK 2003, BAUCK 1994, KEARNS 2003, PEREZ 2003, WERNERY 2004). Among raptors, goshawks (Accipiter gentilis) gyr falcons (Falco rusticolus), immature red-tailed hawks (Buteo jamaicensis), golden eagles (Aquila chrysaetos) and snowy owls (Nyctea scandiaca) are more likely to develop the disease (REDIG 2000). Aspergillosis is the most important cause of morbidity and mortality in falcons trained for falconry in the Middle East (SAMOUR 2000). The disease is caused by Aspergillus fumigatus and less commonly by A. avus and A. niger (BAUCK 1994, KUNKLE 2003, KEARNS 2003). Due to the life threatening nature of these infections and reports of drug resistance, susceptibility testing of pathogenic fungi has become very important in human medicine. As new anti-fungal agents are introduced for the treatment of fungal infections in birds, establishing minimum inhibitory concentrations (MICs) of fungal isolates from birds is necessary.

479

2 MATERIALS AND METHODS

This study was carried out at the Dubai Falcon Hospital, from 2002 to 2004. Biopsy samples were collected during endoscopy from the air sacs of forty-ve falcons diagnosed with fungal diseases of the lower respiratory tract. Falcon species and numbers included; peregrine (F. peregrinus) - 5, saker (F. cherrug) - 5, gyr (F. rusticolus) 15, barbary (F. pelegrinoides) -1 and hybrid falcons - 19. hybrid falcons included gyr x peregrine (F. rusticolus x F. peregrinus) - 13, gyr x saker (F. rusticolus x F. cherrug) - 5, and gyr x lanner (F. rusticolus x F. biarmicus) - 1. Forty-ve fungal isolates were collected on rst presentation before antifungal treatment (Group - 1) was initiated. Fungal isolates were collected during Amphotericin B (AMP) treatment from 8 cases (Group 2), from 7 cases during Itraconazole (ITZ) treatment (Group 3) and from 15 cases during Voriconazole (VOC) treatment (Group 4). Among the treated cases, samples were collected three times from one case during therapy. The antifungal strips used were E strips containing AMP (concentration range, 0.002 to 32g/ml), ITZ (concentration range, 0.002 to 32g/ml) and VOC (concentration range, 0.002 to 32g/ml) supplied by AB Biodisk, Solna, Sweden. The media used for this study was RPMI agar includes RPMI, 1640 with L- glutamine and MOPS buffer.

3 RESULTS AND DISCUSSION

An antifungal study was carried out to determine the MIC of fungi isolated from the air sacs of falcons before (Group - 1) and during (Group 2, 3 & 4) antifungal treatment with amphotericin B (AMP), itraconazole (ITZ) and voriconazole (VOC) respectively. Ninety ve percent of isolates (Group - 1) including Aspergillus fumigatus, A. avus, A. niger and A. terreus were susceptible to VOC at MICs 0.38 g/ml and 100% of the isolates at MICs 1 g /ml. Twenty one percent of isolates (Group - 1) including A. fumigatus (27.6%), A. avus (16.6%), A. niger (100%) and A. terreus (23%) had an MIC to ITZ 1 g/ml. In human medicine fungi with an MIC 1 g/ml are considered resistant. Fifty one percent of isolates (Group - 1) including A. fumigatus (31%), A. avus (78%), A. niger (14%), and A. terreus (77%) had MICs > 1 g/ml to AMP. The MICs of AMP for Aspergillus strains during antifungal therapy (MIC 3 g/ml, median 8 g/ml, range 3-32 g/ml) were signicantly higher than the MICs before therapy. There was no signicant difference between different Aspergillus strains for the MIC of VOC and ITZ before and during antifungal treatment, except one case of A. avus. In-vitro antifungal tests before and during therapy shows that the majority of Aspergillus sp. are susceptible to VOC at low concentrations. In our study, A. niger was resistant to ITZ but, susceptible to AMP at low concentrations. Isolates showed signicant changes of MIC or resistance to AMP, during therapy. Our data indicated that antifungal tests before and during treatment may be helpful in guiding appropriate antifungal therapy in avian species.

480

4 CITATION INDEX
1. BAUCK L. Mycoses. In: BW RITCHIE, GJ HARRISON and LR HARRISON. (eds): Avian Medicine Principles and Application. Lake Worth: Wingers Publishing Inc. 1994; 997 - 1006. DEEM SL. Fungal diseases of birds of prey. Vet Clinics North America: Exotic Animal Practice 2003; 6: 363-76. KUNKLE RA. Fungal infections. In: Y.M. SAIF (ed): Diseases of Poultry, 11th edition. Iowa: Iowa State Press. 2003; 883-895. REDIG P. Fungal diseases: Aspergillosis. In: J. SAMOUR (ed): Avian Medicine. Harcourt Publishers Ltd, London: 2000; 275 - 278. PEREZ J, GARCIA, PM, MENDEZ A, et al. Outbreak of aspergillosis of adult ostriches. Vet Rec 2003; 153: 124 - 125. SAMOUR JH. Veterinary considerations during the hunting trip. In: J.T. LUMEIJ, J.D. REMPLE, P.T. REDIG, et al. (eds): Raptor Biomedicine III. Zoological Education Network, Lake Worth. 2000; 267 - 274. WERNERY R, WERNERY W, KINNE J and SAMOUR J. (eds): Fungal diseases. In: Colour Atlas of Falcon Medicine. Hanover: Schluetersche 2004; 81 91.

2. 3. 4. 5. 6. 7.

5 ACKNOWLEDGEMENTS
We thank HH Sheikh Hamdan bin Rashid al Maktoum, Deputy Ruler of Dubai, Finance and Industry Minister of UAE for his continued support of the Dubai Falcon Hospital. We thank Mr Humaid Obaid al Muhari, the staff of the Dubai Falcon Hospital and Mr. Peter McKinney (Falcon Center, Dubai) for their technical support.

AUTHORS ADRESS
C. Silvanose Dubai Falcon Hospital, P.O.Box 23919, Dubai, United Arab Emirates Email: chris.silvanose@dfh.ae

481

Les Valls Veterinary Hospital, Principality of Andorra

HEALTH CONTROL OF VARIOUS CAPERCAILLIES (TETRAO UROGALLUS) CAPTURED IN THE PRINCIPALITY OF ANDORRA
J. Tena Pera, DVM,

KEYWORDS
Analysis - Capture - Samples - Capercaillies

ABSTRACT
In the principality of Andorra (Central Pyrenees), the capercailles are included in the list of protected species. The framework of health control includes capture operations and clinical analysis. The health of captured individuals was assessed through a comprehensive clinical examination, ectoparasites and endoparasite, blood samples (for haematology, biochemistry and serology for selected infectious diseases). A parallel parasitological study was made by faecal collection in the birds singing sites covering the entire country.

1 INTRODUCTION
The demographic survey has enabled collection of numerous data about the general tendency of the Andorran capercaillies population . The health survey was used to complete the demographic data and to nd new indicators useful for management decisions. Animals caught for radiotracking projects were individually checked for health problems and a parasitic survey was conducted at several lek places.

2 MATERIAL AND METHODS

Capture: Animals were captured using nylon nets on the leks. Physical restraint was used. A complete clinical inspection was performed: external inspection, blood extraction (from the external cubital vein) and collection of a faeceal sample. Fresh faecal samples were collected from several lek sites. A 33% ZnSO4 otation technique was used to give qualitative results and the McMaster technique was used for the quantitative results.

482

3 RESULTS
3.1 Results on captured animals: a total of 80 % of the animals had a Body Condition (BC) of 4, 20% of the animals had a BC of 3. No lesions or injuries were found on external examination. 3.2 Parasitic study External parasites (ticks and lice): no parasites were observed. Internal parasites: see section IV 3.3 Infectious diseases serology Chlamydophila, paramyxovirus and inuenza virus tests were all negative. 3.4 The parasitological survey A 70% frequency of coccidia and a 100% of cestoda was observed in the leks located less than 100 metres of a road. It is possible that human presence, vehicles, capercaillie density might have had an impact on the presence of parasites. Depending on the previous factors cited, optimum leks could be identied.

4 DISCUSSION
The number of individuals that have been studied is low; further capture and marking tasks will produce further data. Allocating thirty minutes to handling enabled marking of the animal and collection of thirty-seven physical and clinical variables. No individuals have died during or post-capture. The data obtained on captured animals has not identied major sanitary problems. Haematological parameters are within normal range. Higher muscular markers (CPK, LDH) than those established for the Gallus domesticus are probably due to stress related to capture or to the mating season (LIUKKONEN-ANTTILA 2000). The liver parameters were within normal range with the exception of one individual. In terms of the parasitology results we consider it likely that a stressor (human presence, vehicles, capercaillie density) has an impact in the presence of parasites (BELLEAU 1991, GORTAZAR 2000). 5 CITATION INDEX References available from the author

AUTHORS ADRESS
J. Tena DVM Les Valls Veterinary Hospital C/Joan Maragall, 2 AD 500 Andorra la Vella, Principality of Andorra, Email: tenavet@andorra.ad

483

Aquila Foundation, ESVA, Asistencia Veterinaria sl Centro de Estudios de Rapaces Ibricas CERI, JCCM Instituto de Investigacin en Recursos Cinegticos IREC Spain

TREATMENT OF CROP FISTULAS DUE TO SEVERE TRICHOMONIASIS IN TWO BOOTED EAGLES (HIERAAETUS PENNATUS)
S. Villaverde; U. He DVM; J.M. Blanco DVM

KEYWORDS
Booted eagles - Hieraaetus pennatus - Crop stula - Trichomoniasis

ABSTRACT
Trichomonas gallinae may provoke the development of large bronecrotic lesions in the upper digestive tract and occasionally other locations of columbiforms and other avian species. Two booted eagles were admitted to the Centro de Estudios de Rapaces Ibricas with large bronecrotic lesions and associated stulas in the crop. Numerous agellate protozoa were observed on direct examination and identied as T. gallinae from morphology and culture and PCR. Both animals had also intestinal parasites and one of them had an infection with Candida albicans. Treatment was medical and also involved surgery achieving satisfactory healing and recovery in both cases.

1 INTRODUCTION
Trichomoniasis is a disease of columbiforms and birds of prey, but also passerines and psittacine species. It is caused by the agellated protozoan Trichomonas gallinae. Trichomoniasis related lesions are usually localized in the oropharynx and crop and may occasionally involve the respiratory tract or other locations such as the infraorbital or supraorbital regions (SAMOUR 2000). Although, especially in pigeons the parasite is frequently present in absence of clinical lesions, bronecrotic lesions that develop in clinical disease may produce impairment of the normal motility of the crop and oesophagus due to the partial obstruction of the crop lumen and destruction of the mucosa and adjacent tissue. These lesions generally lead to death of the affected bird from starvation or secondary infection.

484

2 MATERIAL AND METHODS

Two juvenile booted eagles (Hieraaetus pennatus), a male and a female were admitted to the Centre for Studies on Iberian raptors (CERI) with well developed bronecrotic lesions in the crop that had led to external stulae in both cases. The eagles were received within one month but originated from different regions. Both birds were severely emaciated upon admission and the male presented an additional rm, rounded lesion of about 4mm in diameter on its left eyelid. From both birds swabs were taken from the lesions for direct wet mounts and culture for Trichomonas sp., pathogenic fungi and bacteria. In addition blood samples were obtained from both birds and total white cell counts, differential white cell counts, and electrophoresis of plasma proteins were performed. Also, cultures for Salmonella and other pathogenic Enterobacteriaceae were carried out from the cloaca as well as faecal otation for parasite ova. Both cases were treated with a combined therapy using metronidazole (local) and carnidazole (15mg/kg single dosis). Sistemic antibiotic treatment (Piperacillin 20mg/kg IM BID) and clorhexidine ushing of the crop were used to control secondary infections due to the presence of necrotic tissue. Hydration was maintained with intravenous uids (Ringers lactate). In both cases the bronecrotic mass adhered to the lesion required surgical removal 7-9 days after initiation of medical treatment. In one of the eagles, the tissue damage and necrosis involved the crop mucosa and skin in a region of approximately 2cm around the stula. This stula required continuous cleaning and refreshing during 5 days before excision of the full extent of necrotic tissue and suturing was possible. Closure of the surgery site was obtained using a two-layer suture in one of the cases, the rst layer with a continuous inverted pattern and the second layer using a simple interrupted suture, both with 4-0 Vicryl. In the other case a one-layer continuous inverted suture with 4-0 Vicryl was used and an additional dressing to protect the wound was applied. We used hydrocolloidal pads for this dressing to absorb uids and exudates and to encourage granulation and re-epithelialisation. Additional postoperative care included soft diet and antibiotics for 7-10 days after healing of the lesions. Both birds were re-cultured twice for the presence of T. gallinae throughout a period of two months after surgery and treatment.

3 RESULTS
High numbers of agellated protozoa were observed directly and on culture from the crop lesions and also from the mass on the eyelid of the male booted eagle, and were identied as T. gallinae by morphology and PCR. In both booted eagles other pathogens were found. In the female C. albicans was cultured from the lesion and propagules of Capillaria and were observed on faecal otation (344 propagules/g of faeces) while on direct examination of faecal matter, tapeworm propagules were observed. The male was also positive for the presence of Capillaria and suffered from a low infestation with Leucozytozoon, evident on the blood smear. Both eagles had a moderate to marked leucocytosis, relative and absolute eosinophilia and marked toxicity of the heterophils (Grade II-IV). Upon plasma protein electrophoresis, albumin/globulin rates were decreased due to mainly a marked decrease in albumin and an increase in the alpha 2 and beta fractions. Candidiasis was treated using oral

485

Itraconazole (15mg/kg) and the helminthosis resolved with oral levamisole (4mg/kg) and praziquantel. Healing of the lesions and function of the crop was satisfactory in both eagles. After treatment none of the two birds cultured positive for T. gallinae in the subsequent analysis. Both eagles fully recovered their physical condition, regaining full normal motility of the oesophagus and crop in absence of any constrictions due to scar tissue. Both birds were released back into the wild about six months after admission.

4 DISCUSSION
T. gallinae is generally considered a secondary pathogen, especially in columbiforms were prevalence of the parasite is high and only occasionally associated with disease (KOCAN and KNISLEY 1970). However the disease has been considered an emerging disease in free-living nestlings of birds of prey in Spain in relation to changes in prey availability (HFLE et al. 2000, REAL et al. 2000). In the booted eagles in this case no other pathogen or primary traumatic lesion could be detected, and both birds recovered fully with treatment of trichomoniasis, candidiasis and parasitosis. Potential subjacent viral infections however were not analysed in this case. Summer in central Spain is very dry and hot, and food and water resources are scarce. This and the fact that these juveniles have recently become independent from their parents may have caused them to prey on diseased pigeons or feed on carrion and also rendered them more susceptible to the disease. Direct microscopy would have been sufcient for diagnosis of trichomoniasis in this case, while in others, especially in subclinical infections, culture has proven more effective (KOCAN and KNISLEY 1970). The association of trichomoniasis with infection by C. albicans is consistent with previous observations in Bonellis eagle (Hieraaaetus fasciatus) nestlings (HFLE et al. 2000).

5 CITATION INDEX
1. BOAL CW, MANNAN RW and HUDELSON, KS. Trichomoniasis in Coopers hawks from Arizona. J Wild Dis 1998; 34: 590 - 593. 2. COLE R A. Trichomonosis. In: FRIEND, M. and FRANSON, J. C. (eds): Field Manual of Wildlife Diseases. U.S. Department of Interior. National Geological Survey, 1999, 201 - 206. 3. COOPER J E and PETTY S J. Trichomoniasis in free-living goshawks (Accipiter gentilis gentilis) from Great Britain. J Wildl Dis 1988; 24: 80 - 87. 4. HFLE U, BLANCO J M, PALMA L and MELO P. Trichomoniasis in Bonellis eagle (Hieraaetus fasciatus) nestlings in South-west Portugal. In: LUMEIJ, J.T., REMPLE, J. D., REDIG, P.T. et al. (eds): Raptor Biomedicine III. Lake Worth: Zoological Education Network, Inc 2000, 45 - 52. 5. REAL J, MAOSA S and MUOZ E. Trichomoniasis in a Bonellis eagle population in Spain. J Wildl Dis 2000; 36: 64 - 70. 6. SAMOUR JH. Supraorbital trichomoniasis infection in two saker falcons (Falco cherrug) Vet Rec 2000; 146: 139 - 140.

486

7. SAMOUR JH, BAILEY TA and COOPER JE. Trichomoniasis in birds of prey (order Falconiformes) in Bahrain. Vet Rec 1995; 135: 358-362. 8. KOCAN RM and KNISLEY JO. Challenge infection as a means of determining the rate of disease resistant Trichomonas gallinae free birds in a population. J Wildl Dis 1970; 6: 13 - 15.

AUTHORSS ADRESS
S. Villaverde ESVA, Asistencia Veterinaria s.l., C.E.R.I. Centro de Estudios de Rapaces Ibricas, Sevilleja de la Jara, 45671 Toledo, Spain Email: cerito@jccm.es

487

Laboratoire dAnatomie Pathologique Vtrinaire du Sud-ouest, Toulouse, France

MUCOCELE IN A LESSER SULFUR-CRESTED COCKATOO (CACATUA SULPHUREA)

J.P. Andr, DVM, and F. Degorce-Rubiales, DVM.

KEYWORDS
Parrot Mucocele

ABSTRACT
A 9 year-old lesser sulphur-crested cockatoo (Cacatua sulphurea) was presented with a 3-month history of chronic sinusitis that was non-responsive to treatment. Various treatments and laboratory tests were started over several months without success. Finally a biopsy revealed a mucocele. Surgery alone resolved that apparently unusual case.

1 INTRODUCTION The upper respiratory system of birds is very complex. Both side possess nares, nasal cavities, infra-orbital sinuses (IOS), cervico-cephalic air sacs and choanae (ANDR 2004). The nares are the inlet of nasal cavities. The nasal cavities are separated by an osseous septum (vomer bone). Usually, each nasal cavity is divided into 3 conchae (rostral, middle, caudal). The caudal (or olfactive) concha is not present in the African grey parrot (Psittacus erithacus) (KRAUTWALD-JUNGHANS et al. 1998). The middle concha communicates with the buccal cavity by means of the choana (internal nares). Air enters the nasal cavities via the nares, to be moistened, warmed and ltered in the conchae. It will circulate in the IOS, cervico-cephalic air sacs and cranial pneumatic bones before entering the oropharynx via the choana. The IOS consists of several diverticulae (infra-orbital, pre-orbital, rostral, preauditory, mandibular) and 2 chambers (suborbital, maxillary). In parrots the right and left IOS are in communication. Secretions of the lacrymal glands are drained into the nasal cavity (middle concha) via lacrimal ducti. Each IOS has a very small dorsal opening of the medial concha (0.5 to 3 mm in diameter) into the nasal cavity to allow drainage of secretions. The last concha does not communicate directly with the nasal cavity, making draining of a purulent secretion difcult.

488

2 CLINICAL REPORT
A 9-year old female lesser sulphur crested cockatoo named Jonquille was presented to our veterinary ofce. The chief complaint was a chronic sinusitis for 3 months. Various antibiotics and aerosols had been prescribed by their usual veterinarian, but the sinusitis had not resolved. No diagnostic tests had been performed. All treatment was stopped 10 days before our examination. Jonquille lived in a small inside aviary. She was fed seed, fruits and vegetables. She had laid one egg 4 years before. On physical examination, Jonquille seemed in good body condition, it was alert and weighed 270g. Its face was very swollen all around the eyes. The left nare was mis-shapen (oval). A signicant translucent nasal discharge was associated with this. Neighbouring feathers were moist and matted. The bird was head-shaking as if to eliminate this discharge. BELISA and ELISA tests for Chlamydophila psittaci were negative. A bilateral incision of the infra-orbital diverticulum of the IOS revealed a translucent, slimy, oedematous tissue that was very difcult to clear. Cultures were negative for bacteria and fungi. Cytology showed desquamation cells, very few nonimpaired white cells, but no bacteria nor yeasts. During a 5 month period, many sinus washings with daily cleaning of nares were carried out, with or without anaesthesia. To investigate a possible myxoma, surgery was performed on the right side and a sample of the gelatinous material was taken for histologic analysis. The laboratory report was as follows: Several samples were examined. Under a keratinized Malpighian epithelial tissue, very likely derived from a mucous membrane, one observes, in the underlying brous chorion, numerous puddles of a mucinous bluish product can be observed, often surrounded with a strip of macrophagic inammatory cells. At times, close by these deposits of tissue, one observes glandular structures (more exactly tubular structures) covered with a double layer of epithelial cells reminiscent of salivary or mucous glands excretory ducts. Therefore, histologic appearance is in favour of a breaking or trauma of these excretory structures that accounts for the intrusion of the material secreted in the surrounding chorion, intrusion of which gives rise to a granulomatous inammation, reminiscent of a sialocele or mucocele. Conclusion: a sialocoele (or mucocele), or an inammatory cyst extended around the secretory product of a mucous gland. The diagnosis was conrmed by G. Dorrestein, Veterinary Faculty of Utrecht (personal communication). A week later, surgery was undertaken on the left side to remove gelatinous material. Several surgical operations were still necessary to eliminate the abnormal tissue. Since then, Jonquille has been in good health, without recurrence of swollen IOS.

3 DISCUSSION
The description of a mucocele in a bird appears to be quite unusual. In man it concerns a chronic, cystic lesion of the sinuses which is lined by a pseudostratied or columnar epithelium (PRATER 1997). In birds, one could consider that it is a complication of a chronic sinusitis. The mucus produced by the glands of the hyperplasic respiratory epithelium could account for the retention in connection with a lack of sinus drainage

489

(obstruction of the ostia). In this clinical report, it must be pointed out, the diagnostic error at the outset as an infectious IOS sinusitis was considered, the delayed undertaking of a histologic examination, the duration of disease (8 months) and the difculties encountered for the removal of all the material that completely moulded the IOS diverticulum.

4 CITATION INDEX
1. 2. ANDR JP. Oiseaux de cages et de volires. De la maladie la bonne sant. ANDR JP. (ed) MIPP-F.33130 Bgles 2004. KRAUTWALD-JUNGHANS ME et al. Comparative studies on the diagnostic value of conventional radiography and computed tomography in evaluating the heads of psittacine and raptorial birds. J Avian Med Surg 1998; 12: 149 - 157. PRATER ME. Acute sinusitis. Grand rounds presentations, UTMB, Dept. of Otolaryngology. March 1997; 1-8. http://www.edu/otoref/Grnds/Sinusitisacute-9703/sinus-acute-9703.htm

3.

AUTHORS ADDRESS
J. P. Andr, DVM -Cabinet Vtrinaire, 27bis rue du 14 juillet, F- 33260 La Teste de Buch, France email: jeanpierre.andre9@free.fr

490

Johannesburg Zoo South Africa

ZINC TOXICOSIS IN A WATTLED CRANE (BUGERANUS CARUNCULATUS)

M. Barrows BSc BVMS CertZooMed MRCVS; M. Hartley BVetMed MapplSc (Wildlife Health) MRCVS; J. M. Pittman CVT.

KEYWORDS
Birds - Wattled crane - Toxicosis - Zinc

ABSTRACT
This case report details the diagnosis and treatment of zinc toxicosis in an adult female wattled crane (Bugeranus carunculatus). The report covers a period of several months during which the crane ingested galvanised zinc hog rings used in construction of a new exhibit on two separate occasions. The initial serum zinc level was 53umol/l and the main presenting sign was weight loss. The crane was treated medically with chelation agents and had her ventriculus ushed under anaesthesia.

1 CLINICAL REPORT
A four year old female wattled crane was treated twice over a period of six months for ingestion of heavy metal. The majority of the metal pieces were galvanised zinc hog rings used in construction of the fencing for a new enclosure, which the bird and her mate had been moved to a few weeks prior to the rst episode. Even though the keepers had checked the enclosure with a metal detector before placing the birds in the new exhibit, the female had managed to nd and ingest several hog rings and other pieces of metal discarded by the builders. Interestingly the male bird never ingested any metal. On each occasion the main clinical signs were weight loss in spite of a normal appetite and a depressed attitude. Initially the crane was examined as part of a routine health check and was found to be in poor body condition with a prominent sternum and a body condition index of 2.0/5. She weighed 6.3kg and apart from the weight loss, there were no abnormalities on physical exam. Over the six month period, her weight decreased further to 5.4kg although she eventually regained the weight lost, reaching 6.7kg after successful treatment. Biochemistry results were consistently normal, whereas the white cell count was often but variably raised, to a maximum of 40.68 x 109/l at the time of initial diagnosis. A serum zinc result of 53umol/l (346ug/dl) was obtained initially, with lower levels in the subsequent cases.

491

Zinc levels greater than 200ug/dl are suggestive of zinc toxicosis (LABONDE 1995). Blood lead was 4.0ug/dl. Each time lateral and ventrodorsal radiographs taken under isourane anaesthesia revealed several pieces of metal present in the ventriculus. No other abnormalities were seen on the radiographs. During the rst episode the crane was hospitalised and treatment initiated with 200mg (32mg/kg) sodium calcium edetate (CaEDTA) diluted in sterile water and given intramuscularly into alternate pectoral muscles once daily. Five days later the crane was anaesthetised for repeat radiographs which showed some smaller pieces of wire passing through the intestines but all of the hog rings still present in the ventriculus. The decision was then made to ush the ventriculus under anaesthesia and this procedure was repeated on the subsequent occasion. The crane was fasted overnight and then after anaesthetising and intubating her with a 6.5mm endotracheal tube, a lubricated piece of plastic tubing, 2m long was passed into the ventriculus. The bird was kept in ventral recumbency with the neck streteched out and the body inclined at an angle of approximately 60 degrees. Water at a temperature of approximately 40 C was ushed through the tube into the ventriculus and the birds head held down over a bucket containing a sieve. A piece of gauze was held over the choana during the procedure. Approximately 50ml/kg warmed Lactated Ringers Solution was given intravenously via a catheter in the medial metatarsal vein during the procedure. Analgesics, antibiotics and a course of CaEDTA were started and continued postoperatively as detailed below. On the rst occasion eighteen pieces of metal were removed and on the second occasion twenty pieces. The second episode occurred after the crane had been discharged back to her original enclosure, which had again been searched with a metal detector for any further pieces of metal while she had been hospitalised. In spite of this she continued to nd and ingest the hog rings and so after this a three metre strip of turf was removed from the perimeter of the enclosure before replacing the crane. There has been no recurrence of metal ingestion since. Post-operative care included clavulanate potentiated amoxycillin at 150mg/kg SC sid, meloxicam at 0.1 mg/kg PO sid and a course of CaEDTA at 32mg/kg IM sid. The crane was subdued and anorexic for a day or two after the ushing procedures and so was tube fed with a mixture made from ground trout pellets, raw egg, a maltodextrin and protein concentrate solution (Critical Care Formula, Vetark, UK), an avian probiotic (Avipro, Vetark, Uk) and a calcium/vitamin D3 supplement (ZolCal D, Vetark UK) until her appetite returned. On the rst occasion she was also given cisapride at 1mg/kg PO sid and on the second occasion, after seven days the edetate injections were replaced by 300mg penicillamine given PO for two weeks. Recovery was complicated by a tracheitis necessitating further diagnostic procedures and treatments, however approximately one month after the nal ushing procedure the crane started to gain weight and was discharged back to her enclosure weighing 5.8 kg. Four months later her weight had increased to 6.7 kg.

492

2 DISCUSSION
Cranes are particularly susceptible to heavy metal toxicosis due to their propensity to ingest foreign bodies (OLSEN and CARPENTER 1997). Treatment and diagnosis can be complicated by the stress of hospitalisation and handling in these sensitive birds. Opting for the gizzard ushing procedure at an early stage is likely to be preferable to lengthy courses of chelation therapy and associated handling. The gizzard ushing procedure was found to be straightforward, although repeat radiographs were essential to monitor progress. On one occasion the pieces of metal were ushed quickly out of the ventriculus into the proventriculus requiring an adjustment in tube position before they could be moved up the oesophagus. They then clumped together and got stuck at the base of the neck where repeated ushing attempts were needed to dislodge them. It is important to radiograph the head and neck at the end of the procedure to ensure that there are no pieces of metal left in the oesophagus or oral cavity. In spite of attempting to block the choana during the ushing procedure, on every occasion at least one or two pieces of metal or grit from the ventriculus had to be removed from the choana at the end of the procedure. Zinc is a cumulative toxin and ingestion can result in a variety of clinical signs in birds, including lethargy, depression, weight loss, polyuria, diarrhoea, feather picking, regurgitation, ileus and ataxia (LABONDE 1995, ZDZIARSKI et al. 1994, BAUCK and LABONDE 1997, MAUTINO 1997). Organs affected by zinc toxicity include the pancreas, liver, kidney and gastrointestinal tract. The weight loss in spite of a good appetite seen in some cases of zinc toxicity could at least partly be attributed to maldigestion due to pancreatic insufciency (ZDZIARSKI et al. 1994). It is important to note when relying on serum zinc levels to diagnose toxicity that, as in humans, studies on birds have shown a signicant diurnal variation in blood zinc concentration (ROSENTHAL et al. 2004). CaEDTA is excreted by the kidneys and may be contraindicated in birds with renal compromise. It is recommended that liver and renal functions are monitored during chelation therapy. CaEDTA nephrotoxicity is recognised in humans. However there are no reliable reports of nephrotoxicity in birds as it has been used for up to 23 consecutive days with no ill effects (LABONDE 1991, SAMOUR and NALDO 2002). This case highlights the importance of builders and maintenance workers clearing up meticulously after constructing new exhibits. Stainless steel should also be used instead of galvanised zinc.

3 CITATION INDEX
1. 2. 3. BAUCK L and LABONDE J. Toxic Diseases. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM and QUESENBERRY K (eds): Avian Medicine and Surgery. Philadelphia: W.B. Saunders Co 1997; 604 - 613. LABONDE J. Avian Toxicology. Veterinary Clinics of North America: Small Animal Practice. 1991; 21(6): 1329 - 1342. LABONDE J. Toxicity in pet avian patients. Seminars in Exotic Pet Medicine. 1995; 4: 23 - 31

493

4. 5. 6. 7. 8.

MAUTINO M. Lead and zinc intoxication in zoological medicine: a review. J Zoo Wild Med 1997; 28(1): 28 - 35 OLSEN G and CARPENTER J. Cranes. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM and QUESENBERRY K (eds): Avian Medicine and Surgery. Philadelphia: WB Saunders Co 1997; 973 - 991. ROSENTHAL K, JOHMSTON M, SHOFER F and POPPENGA R. Heavy metal plasma concentration: daily uctuations and clinical implications. Proc Assoc Avian Vet, New Orleans 2004: 33 - 35. SAMOUR J and NALDO J. Diagnosis and therapeutic management of lead toxicosis in falcons in Saudi Arabia. J Avian Med Surg 2002; 16(1): 16 - 20. ZDZIARSKI J, MATTIX R, BUSH M and MONTALI R. Zinc toxicosis in diving ducks. J Zoo Wild Med 1994; 25 (3): 438 - 445.

AUTHORS ADDRESS
M. Barrows, BSc BVMS Cert ZooMed MRCVS Professional Zoolife Consultants, PO Box 956, Houghton 2041, Johannesburg, South Africa Email: Vet@jhbzoo.org.za

494

Strathmore Veterinary Clinic, Andover, United Kingdom

A NEW METHOD FOR THE CYTOLOGICAL SAMPLING OF FEATHER PULP

J.R.Chitty BVetMed CertZooMed MRCVS

KEYWORDS
Cytology - Feather pulp - Pulpitis - Feather plucking - Parrots

ABSTRACT
Feather pulpitis appears to be a common cause or complicating factor in feather plucking parrots. Diagnosis relies on the cytological examination of feather pulp. Most methods for obtaining such samples rely on removing a feather. However these samples are often contaminated with blood from the follicle as the feather is removed. It is also difcult (and painful for the bird) to remove remiges and/or rectrices that may need to be sampled. This method described ne needle aspiration of feather pulp via the superior umbilicus without removal of the entire feather.

1 INTRODUCTION
Feather plucking or chewing are common reasons for psittacine birds being presented to the veterinarian and this is reected by the literature coverage (COOPER and HARRISON 1994, BAUCK 1997, GILL 2001, FORBES and LAWTON 1996, WELLE 1999). These syndromes are seen much less commonly in raptors (MALLEY and WHITBREAD 1996). There are many documented causes of these syndromes and the majority are not directly related to the skin (COOPER and HARRISON 1994, BAUCK 1997, GILL 2001, FORBES and LAWTON 1996, WELLE 1999). Bacterial infections of the feather pulp are described in the texts but are often described as rare (COOPER and HARRISON 1994, BAUCK 1997, GILL 2001, FORBES and LAWTON 1996, WELLE 1999, REAVILL, SCHMIDT RE and FUDGE 1990). Methods used to investigate these infections include culture/ sensitivity (COOPER and HARRISON 1994, BAUCK 1997, FORBES and LAWTON 1996, REAVILL, SCHMIDT RE and FUDGE 1990), biopsy (COOPER and HARRISON 1994, BAUCK 1997, FORBES and LAWTON 1996, WELLE 1999, REAVILL, SCHMIDT RE and FUDGE 1990), and cytology (COOPER and HARRISON 1994, BAUCK 1997, FORBES and LAWTON 1996, WELLE 1999). In addition, a leucocytosis with toxic heterophilia may also be seen (REAVILL, SCHMIDT RE and FUDGE 1990). Of these methods,

495

cytology is simple, non-invasive, sensitive and results are obtained quickly. Some authors recommend cytological investigation of all feather plucking/ chewing cases FORBES NA and LAWTON, WELLE 1995) while others only recommend it where there is obvious inammation or crusting (BAUCK 1997, HARR 2000). Techniques for sampling feather pulp are described by ECHOLS (2000) and CHITTY (2002). However, these techniques require feathers to be removed from the bird and, in the case of body feathers, there is difculty in obtaining a representative sample for the pulp without contamination from blood or from the follicle. Therefore, the following technique has been developed.

2 METHOD
1. 2. 3. 4. The bird should be adequately restrained. The superior umbilicus of the blood feather to be sampled is identied and the area surrounding it cleaned using chlorhexidine on a cotton-tipped swab. A hypodermic needle is passed via the superior umbilicus and into the pulp. The author uses a 0.5 x 16mm needle for body feathers and a 0.6 x 30mm needle for remiges/rectrices. Suction is applied using a 2.5ml syringe and a small aspirate obtained. Smears are made in the usual manner prior to staining.

3 DISCUSSION
Using this technique, cellular harvest seems reasonable. Sampling in this manner does not appear to increase the chances of the bird damaging or removing the sampled feather although that is hard to determine in a feather damaging bird. Interestingly, birds often vocalise when the sample is being aspirated. This may provide an indication that innervation of the growing feather is signicant and that inammatory conditions within the pulp are capable of causing discomfort or pain.

4 CITATION INDEX
1. 2. 3. 4. 5. COOPER JE and HARRISON GJ. Dermatology. In: RITCHIE BW, HARRISON GJ and HARRISON LR, (eds): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing Inc 1994: 607 - 639. BAUCK L. Avian Dermatology. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM and QUESENBERRY K, (eds): Avian Medicine and Surgery. Philadelphia: WB Saunders: 1997: 548 562. GILL, JH. Avian Skin Diseases. Veterinary Clinics of North America: Exotic Animal Practice 2001; 4: 463 492. FORBES NA and LAWTON MPC. Miscellaneous. In: Manual of Psittacine Birds. Shurdington: BSAVA: 1996: 211 - 221 . WELLE K. Clinical Approach to Feather Picking. Proc Assoc Avian Vet, Phoenix 1999: 119 - 124.

496

6.

MALLEY AD and WHITBREAD TJ. The Integument. In: BEYNON PH, FORBES NA and HARCOURT-BROWN NH, (eds): Manual of Raptors, Pigeons and Waterfowl. Shurdington: BSAVA; 1996: 129 - 139. 7. REAVILL DR, SCHMIDT RE and FUDGE AM. Avian Skin and Feather Disorders: A retrospective study. Proc Assoc Avian Vet, New Orleans 1990: 248 255. 8. HARR KE. Avian Integument. Clinical Cytology (Avian Specialty Advanced Program) Assoc Avian Vet, Portland 2000: 21 26. 9. ECHOLS S. Sample Collection, Preparation and Handling. Clinical Cytology (Avian Specialty Advanced Program) Assoc Avian Vet, Portland 2000: 3 16. 10. CHITTY JR. Cytological Sampling in Avian Skin Disease. Proc Assoc Avian Vet, Monterey 2002: 355 358.

AUTHORS ADDRESS
JR Chitty BVetMed CertZooMed MRCVS Strathmore Veterinary Clinic, London Road, Andover, Hants SP10 2PH, United Kingdom Email xmo32@dial.pipex.com

497

Faculty of Veterinary Medicine University of Zagreb, Croatia

KLEBSIELLA OXYTOCA INFECTION IN MONK PARAKEET

D. Horvatek, . Gottstein, I. Ciglar Grozdani, H. Mazija, E. Prukner-Radovi

KEYWORDS
Monk parakeet- Klebsiella oxytoca - Respiratory infection

ABSTRACT
Klebsiella species was isolated from healthy and from birds with upper respiratory tract infection considered to be potential pathogens of the respiratory tract (primary or opportunities), both in healthy and in sick birds under stress or immunosupression. A 17-year-old female monk parakeet (Myiopsitta monachus) came with multiple signs of respiratory distress and loss of weight. Abnormalities shown on physical examination included open-mouthed breathing with severe dyspnoea, coughing, inamed nasal and pharyngeal mucosa with lot of mucopurulent discharge and plugged right nare. Cultures from pharynx and nares revealed heavy growth of Klebsiella oxytoca and Staphylococcus spp. Treatment included enrooxacin orally every 12 ours for 10 days, given by the owner under prescription and supervision. Nasopharyngeal infection resolved completely in 12 days. The presence of K. oxytoca in bird housed in optimal condition, without any contact with birds, with correct feeding and without stress, was unexpected nding.

1 INTRODUCTION
Klebsiella oxytoca is encapsulated, non motile, facultative anaerobic Gram-negative bacteria, present in intestinal tract of man and animals (Kelly et al., 1985). JESUS (1998) reported K. oxytoca and Klebsiella pneumoniae to be potential pathogens of the respiratory tract, under certain circumstances, both in healthy and in sick birds. Klebsiella species are mainly isolated from nches, in which they can act as primary pathogens or can be involved as opportunists in immunosupressed or stressed birds (GERLACH, 1994). COLES (2001) reports about an outbreak of juvenile bird mortality because of pollution of drinking water supply with K. oxytoca.

498

2 CASE REPORT
Bird was kept alone in a big stainless-steel cage, in the living room of owners apartment; most of its time in the cage without any contact with other birds, and was maintained on seed diet with lot of different fruits and vegetables every day. The bird had no previous history of any medical problems. Owner reported the bird hasnt eaten for the last 3 days, had respiratory difculties for two weeks (intensive cough, nasal discharge), in spite of good housing. Prior to our examination, another veterinarian had seen the bird and found nothing signicant, but he prescribed sulfonamides and vitamins, like symptomatic therapy, without performing any other examination. The symptoms worsened despite the therapy. Physical examination revealed poor body condition, weight loss and normal faecal discharge. A faecal Gram stain was normal. Abnormalities included openmouthed breathing with severe dyspnoea, coughing, inamed nasal and pharyngeal mucosa with lot of mucopurulent nasal discharge and plugged right nare. An initial workup preformed included bacteriological and mycological examination of swabs taken from pharynx and nares. Swabs were placed directly on Nutrient Agar (Becton Dickinson, Cockeysville, USA), BPLS-Agar (Merck, Darmstadt, Germany) and Sabourauds Dextrose Agar (Becton Dickinson, Cockeysville, USA), aerobically. The taxonomic classication of isolated bacteria was based on colony morphology, Gram staining, and biochemical characteristic using API 20 E strips (BioMerieux, Lyon, France). Cultures from pharynx and nares revealed heavy growth of K. oxytoca and Staphylococcus spp. Mycological examination was negative. The antimicrobial susceptibility test (Bauer-Kirby test) performed for K. oxytoca revealed, indicated by the zone of inhibition, that K. oxytoca was very sensitive to enrooxacin (5g, Bayer, Germany), semi-sensitive to cefotaxime (30 g, Biorad, France), tetracycline (30g, Biorad, France), sulfonamides (200g, Biorad, France), ampicillin/sulbactam (10/10g, Biorad, France), amoxycillin/clavulanic acid (20/10g, Biorad, France) and sulphonamides (200g, Biorad, France) and resistant to clarithromycine (15g, Biorad, France). Prior to releasing from clinic, rst dose of enrooxacin (5mg/kg IM q12 h; Baytril 2.5%, Bayer, Germany) and multivitamins was administered intramusculary, and Ringers solutiosubcutaneously. Treatment included enrooxacin (30mg/kg p/o q12h; Vetook 10%, Pliva, Zagreb, Croatia) given by the owner (the patient was managed as outpatient) for 10 days, every 12 ours in peace of fruit or cookie, strictly under our instruction and supervision. During the therapy, the bird was physically examined daily. All clinical signs resolved with treatment completely after 12 days. Fourteen days after complete recovery, choanal and pharyngeal swabs were taken and cultures revealed no presence of K. oxytoca.

499

3 CONCLUSION
A 17-year-old female monk parakeet (Myiopsitta monachus) came with multiple signs of respiratory distress and weight loss. The bird had no previous history of medical problems. It was housed in a large stainless-steel cage, without any contact with other birds, kept most of its time in the cage and was maintained on seed diet with lot of different fruits and vegetables every day. First therapy (sulphonamides) given by another veterinarian didnt results with recovery, in a contrary, symptoms worsened. Our recommended therapy with enrooxacin results in complete recovery. This case is presented here because in a last few years K. oxytoca wasnt found in patients in our Clinic, but according to JESUS (1998) Klebsiella species are isolated from healthy birds and from birds with upper respiratory tract infection. Therefore our nding is not unusual, and rise a question why did infection with severe clinical signs appeared, especially because of prior good health status of the bird and no contact what so ever with any other birds species or animals.

4 CITATION INDEX
1. COLES B H. Past Klebsiella oxytoca contamination of a drinking water supply, resulting in an increased juvenile mortality in a variety of avian species in a zoological collection. Proc Euro Assoc Avian Vet, Munich 2001; 6. GERLACH H. Bacteria. In: RITCHIE BW, HARRISON GJ and HARRISON LR (eds): Avian Medicine: Principles and Application. Lake Worth: Wingers Publishing 1994; 949 - 983. DORRESTEIN GM. Bacteriology. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM and QUESENBERRY K (eds): Avian Medicine and Surgery. Philadelphia: WB. Saunders Co 1997, 255 - 280. JESUS O S and DUARTE CORREIA J H. Potential Pathogens Recovered from Upper Respiratory Tract of Psittacine Birds. Proc IVCVM: Diseases of Psittacine Birds 1998 (www.vet.uga.edu) KELLY MT, BRENNER DJ and FARMER III JJ. Enterobacteriaceae. In: LENNETTE EH, BALOWS A, HAUSLER WJ AND SHADOMY HJ (eds): Manual of Clinical Microbiology Fourth Edition. Washington: DC: American Society for Microbiology 1985; 263 - 277. RUPLEY AE. Common Diseases and Treatments. In: RUPLEY AE (ed): Manual of Avian Practice. Philadelphia: WB. Saunders Company 1997; 264 - 309.

2.

3.

4.

5.

6.

AUTHORS ADRESS
D. Horvatek, DVM Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55,10 000 Zagreb, Croatia Email: horvatek@vef.hr

500

Angell Animal Medical Center, Boston, United States of America

CHRONIC POLYCHONDRITIS IN AN AFRICAN GREY PARROT (Psittacus erithacus)

T. K. Ritzman, DVM, Dipl ABVP-Avian Practice

KEYWORDS
Polychondritis - Cartilage - Computed tomography - Psittacine

ABSTRACT
A twenty year old male Congo African grey parrot presented for a chronic history of progressive deformity of the external nares and nasal vestibule. At presentation the bird lacked the external nares and the nasal structures were deformed with severe loss of the normal cartilaginous tissue. The disease condition of chronic atrophic polychondritis was obtained from diagnostics including blood work, fungal and bacterial cultures, CT scan and biopsy of the nasal cartilage for histopathology. Polychondritis is a disease of unknown aetiology characterised by inammation and degeneration of the cartilaginous structures. Polychondritis has been described in humans and feline patients and can involve the ears, nose, larynx, tracheobronchial tree, ribs and pelvis. The condition can involve hyaline cartilage, elastic cartilage or brocartilage. Immune mechanisms are presumed to be of aetiopathogenic importance. This is the rst known case report of polychondritis in a psittacines bird.

1 INTRODUCTION
Polychondritis, also know as relapsing polychondritis (RP) is an uncommon, multisystem disorder of characterised by recurrent episodes of inammation of the cartilaginous tissue. Polychondritis is presumed to be an autoimmune disease. All types of cartilage may be involved, including the elastic cartilage of the ears and nose, the hyaline cartilage of the peripheral joints, the brocartilage at axial sites and the cartilage in the tracheobronchial tree. Relapsing polychondritis can also iname other proteoglycan-rich structures, such as the eye, heart, blood vessels and inner ear. Systemic symptoms including fever, lethargy and weight loss are common. Vasculitis affecting skin or internal organs may occur. Classic features are repeated inammatory episodes involving auricular, nasal and laryngotracheal cartilage and

501

occasionally, joints or ocular tissues. Most human patients develop some degree of disability such as bilateral deafness, impaired vision, phonation difculties, and cardio-respiratory problems. Potentially serious complications include laryngotracheal collapse and or stenosis, valvular heart disease and large-vessel aneurysms. There is no age, gender or ethnic pattern to this disease in humans. Diagnosis in humans is made based on diagnostic criteria rst proposed by MCADAM et al. (1976) and has been modied several times. This criteria state that the patient must have 3 of the following 6 clinical features for diagnosis. The clinical features include: a) bilateral auricular chondritis, b) non-erosive seronegative inammatory polyarthritis, c) nasal chondritis, d) ocular inammation, e) respiratory tract chondritis, f) and/or audiovestibular damage. Biopsy of the affected cartilaginous tissues with histopathology may aid in the diagnosis. Histopathological ndings with polychondritis demonstrate chondrolysis, chondritis, and perichondritis. The cartilage matrix is eventually destroyed and replaced by brous connective tissue. Various studies indicate that circulating antibodies to cartilage-specic collagens types II, IX, and XI are present in 25-70% of human patients with RP. There is no known cure for polychondritis Treatment is directed at reducing the inammation and progressive destruction of the affected tissues. Medical therapy is based on the understanding of other connective tissue or autoimmune diseases, and includes the use of NSAIDs, prednisone and/ or immunosuppressive medications to help control the symptoms. Polychondritis has been described in felines (GERBER et al. 2002) and the condition in cats is usually characterised by a folding deformity elastic cartilage of the pinnas (GERBER et al. 2002). This is the rst known case report of polychondritis in a psittacine bird.

2 CASE REPORT
A twenty year old wild caught male Congo African grey parrot (Psittacus erithacus) presented for a chronic history of deformity of the external nares and nasal vestibule. At presentation the bird lacked external nares and the nasal vestibule was deformed with severe loss of the normal cartilaginous tissue. The inner nasal vestibule was visibly occluded with dried mucopurulent discharge. A nasal ush was performed and this uid sample was collected for aerobic bacterial culture and sensitivity testing, fungal culture and uid cytology. Skull radiographs performed under general anesthesia showed cartilage and bone defect centered at the nasofrontal hinge. Computed tomography (CT) of the head was performed. Contiguous axial 1 mm images were obtained from the rostral premaxilla through the level of the orbit. A large lesion was visibly centered at the nasofrontal hinge with loss of portions of the premaxillary, maxillary and frontal bones, and their enclosed nasal passages. Multiple biopsies were collected from the exposed hyaline cartilage of the naris-nasal vestibule (left and right) and submitted for histopathology. Histopathology showed multifocal degeneration and necrosis of the hyaline cartilage with fracture, brosis and sequestration consistent with polychondritis. The bacterial culture produced abundant growth of E. coli and Citrobacter freundii which was due to secondary bacterial infection of the exposed tissue. Fungal cultures were negative. Bloodwork testing was performed and the results are included in the poster.

502

This patient is being treated currently with non-steroidal anti-inammatory therapy (meloxicam 0.5 mg/kg PO SID) as well as regular nasal ushing and debridement to keep the exposed nasal tissues clean. Antibiotic therapy is provided as indicated for the recurring secondary bacterial infections based on culture and sensitivity results. There has been no appreciable progression of the disease in this patient over the past 20 months. Immunosuppressive therapy was considered but not instituted in this patient due to the concern for increasing risk of secondary infection, such as systemic mycosis in this susceptible species.

3 CITATION INDEX
1. 2. MCADAM LP, OHANLAN MA, BLUESTONE R and PEARSON CM. Relapsing polychondritis: prospective study of 23 patients and a review of the literature. Medicine (Baltimore) 1976; 55(3): 193 - 215. GERBER B, CROTTAZ M. TSCHARNER C VON et al. Feline relapsing polychondritis: two cases and a review of the literature. J Feline Med Surg 2002; 4(4): 189 - 194.

AUTHORS ADDRESS:
Tracey K. Ritzman, DVM, Dipl ABVP-Avian Practice Angell Animal Medical Center, Boston 350 South Huntington Ave. Boston, MA 02130, United States Email: tritzman@angell.org

503

College of Veterinary Medicine University of Georgia, United States of America

RADIATION TOLERANCE DOSES OF CUTANEOUS AND MUCOSAL TISSUES IN PSITTACINE BIRDS USING COBALT-60 TELETHERAPY
G. Heather Wilson, DVM Dipl ABVP-avian, Royce Roberts, DVM, MS, Dipl. ACVR, Nicole C. Northrup, DVM, Diplomate ACVIM (oncology), Stephen HernandezDivers BVetMed, DZoo Med, MRCVS, DACZM, and Kenneth Latimer, DVM, PhD, DACVP

KEYWORDS
Cobalt Radiation Tolerance Ingluvies Skin Ring-necked Parakeets Psittacula krameri

ABSTRACT
The purpose of this study was to demonstrate what level of Cobalt-60 radiation could be tolerated by the cutaneous and mucosal tissues of normal ring-necked parakeets (Psittacula krameri) in order to provide more effective treatment for tumours that have been shown to be radiation responsive in other species. Ingluvial mucosae and the skin over the ingluvies of nine Psittacine birds were irradiated in 4 Gy fractions to a total dose of either 48, 60, or 72 Gy using an isocentric Cobalt-60 teletherapy unit. Acute and chronic radiation effects were measured. Histopathology demonstrated only minimal acute changes in the high dose group. Neither doserelated acute nor chronic radiation effects could be detected grossly in cutaneous or mucosal tissue over a 9 month period.

1 INTRODUCTION
An understanding of the tolerance of normal tissues included in the treatment eld is essential in radiation treatment planning and the initial decision as to whether radiation should be used for cancer treatment. Neoplasia is a common problem in pet birds. In fact, neoplasms are more commonly reported in the budgerigar than in any other domestic animal (BAUCK 1992). Despite this, treatment for avian patients with neoplasia has lagged far behind the technology available for other companion animals. Attempts in the past to use external beam radiotherapy (RT) for cutaneous neoplasms in birds have met with variable, usually poor, success (MANUCY et al.

504

1998, Freeman 1999, WILSON 2000). One study demonstrated that of all domestic species mortality and morbidity due to acute radiation syndromes was lowest in chickens. (ZALLINGER and TEMPLE 1998) Dose response, inuence of fractionation, and normal tissue injury has been evaluated extensively in other companion species over the past 25 years. (ASTASHEVA et al. 2004) However, there have been no controlled studies of external beam radiation therapy in avian species. The goal of this study was to determine the tolerance doses of cutaneous and mucosal tissues of healthy psittacine birds in order to provide more effective treatment for neoplasms that have been shown to be radiation responsive in other species.

2 MATERIAL AND METHODS

Twelve male and female Indian ring-necked parakeets ranging from one to two years of age were divided into 4 randomized groups, each composed of 3 adult, clinically healthy birds. One group served as controls, having crop and skin biopsy, but radiation therapy was not performed. The other 3 groups were irradiated to a total dose of 48, 60, and 72 Gy respectively using an isocentric Cobalt-60 teletherapy unit. A total of 12, 15, or 18 fractions of 4 Gy each were given on a Monday, Wednesday, and Friday until the total prescribed dose was achieved over a 4 to 6 week period. The eld size was 2 x 2 cm for all subjects, centered over the ingluvies. Equally weighted, parallel-opposed, lateral treatment portals were used for each treatment. Lesions were graded according to the Veterinary Radiation Therapy Oncology Group acute and chronic radiation morbidity scoring criteria. (GILLETTE et al. 1995) Biopsies of skin and ingluvial mucosa (3-4 mm) for histologic analysis were obtained at the end of each groups treatment period, or in the case of the control group at week 6. The parakeets were monitored for a total of 9 months for chronic effects.

3 RESULTS
Grossly, all birds received a score of 0 (no change compared to baseline), on the VRTOG radiation scoring scheme at the end of radiation treatment and throughout the duration of the 9 month study. Body weights remained stable throughout the study, as did appetite and attitude. There was no histologic evidence of radiationinduced tissue alterations in either the low (48 Gy) or the medium dose group (60 Gy). In these birds, the histology was similar to that of the control group. However, all 3 birds in the high dose group (72 Gy) had minimal radiation-induced epidermal changes. These lesions were clinically insignicant.

4 DISCUSSION
The principle of radiation therapy is to maximize the radiation dose to the tumour and tumour-harbouring tissues while minimizing radiation exposure to surrounding healthy tissues. This study indicates that doses of 72 Gy given in 4 Gy fractions may be used safely, with no clinically signicant dose-related acute or chronic radiation effects on

505

normal cutaneous or mucosal tissue in Indian ring-necked parakeets. Lower doses of radiation may be insufcient to provide effective tumour control. This project was approved by the University of Georgias Animal Care and Use Committee.

5 CITATION INDEX
1. 2. 3. 4. 5. 6. 7. ASTASHEVA NP, ANNENKO BN, KHRAMTSOVA LK and FINOV VP. Effects of gamma-irradiation on survival, growth, and productivity of the broiler chicken. Radiata Bio Radioecol 2004; 44(1): 43 - 46. BAUCK LA. A clinical approach to neoplastic disease in the pet bird. Seminars in Avian and Exotic Pet Medicine, 1992; 1(2): 65 - 72. FREEMAN KP, HAHN KA, ADMAS WH, et al. Radiation therapy for hemangiosarcoma in a budgerigar. J Assoc Avian Vet 1999; 13(1): 40 - 44. GILLETTE EL, LARUE SM and GILLETTE SM. Normal tissue tolerance and management of radiation injury. Sem Vet Med and Surg 1995; 10(3): 209 - 213. MANUCY TK, BENNETT A, GREENACRE CB, et al. Squamous cell carcinoma of the mandibular beak in a Buffons macaw (Ara ambigua). J Assoc Avian Vet 1998; 12(3):158 - 166. WILSON GH, GRAHAM J, ROBERTS RE, et al. Integumentary neoplasms in psittacine birds: treatment strategies. Proc Assoc Avian Vet, Portland 2000; 211 - 214. ZALLINGER CV and TEMPLE K. The physiologic response of domestic animals to ionizing radiation: a review. Vet Rad & Ultrasound 1998; 39 (6): 495 - 503.

AUTHORS ADRESS
G. Heather Wilson, DVM, Dipl. ABVP (Avian) Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 31522, United States Email: hwilson@vet.uga.edu

506

EAAV 2005 AFSSA - LERRPAS, FRANCE ONCFS, FRANCE

DISEASES FOUND IN FRENCH BIRDS ACCORDING TO SAGIR DATABASE

M.E. TERRIER, DVM, J. BARRAT, DVM, J.R. GAILLET, DVM

KEYWORDS
SAGIR - network diseases birds - France

ABSTRACT
SAGIR is a national surveillance system of wildlife diseases,created in 1986 by the ONC (that became ONCFS, the French Game and Wildlife Agency) and the CNERPAS (that became AFSSA LERRPAS). One of the advantages of SAGIR is its national coverage. The drawback is the sampling method: the necropsy of animals is carried out on a voluntary basis according to the interest of the hunters in each case. Since 1989, 5 500 birds were necropsied by SAGIR. Nowadays, nearly 500 birds are recorded yearly. The presentation rst shows the different species analyzed, their geographical origin, the yearly changes in the analyses. The main causes of death are traumas, intoxications (bromadiolone, chloralose and various inhibitors of cholinesterase), bacterial diseases (botulism, salmonellosis) and parasitic diseases (trichomonasis, aspergillosis). Some pathological ndings are selected: avian tuberculosis, listeriosis, chlamydiosis, fowl pox, drowning, electrocution,

1 INTRODUCTION
Created in 1986 by the ONC (that became ONCFS) and the CNERPAS (that became AFSSA), SAGIR is a national surveillance system for wildlife diseases. It gives informations on the sanitary background of wildlife and on the distribution of pathogens that are common to humans and domestic animals. This network groups terrestrial mammals (about 5/6 of the analysis) and birds (about 1/6). Some 3 000 to 4 000 animals are necropsied each year. All data collected are analyzed and recorded in a database. A yearly report is done and published in the BIPAS (Bulletin dInformation sur la Pathologie des Animaux Sauvages).

507

2 MATERIAL AND METHODS

SAGIR is organized as a network including ONCFS, AFSSA-LERRPAS, the toxicology laboratory of the National Veterinary School in Lyon (ENVL) and other specialized laboratories, the Local (Dpartemental) Veterinary Laboratories (LVD) and the Local (Dpartemental) Federations of Hunters (FDC). The FDC pays for the different routine analyses. One of the advantages of SAGIR is its national coverage; another linked one is the reactivity of the network. The drawback is the sampling method: the necropsy of animals is carried out on a voluntary basis according to the interest of the hunters in each case. Since 1989, more than 5600 birds were necropsied by SAGIR. Their distribution is as follow: web-footed birds, Anseriformes, Columbiformes, Galliformes, diurnal birds of prey, passerines, Charadriiformes, cranes, other various species. Most of the results have been obtained between 1993 and now. The annual number of birds analyzed now is about 500. Most of the registered results come from SAGIR network, but we also get results from the FDC without a SAGIR form, the gamekeepers (ONCFS), the foresters (ONF), the National Parks, scientic teams (for example CNERA of the ONCFS), the Ministry of Agriculture, The birds come from 93 different dpartements, which means that the whole country participates in the network, even if it is with different levels of implication. More than half of birds are game birds, but about 1/3 of them are protected species.

3 RESULTS
3. 1. Causes of death of the birds. The main identied causes of death are - intoxications: bromadiolone, chloralose and various inhibitors of cholinesterase, - traumas: predation and road accidents, - bacterial diseases: botulism, salmonellosis mostly due to Salmonella arizonae , - parasitic diseases: trichomonasis, coccidiosis, aspergillosis. 3. 2. Interesting diseases. Some interesting pathological ndings are selected: avian tuberculosis, pseudotuberculosis, listeriosis, chlamydiosis, fowl pox, drowning, electrocution,

4 DISCUSSION
In about cases, the cause of death stay unexplained. Many lesions are observed, and not necessarily correlated with the identied disease. This may be due to the small number of necropsies of birds done yearly by some laboratories. The SAGIR network needs specialized avian veterinarians to refer difcult cases to. AUTHORS ADDRESS: M.E. Terrier, DVM AFSSA - LERRPAS BP20 54 220 MALZEVILLE / France e-mail: me.terrier@nancy.afssa.fr

508

Clinique vtrinaire de lArche, Valence, France

CLINICAL APPROACH TO OPHTHALMOLOGIC DISORDERS IN BIRDS

F. Rival, DVM

KEYWORDS
Birds Ophthalmology Eye

ABSTRACT
Anatomical and physiological characteristics of the bird eye are described. Common ophthalmological examination procedures will be discussed together with the most frequent ocular disorders. In birds, more often than in mammals, ocular lesions are an expression of systemic diseases. Thus, the eye may be seen as an important diagnostic criterion in avian medicine.

1 INTRODUCTION
The eye is the most important sensory organ in birds. Eye examination should be an integral part of the general clinical examination protocol.

2 PHYSIOLOGICAL AND ANATOMICAL CHARACTERISTICS

The avian eye show a high specialisation as an adaptation to y. Visual acuity is 2-8 times higher compared to mammals. Visual elds are up to 360. Ultraviolet (UV) light perception probably plays an important role in communication based on plumage UV reection. 1. Shape : Three basic shapes are typical: Flat : diurnal birds such as Columbiformes. Conical: diurnal birds such as Falconiformes. Tubular : nocturnal birds such as Strigiformes. Maintained by 10 to 18 scleral ossicles.

509

2. 3. 4. -

Ocular motility limited. Retractor bulbi muscle complex absent, replaced by the pyramidalis and quadratus muscles innervated by cranial nerve VI. Adnexal structures: eyelids: - Lower eyelid with a brous plate (tarsus). - No Meibomian glands. - Filoplumes (mammalian cilia). - Nictitans membrane, thin, transparent and mobile. lacrimal glands: - Small and absent glandulae lacrimaliae - Large Harderian gland (glandula lacrimalis membranae nictitantis) - Two lacrimal puncta and a nasolacrimal duct - Nasal salt gland in some species Complete decussation of chiasma opticus (no consensual papillary reex) Anterior segment: Striated intraocular musculature (no sensitivity to SM or PSL, no reliable pupillary reex corresponding to light stimuli). Iridocorneal angle well developed Lens with annular pad at ist equator (anulus pulvinus) Posterior segment: Vitreous body: large and transparent. Avascular retina. Rods and cones (inclusive special UV cones). No retinal tapetum lucidum. Pecten oculi (vascular prominence of variable shape).

5. 6. 7. -

3 CLINICAL EXAMINATION
A complete ocular examination is composed of three parts : 1.1. History. 2.2 Functional examination: it assesses vision directed behavior. Evaluate the palpebral reex, the direct pupillary light response. 3.3. Morphologic ocular examination : external eye, adnexa, anterior and posterior segments are examined.

510

4. OTHER EXAMINATIONS
Tonometry : standard reference values : 9-22 mm Hg (corneal diameter > 9mm). Schirmer tear test or Tevetest (in study): difcult because of the interspecic variations. Cytology and bacteriology : corneal or conjunctival cytologic specimens may be examined. Physiological bacterial ora contains G+, while G- are indication for pathological conditions. Ophtalmoscopy : induction of mydriasis is indispensable for the examination of the posterior eye segment. Mydriatics have little or no effect in the avian eye due to the striated musculature. Neuromuscular blocking agents such Tubocurarine 3% may be used. However it has to be administered directly into the anterior chamber by paracentesis to be more effective. Mydriasis can be achieved by general anaesthesia with APA (air sac perfusion anaesthesia). This anaesthetic technique is excellent for ophthalmologic examination and head surgery. The combination mdtomidine/ketamine (275 microg/kg, at 10mg/kg) IM is also a good anaesthetic choice for head surgery.

4 EYE DISORDERS IN BIRDS (see poster)

4.1. Classication by aetiology. 2.2 Classication by clinical signs. 2.3 Classication by localisation.

5 CITATION INDEX
1. 2. KERN TJ. Disorders of the special senses: The eye. In: ALTMAN RB, CLUBB SL, DORRESTEIN GM and QUESENBERR YK (eds): Avian medicine and surgery. Philadelphia: WB Saunders 1997, 563 - 589. KORBEL RT. Avian ophthalmology a clinically-oriented approach. Proc Assoc Avian Vet, New Orleans 1999: 335 - 350.

AUTHORS ADRESS F. Rival, DVM, Prsident du GENAC, CES dOphtalmologie Clinique vtrinaire de lArche,

511

Avian and Exotic Animal Clinic of Indianapolis, Indianapolis, United States of America Birch Heath Veterinary Clinic, Cheshire, United Kingdom

POST-INTUBATION STENOSIS IN PSITTACINE BIRDS

A. M. Lennox DVM Dipl. AVBP-Avian, M. D. Stanford BVSc MRCVS

KEYWORDS
Tracheal stenosis - Post-intubation - Psittacine

ABSTRACT
Tracheal stenosis is a commonly reported sequel to endotracheal intubation in humans. The condition is also reported in psittacines with an apparent predilection for macaw and cockatoo species. Histopathological examination of affected tracheas suggests an acute inammatory reaction in birds compared with man where cartilage necrosis is invariably reported. This paper reviews cases of post-intubation stenosis, the possible aetiologies of the condition, treatment and prevention protocols.

1 AETIOLOGY OF POST-INTUBATION STENOSIS IN PSITTACINE BIRDS

Post- intubation stenosis (PIS) is the commonest reason for tracheal resection and anastomosis in man (WAIN 1975). It is usually caused by local ischemic necrosis created by the inated cuff of the endotracheal tube. The condition can develop after very short intubation procedures. Histopathological lesions include degeneration, inammation and necrosis of tracheal cartilage (KLAINER et al. 1975). If the trachea cartilage is damaged then surgical intervention is required with a reported success rate of 93.7%. In mild cases mechanical dilation, laser, medical and stent placement may be successful. The condition is prevented in humans by careful tube selection (both length and diameter), reduction of cuff ination pressure and the use of sterile single use endotracheal tubes. Stenotic tracheal lesions in birds secondary to tracheal intubation typically appear as a severe inammatory response in the supercial tissues rather than cartilage necrosis. This may be a reection that cuffed tubes are contraindicated in avian medicine due to the anatomy of the avian trachea with complete cartilage rings. The use of chemical disinfectants to disinfect tubes between patients has been suggested as an alternative aetiology for stenosis. Other possible aetiologies would include inappropriate tube size, pre-existing inammation in the trachea, rough intubation and

512

mechanical irritation due to the end of the tube touching the tracheal wall. It is useful to note that the condition has not been reported after tracheal endoscopy procedures suggesting the condition is due to prolonged contact between the tube and tracheal mucosa. Of the nine cases reviewed for this paper 5 were macaw species and three cockatoos. The diameter of the normal trachea in both species reduces by up to 55% along its length (LENNOX 2004). The selection of an endotracheal tube based on glottis diameter might therefore be too large in these birds. This might also reect the difculty in diagnosing trachea stenosis in the smaller psittacine birds leading to under reporting of the condition.

2 MANAGEMENT OF POST-INTUBATION STENOSIS IN PSITTACINE BIRDS

Treatment of tracheal stenosis can range from conservative therapy, including antibiotics and anti-inammatory medications, to complete tracheal resection and anastomosis depending on the severity of the condition (BENNETT 2001). Adequate control of inammation is a prerequisite for successful management of tracheal trauma. Tracheotomy, resection of the stenosis followed by anastomosis is normally required (CLIPPINGER and BENNETT 1998). Incisions should be made between tracheal rings and closed with 4-0 to 8-0 monolament absorbable suture. Two rings on either side of the tracheotomy site should be included in each suture, placing knots externally to the tracheal lumen. Stenosis at the point of anastomosis is reported as a complication of surgery. Successful surgery depends on atraumatic tissue handling, careful placement of sutures, minimal number of sutures with adequate control of secondary infection and inammation (PYE 2000). Permanent tracheotomy has been described in psittacine birds and may be considered in unsuccessful resection cases. Of the nine cases reviewed 5 died or were euthanased within 6 months of repair. In the opinion of the authors PIS could be avoided by following these simple principals. The use of cuffed tubes should be avoided in birds. Always carefully select the tube diameter based on where the tube tip will rest in the trachea. Use sterile or adequately rinsed tubes. Intubate carefully under the appropriate plane of anaesthesia. Ventilate gently using the minimal pressure to achieve adequate ventilation. Avoid long intubation or anaesthetic times. Avoid bending the tube or trachea during the procedure.

3 CITATION INDEX
1. 2. 3. 4. 5. WAIN JC. Post-intubation tracheal stenosis. Chest Surg Clin N Am 2003; 13(2): 231 - 246. KLAINER AS, TURNDORF H, WU WH, et al. Surface alterations due to endotracheal intubation. Am J Med. 1975; 58(5): 674 - 683. LENNOX AM. Management of tracheal trauma in birds. Proc Assoc Avian Vet, New Orleans 2004, 339 - 343. BENNETT RA. Avian respiratory tract surgery. Proc Australian Assoc Avian Med 2001; 103-108. PYE GW. Surgery of the avian respiratory system. Vet Clinics North Am 2003; 3: 693 - 713.

513

6.

CLIPPINGER TL and BENNETT RA. Successful treatment of a traumatic tracheal stenosis in a goose by surgical resection and anastomosis. J Av Med Surg 1998; 12: 243 - 247.

AUTHOR ADDRESS
M. Lennox Avian and Exotic Animal Clinic of Indianapolis, Indianapolis, United States of America Email: BirdDr@aol.com

514

Great Western Referrals, Swindon, Shernacre Cottage, Malvern, United Kingdom

IXODES FRONTALIS AS A CAUSE OF AVIAN DISEASE IN THE UNITED KINGDOM

D. Monks BVSc (Hons) MACVSc (Avian Health) CertZooMed MRCVS N. Forbes BVetMed CBiol MIBiol DipECAMS FRCVS M. Fisher BVetMed CBiol MIBiol DipEVPC MRCVS

KEYWORDS
Ixodes frontalis - Tick - Avian - Haemorrhage - Oedema

ABSTRACT
This study obtained samples from 70 birds with attached ticks or signs consistent with tick related syndromes. It aimed to characterise ticks infesting birds in the UK; to test (via PCR) for tick-borne infections; and to determine the correlation between tick infestation and disease. The most frequently identied tick species was Ixodes frontalis. Forty-two birds (60%) had adult female I. frontalis ticks present, of which 25 birds (59.5%) had signs referable to tick related syndromes. PCR tests were uniformly negative for Ehrlichia, Bartonella, Babesia and Borrelia.

1 INTRODUCTION
Avian tick-related syndrome (TRS) is a syndrome in which birds with an attached tick present acutely lethargic and depressed or dead with mild to severe subcutaneous haemorrhage and oedema on the head or neck. This report aims to discuss the species of ticks infesting birds in the UK; test tick-borne infections via PCR; and to examine the correlation between tick infestation and disease.

2 MATERIALS AND METHODS

Submission kits and questionnaires were sent throughout the UK, requesting samples from wild and captive birds. Clinical presentations were categorised as associated with TRS or not (trauma, infection, parasitic, ocular, miscellaneous, unknown). Ticks were identied according to standard keys. PCR was performed on ticks, blood, spleen and aspirate from tick attachment site using a silica cartridge-based kit (MacheryNagel) and tested at the AcarusTM laboratory (Langford, UK).

515

3 RESULTS
Submissions were solicited from April 2000 to January 2004. The majority of submissions occurred in August and September, while no submissions were received in January or February. In total, 70 samples were tested. Sixty-eight of those were from birds with attached ticks. Most birds presented with one or two ticks. In total, 161 ticks were examined. All of the adult and nymph ticks were female. Adult female I. frontalis were present on 42 birds, of which 25 (59.5%) had signs of TRS. There was no correlation between numbers of attached, engorged adult I. frontalis females and clinical syndrome or outcome. Signs of TRS were also found with the presence of three I. frontalis nymphs and an unspeciated larva. All samples submitted to PCR tested negative for DNA of Borrelia burgdorferi sensu lato, Babesia species, Bartonella species and Ehrlichia species with the AcarusTM battery of tests.

4 DISCUSSION
Ixodes frontalis is a specic avian parasite and has been documented to cause TRS in birds (CHASTEL et al. 1991). Our study would seem to indicate that there is a strong correlation between attachment of adult female I. frontalis and TRS. Tissue oedema and haemorrhage associated with I. frontalis attachment has only been previously reported with multiple engorged adult female ticks (CHASTEL et al. 1991). However, in this study one case of swelling was associated with three I. frontalis nymphs while another was associated with an unspeciated larva. Although it is possible that fullyengorged adult I. frontalis had previously detached from the birds, the absence of an adult tick on these cases raises the possibility that other life cycle stages may be capable of causing disease in certain hosts. On PCR no sample tested positive for B.burgdorferi sensu lato, Bartonella, Ehrlichia and Babesia in our study. It is possible that other pathogens, including viruses, may be responsible for TRS. Additionally, some ixodid species produce specic salivary toxins (LUTTRELL et al. 1996). The host response to tick infestation may contribute to the aetiology of TRS. Avian tick-related syndrome is clearly an area requiring further research.

5 ACKNOWLEDGEMENTS
The project was supported by grant funding from BSAVA Petsavers. PCR testing was subsidized by the AcarusTM laboratory and their staff, especially Drs Martin Kenny and Susan Shaw, are thanked for their work on this project.

516

6 CITATION INDEX
1. CHASTEL C, GUIGUEN C, CHASTEL O and BEAUCOURNU JC. Pathological effects, vector role and new host of Ixodes Pari (= Ixodes frontalis) Acari Ixodoidea Ixodidae, Annales de Parasitologie Humaine et Comparee 1991; 66(1): 27 - 32. LUTTRELL MP, CREEKMORE LH, AND MERTINS JW. Avian tick paralysis caused by Ixodes brunneus in the southeastern United States. J Wildl Dis 1996; 32(1): 133 - 136.

2.

AUTHORS ADDRESS
Deborah Monks Great Western Referrals, County Park Business Park, Shrivenham Road, Swindon, SN1 2NR, United Kingdom, Email: d.monks@gwreferrals.co.uk

517

Department of Animal Production, Epidemiology and Ecology1, University of Turin, Italy; National Ornithological Association of Cuba2; Dep. of Animal Health, Agricultural University of La Havana Cuba3; Zapata Swamp National Park4 National Organization of Flora and Fauna Conservation5 Cuba.

THE ROLE OF AVIAN MEDICINE IN A PARROT CONSERVATION PROJECT ON AMAZONA LEUCOCEPHALA IN CUBA : PRELIMINARY RESULTS
E. Bert1, L. Tomassone1, G. Forneris1, C. J. Soto Pieiro2, I. Acosta Guevara1, E. Cruz Lpez1, M. Gonzles Navarrete3, S. Alvarez4 , X. Glvez Aguilera5

KEYWORDS
Amazona l. leucocephala - Conservation - Breeding - Wild populations Haematology Parasitology - Chlamydial antibodies.

ABSTRACT
Research on three different Cuban Amazon (Amazona l. leucocephala) populations within a conservation project in Cuba, is described. Haematological parameters, presence of parasites and prevalence of Chlamydial antibodies were evaluated. No Chlamydial antibodies were detected, and very few endo and ectoparasites were found (5 cases of Ascaridia spp., 12 of Dermanissus spp.).

1 INTRODUCTION
Amazona l. leucocephala is a Cuban endemic subspecies, included in the IUCN Red List of the endangered species (GRAJAL 2000). These parrots are traditionally considered companion birds in the houses of Cubans. Despite the international laws that forbid the capture and trade of these birds, poaching still occurs, in particular of nestlings, which are sold in Cuba (WILEY et al. 1995; WRIGHT 2001). Farming these birds is an interesting alternative to the capture of wild parrots; birds raised in captivity could satisfy the demand for domestic birds without affecting the size of wild populations. Veterinary medicine can have an important role in species preservation, monitoring health conditions and supporting breeders. Within a conservation project, we investigated the health status of A. leucocephala parrots in Cuba from three distinct populations.

518

2 MATERIALS AND METHODS

From March to September 2004, three parrot populations were studied. Group 1 comprised 15 nestlings from a wild population living in the Los Indios Ecological Reserve (LIER) of Juventud Island. Group 2 comprised 50 birds (18 juveniles and 32 adults) living in a breeding station in the Zapata Swamp National Park (ZSNP). In this centre, the aviaries were located in an open space, at the limits of the tropical forest and were in contact with wild birds. Group 3 comprised 55 Cuban Amazons (21 juveniles and 34 adults) kept in captivity in private houses in La Havana. Blood, faecal and ectoparasite samples were collected. Haematocrit and red blood cells count were carried out in a eld laboratory. Chlamydial antibodies were investigated with the ImmunoComb Avian Chlamydophila psittaci Antibody test kit, thanks to a donation by the manufacturers (Biogal Galed labs, Israel).

3 RESULTS
No Chlamydial antibodies were detected. Few endo and ecto-parasites were found: including no cases of haemoparasites, 5 cases of Ascaridia spp., and 12 cases of Dermanissus spp. Average haematological values corresponded to published values for other Amazon species. No differences in haematological parameters were noted between the second and third groups (Zapata Swamp National Park and La Havana captive birds respectively) or between juveniles and adults. The nestlings of the wild population in Los Indios showed lower blood parameters (Table 1).

4 DISCUSSION
Hematological parameters of adult and juvenile Cuba Amazons kept in captivity correspond to those reported for other Amazon species (FUDGE 2000). In the wild population, in which only nestlings have been sampled, results are difcult to interpret; moreover, they cannot be considered denitive and a reference range could not be established because of the small sample size. According to our data, endoparasitic infections do not appear to be very frequent in this species and cause little damage. On the contrary, ectoparasites seem to have a signicant impact. In the case of the LIER population, in nests with intense infection, chick condition was poor and an intense anemia was noted. The presence of ectoparasites was noted in the ZSNP, probably due to the location of the aviaries. A control plan for parasites is advisable in breeding centres. Our study is the rst of the kind carried out so far on A. leucocephala in Cuba. We believe that the role of veterinarians is important in a parrot conservation project, both to study the physiological characteristics of the species and to recognize the situations when intervention may be necessary. Another important aspect is providing suggestions to owners during the clinical visits, in order to increase the life expectancies of subjects kept in captivity.

519

5 LITERATURE CITED
1. 2. 3. 4. FUDGE AM. Laboratory Medicine - Avian and Exotic Pets. Sound Company, Philadelphia, USA; 2000: 486. GRAJAL A. Neotropics. In: SNYDER NMC et al. (eds): Parrots Action Plan. Status survey and conservation Plan 2000-2004. IUNC, Gland, Switzerland and Cambridge, UK. 2000: 2 - 55 WILEY JW, GNAM RS, GLVEZ X. Nest site ecology of the Cuban Parrot. Report of activities during 1995. WPTI Grant 94/95. N.010: 17. WRIGHT T. et al. Nest poaching in neotropical parrots. Cons Biol 2001, 3: 710 - 720.

AUTHORS ADDRESS
Dr. Elena Bert, DVM, PhD Department of Animal Production, Epidemiology and Ecology, University of Turin, Via Leonardo da Vinci 44, 10095 Grugliasco (Torino), Italy Email: Elena.bert@unito.it

520

Table 1. Haematological parameters of the wild population of A. leucocephala (n=15) (Reference values from FUDGE 2000). RBC (x103/ L) 1.70 1.26 2.10 2.45-3.18 160-175 5000-17000 125-250 6020-4408 176 5312 48.71 35-72 31-71 42 26-63 20-67 MCV (fL) WBC /L Heter (%) Linf (%) Mon (%) 1.69 1-2 0-2 Bas (%) 0.62 0-1 0-2 Eos (%) 0 0 0

Htc (%)

Nestlings

29.29

521

Range

25.5 - 34.5

Reference

41-53

Practical laboratories, European Association of Avian Veterinarians Conference

The texts will be given to the participants during the practical labs

L. Crosta, J.M. Pricard. Avian endoscopy N.A. Forbes. Avian soft tissue surgery S. Orosz, J.M. Hatt. Orthopaedic surgery in birds N. Harcourt-Brown, J.P. Andr Avian clinical imaging, basic and advanced R.T. Korbel, N. Bohnet. Avian ophthalmology: principles and applications G.M. Dorrestein, H. Pendl. Basic and advanced avian cytology and haematology M. Lithtenberger, J. Morrissey. Emergency and critical care in birds J. Hooimeijer, B. Speer. Behavior, Handling and Restraint of Parrots M. Lierz. Raptor biomedicine

523