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UNIVERSITATEADETIINEAGRICOLE

IMEDICINVETERINAR
IONIONESCUDELABRADIAI

LUCRRITIINIFICE
VOL.53(12)

MEDICINVETERINAR
PARTEA4

LucrrileSimpozionului
PROGRESSANDPERSPECTIVESIN
VETERINARYMEDICINE
Iai,June10th11th2010

EDITURAIONIONESCUDELABRAD
IAI2010

COLEGIULDEREDACIE

Redactorresponsabil:
Prof.univ.dr.GheorgheSOLCAN

Redactoradjunct:
Prof.univ.dr.OctavianZaharieOPREAN

Membri:
Prof.univ.dr.CorneliuCOTEA
Prof.univ.dr.VasileVULPE
Prof.univ.dr.MihaiCARPCRARE
Prof.univ.dr.DanDRUGOCIU

COMISIADEREFERENITIINIFICI
Prof.univ.dr.OctavianZaharieOPREAN
Prof.univ.dr.AbdelfatahNOURUniversitateaPudue,SUA
Prof.univ.dr.H.C.FrancoisCRESPEAUENVAlfort,France
Prof.univ.dr.GheorgheSOLCAN
Prof.univ.dr.LiviuMIRON
Prof.univ.dr.GheorgheSVUA
Prof.univ.dr.GabrielPREDOIFMVBucureti
Prof.univ.dr.IoantefanGROZAFMVClujNapoca
Prof.univ.dr.GheorgheDRBUFMVTimioara
Prof.univ.dr.CorneliuCOTEA
Prof.univ.dr.MihaiCARPCRARE
Conf.univ.dr.erbanMOROANINSERMParis
Prof.univ.dr.H.C.LiviuRUNCEANU
Prof.univ.dr.TudorPERIANU
Prof.univ.dr.IoanCOMAN
Prof.univ.dr.ElenaVELESCU

Volumulafosteditatcusprijinulfinanciaral
MinisteruluiEducaiei,Cercetrii,TineretuluiiSportului

ISSN:14547406

UniversitateadetiineAgricoleiMedicinVeterinarIai

CUPRINS
PARTEA3
PRECLINICS

DETERMINATIONOFTESTOSTERONEPLASMALEVELSINRAMSBYGASCHROMATOGRAPHY
SandaANDREI,BOGDANL.,FOCSANEANUV.,GROZAI.,DianaCRAINIC,AnaMariaPETREAN

EVALUATIONOFOXIDATIVECHANGESINDUCEDBYSUBCLINICALMASTITISINCOWSON
MILKLIPIDS
SandaANDREI,PinteaAdela,GrozaI.,CrainicDiana,MateiSorana,CiupeSimona,Bogdan
Sidonia

THEIMMUNOSTIMULATORYEFFECTOFTHELEPTINONTHEMESENTERICLYMPHNODEIN
WISTARRAT
G.R.BOTEZATU,C.COMAN,ManuellaMILITARU,N.CONSTANTIN

THEDYNAMICSOFTHESHELLMEMBRANESFORMATIONINTHEISTHMUSOFTHE
LOHMANNBROWNLAYINGHENS
C.TODIREANU,C.COTEA,CarmenSOLCAN

THEMORPHOLOGYANDHISTOCHEMISTRYOFTHEMAGNUMDURINGTHEEGGPASSAGE
THROUGHTHEOVIDUCT,INHENSDURINGTHELAYINGCYCLE
C.TODIREANU,C.COTEA,CarmenSOLCAN

HISTOLOGICALRESEARCHESINMALEPSEUDOHERMAPHRODITISMINDOMESTICSWINES
(SUSSCROFADOMESTICA)
CIORNEICristina,COTEAC.V.,SOLCANCarmen

HISTOLOGYCALRESEARCHESOFTRUEBILATERALHERMAPHRODITISMINDOMESTIC
SWINES(SUSSCROFADOMESTICA)
CIORNEICristina,COTEAC.V.,SOLCANCarmen

COMPUTERIZEDMICROSCOPYRESEARCHONSTRUCTURALBIOLOGYOFTHESEMINIFEROUS
TUBULESANDINTERSTITIALGLANDATTHECOCKS6090DAYSOLD
ValericaDNACU,N.CORNIL,NicoletaMOCANU,tefaniaPREDOI,M.CORNIL.

COMPARATIVESTUDYOFTHETHORACICLIMBSKELETONINTHELION(PANTHERALEO)
ANDINTHETIGER(PANTHERATIGRIS)
C.DEZDROBITU,MelaniaCRIAN,Al.GUDEA,A.DAMIAN,Fl.STAN,IoanaCHIRILEAN,
Fl.TUNS,IrinaIRIMESCU

ANTIOXIDANTACTIVITYOFWILDPANSY(VIOLATRICOLOR)ANDBLESSEDTHISTLE
(CNICUSBENEDICTUS)EXTRACTSONLINOLEICACIDSISTEM
CorinaDURDUN,MariaCRIVINEANU,V.NICORESCU

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ORGANOGENESISOFTHEHARDERIANGLANDINRABBITS(ORYCTOLAGUSCUNICULUS)
ElenaCtlinaFLOREA,C.V.COTEA,CarmenSOLCAN

RESEARCHONTHEMORPHOLOGYOFTHECAROTIDARTERYATGOAT(CAPRAHIRCUS)
B.GEORGESCU,G.PREDOI,C.BELU,N.CORNIL,I.DUMITRESCU,PetronelaROU,
CarmenBIOIU,AncaEICARU

THEINFLUENCEOFREMEDYBIORONPROTEINMETABOLISMINYOUNGRABBITS
NataliaIACUB

EFFECTSOFTHEREMEDYBIORONCERTAINHEPATICINDEXESINYOUNGRABBITS
V.MACARI,NataliaIACUB,D.MAENCU,AnaMACARI,NataliaPAVLICENCO

MANIFESTATIONSOFALKALINEPHOSPHATASEINBLOODSERUMOFBROILERCHICKS
TREATEDWITHTHEBIORREMEDY
V.MACARI,V.PUTIN,CristinaGAVRIL,AnaMACARI,A.POPESCO

MACROSCOPICANATOMOPATHOLOGICALCHANGESINNATURALINFESTATIONWITH
ARGASPERSICUS
S.MORARIU,Gh.DRBU,P.BRIL,M.ILIE,FloricaMORARIU,M.CRSTEA,

SPONTANEOUSCASESOFCHRONICHEPATOPATHIESINDOGS
NAGYA.L.,C.CTOI,A.GAL,P.BOLF,M.TAULESCU,F.TBRAN,COSMINACUC,G.BORZA,
R.MOUSSA

USEOFBLOODPARAMETERSINHEMATOLOGYANDCYTOLOGYHEALTHCRECKCARPS
(CYPRINUSCARPIO)REAREDINSYSTEMSEMIINTENSIVE
L.OGNEAN,AniAlinaRodica,CristinaCERNEA,M.CERNEA,C.PESTEAN,MedaMaria
MOLDOVAN

THEUSEOFSOMEQUICKTESTSFORBLOODTYPINGINDOGSANDCATS
L.OGNEAN,S.HABAGO,MedaMOLDOVAN,I.MORAR

MORPHOPATHOLOGICALCHANGESINAVIANINFECTIOUSBRONCHITIS,INTHE
RESPIRATORYFORM,INCHICKENSANDASPECTSOFHISTOPATOLOGICALDIAGNOSISOF
DISEASE
A.OLARIUJURCA,M.COMAN,I.OLARIUJURCA,A.STANCU,A.LAZU,E.AVRAM

POTASSIUMDICHROMATEIMPACTONSOMEBIOMARKERSOFFEMALEPHYSICAL
DEVELOPMENT(VAGINALOPENING)INRATS(THREEGENERATIONSTUDY)
SnejanaPETROVICI,AlexandraTRIF,Muselin,F.,Cristina,R.T.,MilcaPETROVICI

CONSEQUENCESOFPOTASSIUMDICHROMATEINTAKEONENERGETICPROFILEINFEMALE
RATS(SIXMONTHSEXPOSURE)
SnejanaPETROVICI,AlexandraTRIF,EugeniaDUMITRESCU,MilcaPETROVICICameliaTULCAN

MORPHOLOGICALOBSERVATIONSOFNEURONSINTHESPINALNUCLEUSBULBATSHEEP
AlinaPOPA,C.V.COTEA,CarmenSOLCAN,StefaniaANDERCO

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THEFORMANDTHELOCATIONOFNUCLEIOFTHERAHIDIANBULBATSHEEP
AlinaCoraliaPOPA,CorneliuV.COTEA,MihaelaSPATARU

HEPATICLESIONSINDOLPHINSSTRANDEDFROMTHEROMANIANBLACKSEACOAST
OanaMelaniaPOPA,M.COMAN,AlexandraTRIF,A.STANCU,DianaARGHERIE

MONITORINGTHEANTITOXICEFFECTOFSOMEGLUTATHIONERICHVEGETALPRODUCTS
CorneliaPRISCARU

STUDYOFSOMEOXIDATIVESTRESSPARAMETERSDYNAMICSUNDERTHEINFLUENCEOF
THEEXOGENASCORBICACIDCONTRIBUTIONINSUBACUTEACRYLAMIDEINTOXICATION
AncaIrinaPRISCARU,CorneliaPRISCARU

EFFECTSOFTHEREMEDYBIORDRUGONTHETRYPSINANTITRYPSINSYSTEMINBROILER
ChicksV.PUTIN

CLINICAL,ANATOMOPATHOLOGICALANDHISTOLOGICALASPECTSINGUINEAPIGS
SENSITIZEDWITHM.bovisAN,USEDTOBOVINEPPDSTANDARDIZATION
MariaMioaraRDU,P.TIUBE,VioricaCHIURCIU,t.POPESCU

THECONSEQUENCESOFHEXAVALENTCHROMIUMCOMPOUNDSIXMONTHSEXPOSURE
ONSOMEMORPHOLOGICALBIOMARKERSINMALERATS
JelenaRANKOV,AlexandraTRIF

SIXMONTHSEXPOSURETOPOTASSIUMDICHROMATEOUTCOMESONHISTOARCHITECTUR
EOFGENITALORGANSANDSEXUALACCESSORYGLANDSINMALERATS
JelenaRANKOV,AlexandraTRIF,DianaBREZOVAN

THEPECULIARITIESOFTHEPELVICLIMBMUSCLESINREDSQUIRREL(SCIURUSVULGARIS)
MihaelaSPATARU,C.SPATARU,V.COTOFAN,M.LAZAR,A.MUNTEANU

MORPHOFUNCTIONALPARTICULARITIESOFTHEAXIALSKELETONATREDSQUIRREL
(SCIURUSVULGARIS)
C.SPATARU,MihaelaSPATARU,V.COTOFAN,V.VULPE,M.LAZAR,A.MUNTEANU

MORPHOFUNCTIONALPECULIARITIESOFTHETHORACICLIMBJOINTSATREDSQUIRREL
(SCIURUSVULGARIS)
C.SPATARU,MihaelaSPATARU,M.LAZAR,A.MUNTEANU

CLINICS

ANEOPLASICENCEPHALOPATHYINDOGCASEREPORT
MihaelaARMAU,M.MUSTEA,CarmenSOLCAN,Gh.SOLCAN

RESEARCHREGARDINGTHEINCIDENCE,DIAGNOSISANDTREATMENTOFRETAINED
PLACENTAINAUSTRIANBROWNCOWS
L.BOGDAN,I.S.GROZA,SimonaCIUPE,M.CENARIU,I.PACA,SandaANDREI,
AnamariaPETREAN,SoranaMATEI,SidoniaBOGDAN

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DIETARY,MAINTENANCEANDPHYSIOPATHOLOGYFACTORSINVOLVEDINTHEETIOLOGY
OFKETOSISINDAIRYCOWS
V.BOGHIAN

CORRELATIONBETWEENMORPHOPATHOLOGICALEXAMANDEVOLUTIVESTAGEIN
NATURALINFECTIONWITHEQUINEINFECTIOUSANEMIAVIRUS
P.BOLF,C.CTOI,M.TAULESCU,A.GAL,CosminaCUC,R.MOUSSA,F.TBRAN,
A.NAGY,G.BORZA,C.MUREAN,MarinaSPNU

USINGNUTRITIONALMETABOLICPROFILEASATOOLFORPROGNOSISGROUPOFDAIRY
COWSINFARM
S.I.BOR,L.RUNCEANU,Gh.SOLCAN,D.DRUGOCIU,P.ROCA,R.MLNCU

RESEARCHABOUTMORPHOLOGICALASPECTSWITHABACTERIALINFECTIONONBROILERS
RAISEDONGROUND
OanaBUSUIOC

THEEFFICACYOFMACROCYCLICLACTONESANDBENZIMIDAZOLESCOMBINATIONIN
EQUINESTRONGYLIDOSIS
LauraCristinaCERNEA,M.CERNEA,L.OGNEAN,V.NSTASA,.RILEANU,M.MARE,
AncaCHEREJIL.M.MadeiradeCARVALHO

THEEFFICACYOFTETRAHYDROPYRIMIDINESANDBENZIMIDAZOLESCOMBINATIONIN
EQUINESTRONGYLIDOSIS
LauraCristinaCERNEA,M.CERNEA,L.OGNEAN,V.NSTASA,.RILEANU,M.MARE,
AncaCHEREJIL.M.MadeiradeCARVALHO

BIOLOGICALCONTROLOFPASTURESINFESTEDWITHEQUINESTRONGYLSLARVAE
M.CERNEA,LauraCristinaCERNEA,.RILEANU,V.NSTASA,M.MARE,L.OGNEAN,
L.M.MadeiradeCARVALHO

THEEFFICACYOFHERBALEXTRACTONEQUINESTRONGYLIDOSIS
M.CERNEA,LauraCristinaCERNEA,.RILEANU,V.NSTASA,M.MARE,L.OGNEAN,
L.M.MadeiradeCARVALHO

CONTRIBUTIONSTOTHESTUDYOFECTOPARASITESDEMODEXCANISLOCALIZATIONON
DOGS`BODYSURFACE
Al.D.CHESLER,M.D.CODREANU,IulianaCODREANU,D.CRNGANU,A.BLB

INVESTIGATIONCONCERNINGTHEINFLUENCEOFBIOTICANDABIOTICFACTORSONTHE
INCIDENCEANDPREVALENCEOFCANINEDEMODICOSIS
Al.D.CHESLER,M.D.CODREANU,I.DUCA,IulianaCODREANU,A.BLB,

RESEARCHONEFFICIENCYOFPRESYNCHOVSYNCHPROTOCOLONCOWSFROM
HOLSTEINBREED
C.A.CHIRU,

DIROFILARIAREPENSINFECTIONPREVALENCEINHUNEDOARACOUNTY
RobertaCIOCAN,Gh.DRBU,M.S.Ilie,MirelaIMRE,IonelaHOTEA

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SALPINXDISORDERSASAFACTOROFINFERTILITYINCOWSFORMILKPRODUCTION
t.Gr.CIORNEI,L.RUNCEANU,D.DRUGOCIU,P.ROCA,V.D.PDURARU,C.BLU,
GALANC.,I.DOROBANU

BIOTECHNOLOGYRESEARCHONRECOVERY,MORPHOLOGICALASSESSMENTANDVIABILITY
TESTINGOFCATOOCYTESBEFOREANDAFTERINVITROMATURATION
MariaSimonaCIUPE,I.t.GROZA,L.BOGDAN,EmokePALL,Al.POP,AnamariaPETREAN,
SandaANDREI,SoranaMATEI,AndreaCRIAN

CORRELATIONBETWEENTHECLINICALANDEVOLUTIVEEXPRESSIONANDTYPEOF
ULTRASOUNDCHANGESINTHEDIAGNOSISOFCAVITARYORGANS`DISEASESINDOG
M.CODREANU,CristinaFERNOAG,M.CORNIL,IulianaCODREANU,D.CRNGANU,
M.TURCITU

STUDYREGARDINGTHECORRELATIONBETWEENTHECLINICALFEATURESAND/ORTHE
TYPEOFULTRASOUNDCHANGESINTHEDIAGNOSISOFTHEPARENCHYMATOUSDISEASES
INDOG
M.CODREANU,CristinaFERNOAG,M.CORNIL,IulianaCODREANU,C.ERDEAN,
M.TURCITU,

PROFILACTICOTHERAPEUTICSTRATEGIESINTHEDOGDISORDERSMALIGNANTPROSTATE
D.CRINGANU,ManuelaMILITARU,M.CODREANU,Al.DIACONESCU,CristinaPREDA,
BogdanGHEONGHEOS

THEDIAGNOSTICANDTHERAPYAPPROACHINTHEURINARYTUMORALPROCESSESINDOG
D.CRNGANU,CristinaPREDA,M.CODREANU,RalucaCRNGANU,V.NICORESCU

FREERADICALSSCAVENGINGACTIVITYOFSOMEHYDROALCOHOLICPHYTOCOMPOUNDS
MariaCRIVINEANU,CameliaPAPUC,CorinaDURDUN,V.NICORESCU

THEEFFECTSOFTHIAMPHENICOLADMINISTRATIONUPONKIDNEYSINRATS
MariaCRIVINEANU,V.NICORESCU,CameliaPAPUC,MariaVOICU(MIU),ElenaROTARU

MORPHOLOGICALASPETCSOFTHEMAJORSALIVARYGLANDSINTHERAT
A.DAMIAN,V.MICLU,I.PAPUC

EYEFUNDUSVASCULARPATTERNINSOMEDOMESTICANIMALS
AlinaDONISA,A.MUSTE,I.PAPUC,F.BETEG,M.MUSTE

MORPHOLOGYCALASPECTSOFTHEEYEFUNDUSINDOMESTICRABBIT(ORYCTOLAGUS
CUNICULUSDOMESTICUS)
AlinaDONISA,A.MUSTE,F.BETEG,A.KRUPACi,M.MUSTE

MEDICINESFORBEESCURRENTSITUATIONANDFUTUREASPECTS,ANIMPORTANT
SUBJECTDEBATEDATEUROPEANLEVEL
AlinaKarinaDRAGHICI,AncaBITOIU,LollitaTABAN

ANTIHELMINTICSRESISTANCEINCATTLEANATIONALANDEUROPEANPROBLEM
AlinaKarinaDRAGHICI,I.DUCA,MirelaPREDA,D.PREDA

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RESEARCHREGARDINGTHEEFFICIENCYOFROMFENBENDAZOL0%SUSPENSION
TREATMENTINSOMEHELMINTOSISINSHEEP
ElenaDRAGOSTIN,C.TUDORAN,CtinCHIURCIU,I.IACOB

OBSERVATIONREGARDINGOVARIANDISORDESATDAIRYCOWSANDTHEIRINFLUENCE
ONREPRODUCTIVEFUNCTION
DanaSimonaDRUGOCIU,AlinBIROIU,CristinaLoredanaPANAITE,M.Fl.FOICA,
ManuelaSTNESCU,

LEADACETATEIMPACTONFUNDAMENTALBIOMARKERSOFREPRODUCTIVE
FUNCTIONALITYINFEMALERATS(INUTEROEXPOSURE)
EugeniaDUMITRESCU,AlexandraTRIF,R.T.CRISTINA,Fl.Muselin,

INDEXOFPARASITICIMPACTUPONTHEPHYSIOLOGICINDICESINBOVINES
D.ERHAN,Ol.CHIHAI,GalinaMELNIC,t.RUSU,MariaZAMORNEA,NinaTLMBU,
V.BUZA,T.ANGHEL

ANTIBACTERIALANDPROTECTIVEEFFECTOFALLIUMSATIVUMONINTESTINALFLORAIN
RATS(RATTUSNORVEGICUS)
N.FI,Fl.CHIRIL,G.NAD,AdrianaCRISTE

STUDIESABOUTMICROBIALETIOLOGYANDANTIBIOTICRESISTANCEINSUBCLINICAL
MASTITISINCOWS
N.FI,F.CHIRIL,S.RPUNTEAN,G.NAD,SandaANDREI,SoranaTeodoraMATEI

RESEARCHONAVIANCOLIBACILLOSISINTHECASEOFINTENSIVEBREEDING
DianaGALAANU,T.PERIANU,OanaTNASE

CLINICALASPECTSOFSOMEPODALDISEASESINBOVINEFROMDIFFERENTBREEDING
SYSTEMS
DanGSC,

INSULINDETERMINATIONINCATSWITHDIABETESMELLITUS
LuminitaDianaHRICU

NEBULISATIONCOMPLEMENTARYTHERAPYFORRESPIRATORYDISEASESINCATS
LuminitaDianaHricu

PERCUTANEOUSNORMOGRADEINTRAMEDULLARYPINNINGOFFELINEFEMURFRACTURES
C.IGNA,LarisaSCHUSZLER,RoxanaDASCLU,M.SABU,C.LUCA

OSTEOGENESISCAPACITYOFFREEVASCULARIZEDCORTICOPERIOSTEALFLAPINDOGS
C.IGNA,C.LUCA,RoxanaDASCLU,LarisaSCHUSZLER,M.SABU

RFIDTECHNOLOGYUSEDFORMONITORINGESTRUSTEMPERATUREINCOW
Fl.IONESCU,R.HUU,A.CIMPONERIU,M.CHILINANR.URIAN,I.HUU

VENTRALOSTEOTOMYINTHETREATMENTOFOTITISMEDIAINDOG
AndreiKRUPACI,AurelMUSTE,FlorinBETEG,RaduLACATUS,AlinaDONISA,
FlorinaAlexandraKRUPACI

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EXPERIMENTALPERFORATORDORSALTEGUMENTARYPIGCOETANEOUSPEELBYUSING
NONIONICCONTRASTMEDIA
R.LCTU,I.PAPUC,A.MUSTE,B.CHIROIU,IleanaMATEI,F.ARDELEAN,R.C.PURDOIU,
AlexandraNicoletaPVLOIU

USAGEOFNONIONICCONTRASTMEDIAFORTHEIDENTIFICATIONOFPERFORATING
CUTANEOUSARTERIESINRATSWITHAPPLICATIONSINPLASTICSURGERY
R.LCTU,I.PAPUC,F.ARDELEAN,B.CHIROIU,IleanaMATEI,A.MUSTE,R.C.PURDOIU,
AlexandraNicoletaPVLOIU

ENDOSCOPICALANDSURGYCALCOMPLEXTECHNIQUESFORTHEDIAGNOSTICAND
TREATMENTOFBONEYFOREIGNBODYRETAINEDINTHORACICESOPHAGEALSEGMENT
D.C.LESCAI,F.DUMITRESCU,ADELAMENDEA,L.HARBUZ,I.BURTAN

OBSERVATIONSONTHEELECTROTHERAPYUSEFORTHEPOSTOPERATIVEFRACTURE
RECOVERYINDOG
LAURALIVITCHI,A.MUSTE,F.BETEG,I.SCURTU

THEUSEOFULTRASONOGRAPHYINGASTROINTESTINALDISEASEINDOGS
R.N.MLNCU,Gh.SOLCAN,CristinaTOFAN(MALANCUS)

IMPORTANCEOFDIAGNOSTICIMAGINGINHYDRONEPHROTICKIDNEYSURGERY
ADELAMENDEA,N.HAGIU

COMPARATIVECHARACTERISTICSOFDUROCANDWHITELARGEDILUTEDBOARSEMEN
STOREDFORFIVEDAYSAT7CINRELATIONTOFERTILITY
C.MIRCU,VioletaIGNA,CameliaTULCAN,H.CERNESCU,AncaLELESCU,SimonaZARCULA,
R.I.URIANSUESCA,G.OTAVA,V.ARDELEAN,Gh.BONCA

DENTALFRACTUREINDOGS:INCIDENCE,CLINICALANDTHERAPEUTICALLYASPECTS
A.MUSTE,FL.BETEG,I.PAPUC,ALINADONISA,R.LACATUS,M.MUSTE

SURGICALPROTOCOLINDOGPERINEALHERNIA
A.MUSTE,BETEGFL.,TNASEA.,DONISALINA,MUSTEM.

SOMEOBSERVATIONSREGARDINGSTANDINGCASTRATIONINHORSES
L.OANA,C.OBER,C.PESTEAN,V.MICLAUS,A.N.OROS,O.NEGREA,DanielaOROS

ASPECTSREGARDINGTHETECHNIQUEOFORCHIECTOMYINROOSTERS(GALLUSGALLUS)
L.OANA,C.OBER,C.PETEAN,V.MICLU,O.NEGREA,A.N.OROS,L.OGNEAN,
DanielaOROS

THECORRELATIONBETWEENTHEPROGESTERONELEVELS67DAYSPOSTINSEMINATION
ANDTHEPREGNANCYLOSSINCOWS
G.OTAVA

CORELATIONSBETWEENBIOLOGICALVALUEANDFERTILITYINBEEFBULLBREEDS
V.D.PDURARU,D.DRUGOCIU,D.ALEXA,M.GRIGORA,CO.A.OJOCARU

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HEMATOLOGICALANDBIOCHEMICALMODIFICATIONSOFBLOODANDCEREBRALSPINAL
FLUIDINDOGMYELOGRAPHYUSINGNONIONICCONTRASTSUBSTANCESOPTIRAY0AND
ULTRAVIST70
I.PAPUC,R.LCTU,A.MUSTE,R.C.PURDOIU,S.BORBIL,AlexandraNicoletaPVLOIU

CLINICALANDPARACLINICALDIAGNOSISINSPINALCHORDCONDITIONSINDOGS
I.PAPUC,R.LCTU,C.MUREAN,AlexandraNicoletaPVLOIU,R.C.PURDOIU

ADVANCEDBIOTECHNOLOGIESAPPLIEDINCARNIVORESREPRODUCTIONINORDERTO
IMPROVEFERTILITY
C.PAVLI,OanaTANASE

PROSTATEPATHOLOGYINDOG,DIFFERENTIALDIAGNOSTICBETWEENCYSTS,ABSCESSAND
NEOPLASM
ConstantinPAVLI,OanaTANASE

GONIOSCOPYWITHIMAGEACQUISITIONATDOGSANDCATS
B..RUGIN,I.BURTAN,L.C.BURTAN,DianaIAMANDI

STUDIESREGARDINGTHEPREVALENCEANDDETERMINANTFACTORSOFTHEMAINDISEAS
ESINDAIRYCOWS
ElenaRUGINOSU,t.CREANG,AncaPLVNESCU,L.DASCLUD.ANI,AdrianaANI,Cr
istinaREBEGEA,Gh.SOLCAN

THEROLEOFLIDOCAINECONTINUOUSINFUSIONINPAINCONTROLAFTERORTHOPEDIC
SURGERY
LarisaSCHUSZLER,C.IGNA,M.SABU,RoxanaDASCLU,A.SALA,C.LUCA

ATTEMPTTOINDUCEIMMUNOTOLERANCEINXENOTRANSPLANTUSINGSKINCELLS
MonicaERE,E.TRZIU,IleanaNICHITA,C.CUMPNOIU,D.CIOCA

NEWANDIMPROVEDOSTEOSYNTHESISTECHNIQUESUSEDINFOREARMFRACTURE
V.INDILARE,V.VULPE,S.PACA,S.GRMAD

RESTORATIVEMODERNMATERIALSUSEDINVETERINARYDENTALMEDICINE
V.INDILARE,S.GRMAD

AGEDEPENDENTEFFECTSOFCERTAINVEGETALEXTRACTSONTHEINNATECELLMEDIATED
IMMUNITYINCHICKENS
MarinaSPINU,CarmenSANDRU,Gh.F.Brudac,MihaelaNICULAE,D.CADAR,Krisztina
RINDT,TimeaKISS,ArmelaBORDEANU,FlorinaAlexandraKRUPACI,SilvanaPOPESCU,
R.TEFAN

VEGETALEXTRACTSINTERACTWITHPHYSIOLOGICALCELLMEDIATEDIMMUNEACTIVITYIN
FARMEDHERBIVORES
MarinaSPINU,CarmenSANDRU,Gh.F.Brudac,SilvanaPOPESCU,MihaelaNICULAE,
D.CADAR,KrisztinaRINDT,TimeaKISS,FlorinaAlexandraKRUPACI,ArmelaBORDEANU,
R.TEFAN

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FREQUENCYANDETIOPATHOGENESISOFHIPDYSPLASIAINDOGS
R.E.STECYK,L.C.BURTAN,IoanaBURCOVEANU

RESEARCHOFNEGATIVEENERGYBALANCEANDITSINFLUENCEONTHEREPRODUCTION
FUNCTIONINDAIRYCOWS
CristinaMariaTOFAN(MLNCU),D.Gh.DRUGOCIU

PARACLINICALDIAGNOSISOFHEPATICDISEASEINCATTLE
CristinaTOFAN(MLNCU),Gh.SOLCAN,D.DRUGOCIU,R.N.MLNCU

ULTRASOUNDDIAGNOSISINEARLYPREGNANCYTOBUFFALO
G.TOMAI,I.MORAR,I.St.GROZA

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PARTEA4
INCIDENCEOFHUMANCRYPTOSPORIDIOSISINBUCHARESTBETWEEN20002005PERIOD
AdrianaAMFIM,MonicaPRVU,VioletaElenaSIMION

EPIDEMIOLOGICALRESEARCHONOUTBREAKSOFAVIANINFLUENZAINLETEAANDPLAUR
FROMTULCEACOUNTY
M.ARSENE,ARSENEMarinela,TERTISMarinela,P.BRIA,D.MAFTEI,LaurentaBOICIUC

ETIOLOGYANDPATHOLOGYINPORCINERESPIRATORYDISEASECOMPLEX(PRDC)IN
FINISHINGPIGS
S.BRITREANU,MihaelaBEZMAN,S.MARINACHE,DCOBZARIU,GabrielaBAGRINOVSCHI,
MariaOELEA,M.V.CMPEANU,SimonaIVANA,DoinaDANE

SIGNSANDLESIONSOFTHESPONTANEOUSACUTEPNEUMONICPASTEURELLOSISINPET
RABBITS
S.BRITREANU,D.COBZARIU,MariaOELEA,M.V.CMPEANU,SimonaIVANA,
DoinaDANE

TREATMENTEFFICACYINUDDERLOCALISATIONOFCONTAGIOUSECTHYMAINSHEEPAND
GOATS
R.BIA,G.RPUNTEAN,S.RPUNTEAN

EFFECTIVENESSOFTREATMENTFORMULAINCONTAGIOUSECTHYMAINLAMBSFROM
INTENSIVEBREEDINGSYSTEM
R.BIA,S.RPUNTEAN,G.RPUNTEAN

CLINICALANDANATOMOPATHOLOGICALRESEARCHINPORCINECIRCOVIRUSTYPE
INFECTIONINYOUNGSWINE
N.CTANA,V.HERMAN,IonicaFODOR,V.PETRUSE

SEROLOGICALANDPCRRESEARCHDIAGNOSISOFTHEINFECTIONWITHPORCINECIRCOVIR
USTYPEINYOUNGSWINE
N.CTANA,VIRGILIAPOPA,V.HERMAN,IonicaFODOR

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RESEARCHCONCERNINGTHEANTIBACTERIALACTIVITYOFHONEYAGAINSTCOAGULASE
NEGATIVESTAPHYLOCOCCIWITHUSEINVETERINARYMEDICINE
C.CEAUI,I.OGOE,ANCAMARIAGALI,L.TUDOR,I.L.ILIE,

RESEARCHESREGARDINGTHEEFFICIENCYOFPLANTSESSENTIALOILSEXTRACTSONSOME
BACTERIALSTRAINSISOLATEDFROMMASTITISCOWMILK
F.CHIRIL,N.FI,S.RAPUNTEAN,G.NAD,O.NEGREA

RESEARCHONTHEFREQUENCYOFBACILLUSCEREUSINMILKASRAWMATERIAL
C.CIOTU,E.V.INDILAR

OBSERVATIONSREGARDINGTHEHISTOLOGICALSTRUCTUREOFTHEFOOTMUSCLEOFTHE
FOODSNAILH.pomatia
AndreeaFlaviaCIRLAN,E.SINDILAR,

OBSERVATIONSREGARDINGTHEHISTOLOGICALSTRUCTUREOFTHEGENITALSYSTEMOF
THEGARDENSNAILH.POMATIA
AndreeaFlaviaCIRLAN,E.SINDILAR,

THECONCORDANCEBETWEENTHETOTALGERMSNUMBER(TGN)FROMTHESHELLAND
THETOTALGERMSNUMBERFROMTHEFOOTOFTHEFOODSNAILHELIXPOMATIA
AndreeaFlaviaCIRLAN,ESINDILAR,

MAINANTIBIOTICSUSEDINTHETREATMENTOFUDDERINFECTIONSINHUMAN
CONSUMPTIONMILKSUPPLIERANIMALSPECIESFROMSOUTHEASTERNSIBIUCOUNTY
M.CRSTEA,R.TRIF,S.MORARIU,FloricaMORARIU

PREVALENCEOFMASTITISINCOWS,SHEEPANDBUFFALOESINFIVELOCALITIESFROM
SIBIUCOUNTY
M.CRSTEA,R.TRIF,S.MORARIU,FloricaMORARIU

RESEARCHRELATEDTOTHEREFINEMENENTOFTHEPOULTRYASPRODUCTSWITHOUT
PROTECTIVEMEMBRANE
LilicaCOCLEA,AngelaGAVRILA,D.SIMEANU,CristinaSIMEANU

RESEARCHRELATEDTOTHEREFINEMENENTOFTHEPOULTRYASPRODUCTSWITH
PROTECTIVEMEMBRANE
LilicaCOCLEA,AngelaGAVRILA,D.SIMEANU,CristinaSIMEANU

INVITROACTIVITYOFSOMENATURALESSENTIALOILSAGAINSTTHEYEASTLIKEALGA
PROTOTHECA
CosminaBOUARI(CUC),
Gh.RPUNTEAN,C.CTOI,N.FI,G.NAD,P.BOLF,M.TAULESCU,A.GAL,R.Moussa,A.
NAGY,F.TBRAN

ORGANOLEPTICRESEARCHOFSALAMIDURINGTHEVALIDITYPERIOD(FOOD
DETERIORATION)
F.M.FOICA,M.CARPCRARE,DanaSimonaDRUGOCIU

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RESEARCHCONCERNINGDIARYCOWSWELFAREINAFARMNEARBUCHAREST
F.FURNARIS,ElenaMITRANESCU,L.TUDOR,VioletaSIMION,C.TOGAN,B.TASBAC,
S.BUSTANI

RESEARCHESREGARDINGAIRQUALITYINNEAMTCOUNTY
F.FURNARIS,ElenaMITRANESCU,CatalinaLALA,L.TUDOR,MagdalenaGONCEAROV,
MarianaERBEA,L.I.ILIE

RESEARCHONAVIANCOLIBACILLOSISCONDITIONSININTENSIVEREARING
DianaGALAANU,T.PERIANU,OanaTNASE

RESEARCHCONCERNINGTHEINFLUENCEOFTHERMALTREATMENTONTHEQUALITYOF
FLORALHONEYPRODUCEDINROMANIA
ANCAMARIAGALI,I.OGOE,L.TUDOR,I.L.ILIE,MITRNESCUElena

RESEARCHCONCERNINGTHEMOISTURECONTENTANDVISCOSITYOFROMANIANHONEY
STOREDATDIFFERENTTEMPERATURES
ANCAMARIAGALI,I.OGOE,L.TUDOR,I.L.ILIE,MITRNESCUElena

AUDITSYSTEMATICANDINDEPENDENTEXAMINATIONOFEFFECTIVEIMPLEMENTATIONOF
SANITATIONPROGRAMS
MAGDAGONCIAROV,R.POPA,L.TUDOR,DOINALUPU,ELENAMITRNESCU

OFFICIALCONTROLCONTROLMETHODFORCHECKINGCOMPLIANCEWITHFOODLAW
MAGDAGONCIAROV,R.POPA,L.TUDOR,DOINALUPU,ELENAMITRNESCU

ANTIMICROBIALSENSITIVITYOFE.COLISTRAINSISOLATEDFROMPIGSEPTICEMIC
COLIBACILOSIS
V.HERMAN,CorinaPASCU,LuminiaCOSTINAR,B.FAUR,IoanaVDUVA,AncaSURPAT,Sorin
aIRIMIE

STUDYCONCERNINGTHESEASONALVARIATIONOFCATTLEMILKPHVALUESANDTHE
OVERALLINFLUENCEONMILKQUALITY
L.I.ILIE,L.TUDOR,AncaMariaGALI,ElenaMITRNESCU,R.F.POPA

RESEARCHCONCERNINGTHESEASONALVARIATIONOFCATTLEMILKDENSITYVALUES
L.I.ILIE,L.TUDOR,AncaMariaGALI,ElenaMITRNESCU,F.FURNARIS

ASTUDYONTHEBEHAVIOROFBROWNBEAR(URSUSARCTOS)FROMZOOLOGICAL
GARDENTIMISOARA
C.Fl.LZRESCU,AdinaBAIAS,M.AFRENIE

RESEARCHESREGARDINGIMPLEMENTATIONOFACOMPUTERIZEDSURVEILLANCE
PROGRAMOFMAMMARYGLANDINADAIRYCOWFARM
SoranaTeodoraMATEI,I.GROZA,L.BOGDAN,SimonaCIUPE,AnamariaPETREAN,
SandaANDREI

SPECTROPHOTOMETRICDETERMINATIONOFNITRITEANDNITRATEINMEATPRODUCTS
MirelaMICLEAN,CeciliaROMAN,LauraPARLAPAN,IoanStefanGROZA

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STUDIESCONCERNINGLEPTINSINFLUENCEONTHESCCINBUFFALOMILK
M.MIHAIU,AlexandraLAPUSAN,,CrinaTeodoraCARSAI,CarmenJECAN,RomolicaMIHAIU,
S.D.DAN

STUDYCONCERNINGMOLDOVARIVERWATERQUALITYINNEAMTCOUNTY
ElenaMITRANESCU,CatalinaLALA,F.FURNARIS,L.TUDOR,MarianaERBEA,D.MITRNESCU
,VioletaSIMION

RESEARCHESCONCERNINGFISHWELFAREINAFARMFROMSOUTHERNAREAOFROMANIA
ElenaMITRANESCU,F.FURNARIS,L.TUDOR,MagdaGONCIAROV,VioletaSIMION,
S.BUSTANI,AdrianaORASANU

RESEARCHESREGARDINGTHESENSITIVITYTOANTIBIOTICSOFSOMEBACTERIALSTRAINS
ISOLATEDFROMMASTITISCOWMILK
G.C.NAD,N.Fi,F.CHIRIL,S.RPUNTEAN,V.RUS

EVALUATIONOFPHOSPHORUSLEVELSINLAMBBYLUCERNEHAYFEEDINGTESTS
AritinaNEAGU,V.CRIVINEANU,G.V.GORAN

SOMEINVESTIGATIONSINCONSUMPTIONSEAFISH(HERRING,MACKEREL)
O.NEGREA,CameliaRADUCU,L.OANA,VioaraMIRESAN,Z.MARCHIS,Gh.MIHAI,F.CHIRILA,
AncuaROTAR

ASPECTSREGARDINGEPIZOOTICMETALINGUATULOSAINMAINDOMESTICRUMINANT
SPECIES
O.NEGREA,CameliaRADUCU,L.OANA,VioaraMIRESAN,Z.MARCHIS,V.MICLAUS,
F.CHIRILA,AncuaROTAR

APPLICATIONSOFBIOMEDICALINFORMATICSANDTELEMEDICINE
T.NICA,V.CRIVINEANU

BUILDINGANDMANAGINGANONLINEDATABASEOFTHEMOSTIMPORTANT
TOXICOSESINVETERINARYMEDICINE
T.NICA,V.CRIVINEANU

ANTIMICROBIALRESISTANCEOFCAMPYLOBACTERJEJUNIANDCAMPYLOBACTER
COLISTRAINSISOLATEDFROMBROILERSSKIN
IsabelaNICORESCU,MariaCRIVINEANU

INCIDENCEOFSALMONELLASPP.INAPOULTRYSLAUGHTERINGUNIT
M.OBAD,AlinaVLADSABIE,M.CARPCARARE

THEPROTECTINGEFFECTOFAPOLYPHENOLICEXTRACTOBTAINEDFROMSEABUCKTHORN
(HIPPOPHAERHAMNOIDES)FRUITSAGAINSTPHOTOOXIDATIONOFDIFFERENTVEGETAL
OILS
CameliaPAPUC,V.NICORESCU,CorinaDURDUN,Gh.GORAN,CarmenCRIVINEANU

OBSERVATIONSONAVIANINFLUENZA
T.C.PATACHE,M.ARSENE,ARSENEMARINELA

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MORPHOLOGICALQUALITYINFLUENCEOFSHEEPOOCYTESONINVITROMATURATION
AnamariaPetrean,I.Groza,L.Bogdan,SimonaCiupe,SoranaMatei,AL.Pop

CREATINGBESTPRACTICEGUIDESBASICREQUIREMENTFORACHIEVINGSAFEAND
HEALTHYFOOD
R.POPA,MAGDAGONCIAROV,L.TUDOR,DOINALUPU

STUDYONTHERISKANALYSIS(IDENTIFICATIONOFHAZARDS,THEIREVALUATION,
IDENTIFYINGPREVENTIVEMEASURESTOCONTROLRISK)
RAREPOPA,MAGDAGONCIAROV,LAURENIUTUDOR,DOINALUPU

THEASSESSMENTOFHYGIENEINDAIRYCOWSHEDSWITHTIESTALLSINTRANSYLVANIA
SilvanaPOPESCU,CristinBORDA,MarinaSPINU,R.STEFAN,CarmenD.SANDRU,
EvaA.LAZAR

THELEVELOFMICROCLIMATEPARAMETERSINDAIRYCATTLEBARNSWITHTIESTALLS
SilvanaPOPESCU,CristinBORDA,IulianaCHEGEDUS,R.STEFAN,EvaA.LAZAR

STUDIESCONCERNINGHYGIENICBODYSCORESANDTHERISKOFINTRAMAMMARY
INFECTIONSINCOW
ElenaROTARU,B.TABAC,ElenaMITRNESCU,MariaCRIVINEANU,V.NICORESCU,MagdaG
ONCIAROV

KEYBIOCHEMICALPARAMETERSCHANGESOFTHEAPISMELLIFERACARPATHICABEEIN
STRESSFULCONDITIONS
apcaliuAgripina,I.Rdoi,TudorPoliana,TutunaruAlexandru,MgdiciMaria,CuiaEliza,
N.Tudor

THEEFFECTOFXAGO(X)[BOCAOPO]OXIDEPOWDERSONGRAMPOSITIVEBACTERIA
R.STEFAN,SilvanaPOPESCU,MarinaSPINU,N.FIT,A.MACRI,G.NADAS

THEANTIBACTERIALEFFECTOFSILVERCONTAININGBOCAOPOGLASMATRIXON
GRAMNEGATIVEBACTERIA
R.STEFAN,MarinaSPINU,SilvanaPOPESCU,N.FIT,MariaBINDEA,FloreCHIRILA

MANGANESEIMPACTONMALEREPRODUCTIVESYSTEMINTEGRITYANDPERFORMANCES
BIOMARKERS
NicoletaSimonaSteliac(Munteanu),AlexandraTrif

ANATOMOPATHOLOGICALANDEPIDEMIOLOGICALSTUDYOFVISCERALANDNONVISCERAL
HEMANGIOSARCOMAINDOGS
TBRANA.F.,C.CTOI,A.GAL,P.BOLF,M.TAULESCU,A.L.NAGY,COSMINACUC,
G.BORZA,R.MOUSSA

ARGULUSSPP.(FISHLOUSE)INFECTIONINACOMMONCARP(CYPRINUSCARPIO)FROMA
FISHFARMINCLUJCOUNTY
M.TAULESCU,C.CTOI,G.BORZA,P.BOLF,A.NAGY,A.GAL,F.TABARAN,COSMINACUC,
R.MOUSSA,A.BARBU

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LOSSOFBODYWEIGHTASANINDICATORFOREVALUATINGLAMBSWELFAREDURINGTRA
NSPORTATION
Marianaerbea,ElenaMitrnescu

COMPARATIONOFTWOMETHODSFORDETERMININGSALMONELLAspp.
I.IBRU,SORINAMARIAIRIMIE

THEDETERMINATIONOFCOLISTINRESIDUESFROMPORKMEATANDORGANSUSING
MICROBIOLOGICALANDHPLCANALYSIS
V.TRIFAN,MariaCRIVINEANU,A.iOANCEA,C.LUPESCU,V.NICORESCU

RESEARCHCONCERNINGTHEEXTRACTIONMETHODSFORPATHOGENICYERSINIA
ENTEROCOLITICAFROMPORKMEAT
La.TUDOR,I.OGOE,I.L.ILIE,ANCAMARIAGALI,ANETALAURATUDOR

RESEARCHCONCERNINGTHEIDENTIFICATIONOFSEROTYPEO:9OFYERSINIA
ENTEROCOLITICAFROMMEAT
L.TUDOR,I.OGOE,I.L.ILIE,ANETALAURATUDOR,ANCAMARIAGALI

DIROFILARIAIMMITISINFECTIONANEWCHALLENGEFORVETERINARYPRACTITIONERSIN
IASICOUNTY
D.ACATRINEI,L.MIRON,SimonaDIMITRIU,AnaMUSTEA,LarisaPARASCA,RamonaORIC

STUDIESCONCERNINGTHEHUMORALIMMUNERESPONSEATGOATSAFTERVACCINATION
AGAINSTGANGRENOUSMASTITIS
A.Tudose,TurcuD.,T.Perianu,MarianaOporanu,P.Grigorescu,D.Condur,T.Petru

THEEVOLUTIONOFRABIESINTHENEIGHBORINGCOUNTRIESOFROMANIAINTHEPERIOD
20052008
IuliaAdelinaTURIAC,F.MAZDRAG,C.VASIU.

RESEARCHONRABIESEPIDEMIOLOGYOFANIMALSINMUNTENIA,DURING20052009
IuliaAdelinaTURIAC,C.VASIU,F.MAZDRAG

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INCIDENCEOFHUMANCRYPTOSPORIDIOSISINBUCHAREST
BETWEEN20002005PERIOD

AdrianaAMFIM,MonicaPRVU,VioletaElenaSIMION
FacultateadeMedicinVeterinar,UnversitateaSpiruHaret,
Str.MainadePine,nr.7,Bucureti,Romnia
adrianaamfim@yahoo.com
Abstract
At present both in Romania and internationally, is a parasitic disease of public health issues
carefullymonitoredbecausehighprevalence,healthproblemsthelackofdiagnosisandcorrect
treatment,problemsofdifferentialdiagnosisandnotleastmanyeconomicimplications.
Thepurposeofthisstudyistodeterminetheincidenceofcryptosporidiosisinhumansdynamics
in Romania over a period of time and the category of age at highest risk of contamination
Criptosporidiumsp.
Setting annual growth rate of new cases of cryptosporidiosis in human populations was done
usingthedatabaseprovidedbytheNationalCenterforOrganizationandInformationSystemand
Information Assurance in Health, Bucharest, Romania. The database is the number of cases of
illnessoccurredannuallyduring20002005,inBucharest,theratiooffamilydoctorsandhospital
doctorsinspecialtyreporting.Epidemiologicalassessmenttookaccountofcryptosporidiosisand
age group affected, sothat data interpretation was made for four age groups (under one year
old, 114 years, 1564 years, over 64 years). Research methodology consisted of retrospective
analysis,basedonanalyticalinvestigationsusingstatisticalprocessingofdatafromquantitative
assessmentofepidemiologicalparameters(incidence).
Thus were established as the age category with the highest degree of contamination
Criptosporidiumparvumandtemporaldistributionofnewcasesofdiseaseoccurredduringthe
six years of epidemiological monitoring. Also epidemiological assessment of cryptosporidiosis,
given the time and age group, according to the source reporting has shown a consistency of
epidemiologicalinformationfromsecondarysourcesreporting.

Keywords:cryptosporidiosis,zoonosis,incidence

INTRODUCTION

Cryptosporidiosis is a parasitic zoonosis caused by a protozoan of the genus


Cryptosporidium. By eliminating a large number of oocysts in the environment that can
contaminate many animals, cryptosporidiosis is a public health problem. Indeveloped
countries 0.20.4% of oocysts excreted healthy and diarrhea sickness oocysts secretion is
reported in 22.5% of the population (3). Cryptosporidiosis incidence in patiens with BDA,
around the world is: 0.619.3% in Chile, 13% in India, 9% Liberia, 9.3% in Finland, 4.8% in
Australia,2.8%intheU.S.,0.6%inCanada(3).
TheprevalenceofcaseswithC.hominisis62%anditisspecifictoSouthAmerica,
Australia and Africa. Criprosporidiosis in Europe is produced by C. parvum and disease
prevalence is 57% (1). It has developed severe disease especially in immunosuppressed
persons(HIVpatients)(1).
Cryptosporidiosisbeingacommondiseaseofyounganimals,especiallyfarmanimals,
causing high levels of mortality and morbidity, is that it is considered among the most
important parasitic zoonoses in nature (4). Highest frequency of cases characterized rural
areas.Theprevalenceinanimalsisgenerallyestablishedforfarmanimals,being55.6%(2).

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Lucrritiinificevol53seriaMedicinVeterinar
AlthoughtheetiologicagentofdiseaseisincludedintheO.I.E.ListBand"Zoonoses
directive 2003/99/EC, cryptosporidiosis is declared only a few countries (Germany and
Sweden) and recorded only sporadic reports in the Netherlands and Spain. Surveillance
monitoring of water and therefore casuistry with Criptosporidium sp., is adopted by United
Kingdom law only. Drinking Water Directive 98/83/EC, requires European countries to take
measurementsofwaterpollutionbyCryptosporidiumsp.,wherethewatersamplesexamined
wereidentifiedClostridiumperfringens(RealDecreto140/2003,BOE21/02/2003)(5).W.H.O.
report for to ouer country 20 children under one year decease/1000 by B.D.A, also 14% of
deathsinHIVpatientsisduetoinfestationwithCryptosporidiumsp.(3).

MATERIALANDMETHODS

This studywasconductedtomonitorthehealthofhumanpopulationinBucharest,
within a specified time period (2000 2005) in the incidence of cryptosporidiosis, using two
sources of data provision.The study was the number of new cases of cryptosporidiosis in
humans during 20002005, reported by family practitioners and specialist outpatient,
Bucharest. Centralization of data was the National Center for Organization and Information
SystemandInformationAssuranceinHealthinBucharest.
Themodeconsistedofasurveyofdescriptiveepidemiologyusingtransversal
descriptivestudy,withapplicationsinassessingandmonitoringthehealthofhuman
populationbetween2000to2005.Inpreparingthedatabasehavecomefollowingsteps:
1.selectionofdatasources;
2.preparationofthedatabase;
3.establishmentofcriteriabywhichtomakeinterpretationofdata;
4.analysisandstatisticalprocessingofdata;
5.disseminationofresults.

1. Selection of data sources was consistent with this model in descriptive


epidemiologicalinvestigation.Ittooksoonlydatasourcesprovidedbysecondarycare(family
offices / outpatient specialist) using medical records. Centralization of data was made by a
nationalbodyempoweredunderArt.108oftheRomanianConstitution,republished,art.14
andart.43para.(2)ofLawno.95/2006,representedbytheNationalCenterforOrganization
andInformationSystemandInformationAssuranceinHealth,Bucharest,Romania.

2.Inpreparingthedatabasethefollowingstepshavebeentaken:
evaluation of data for monitoring the human population in Bucharest
criptosporidiosisthroughout6years;
datacollection;
enteringdataintothedatabase;
datamanagement

3.Thecriteriaaccordingtowhichthedatabasehavebeenprocessed:
epidemiologicalindextoassesstheepidemiologicalsituation(incidenceorfrequency
ofnewcasesofcriptosporidiosisinhumans);
sourcesreportingnewcasesofdisease(familyofficesandoutpatientspecialist);
temporaldistributionofdisease(timewithestablishedscope2000to2005);
measurementofdiseasedistributionbyage(lessthanoneyear,114years,15to64,
over64.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
4.Dataanalysiswasdoneusingdescriptivevariablessuchquantitytheincidenceof
criptosporidiosis in the human population per calendar year, age group and source of data
reporting.Itwasobtainedbycomparingtheincidenceofnewcasesofdiseaseoccurringeach
year from 100,000 during the study. Most convenient summary format and presentation of
datawasbyfrequencydistributioncharts(linegraphs,chartstructure,Chart,ScatterDiagram,
etc.). Epidemiological analysis of criptosporidiosis in humans was done by linking incidence
datawithcalendarperiod,agegroupandcomparedthetworeportingsources.
5.Disseminationofresultsmaybeintheformofreportsaimedtoprovideknowledge
andraiseawarenessofhealthproblemsinhumans.

RESULTSANDDISCUSSION

Afterprocessingandinterpretingstatisticaldatabasefortheperiod20002005could
make the following observations about the evolution of human cryptosporidiosis in the
populationmeasuredintermsoftemporaldistributionofdiseaseandagegroup,inBucharest
fromdoctorsreportingfamily(Table1).

Table1
Cryptosporidiosisincidencepercalendaryearandagegroup(reportingbyphysicians)
(indexedat100000inhabitants)
I%(100.000
Under1
1564
Year
inhabitams)
year
114years
years
64years
2000

0.54

0.00

0.00

0.47

1.46

2001

1.55

0.00

0.81

1.71

1.43

2002

1.24

7,23

0.43

1.34

1.10

2003

0.36

0.00

0.00

0.42

0.36

2004

1.81

0.00

1.44

1.89

1.79

2005
1.35
11.18
2.01
1.21
1.42

Between20002005theincidenceofcryptosporidiosisinthehumanpopulationhad
valuesrangingfrom0.361.81%.Highestincidencewasrecordedin2004(I1.81%),andthe
minimum value recorded in 2003 (I 0.36%). By analyzing the graph is observed that the
differencebetweenincidencevaluesthroughoutthestudyperiodisonly0.99%(Chart1).

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Graphics1.TemporaldistributionoftheincidenceofcryptosporidiosisinBucharestinthe
period20002005(referencefamilydoctors)

Distribution of new cases of disease occurring in each calendar year in the study
accordingtoageofpatientsisasfollows(Chart2):

Graphics2Measurementofdistributionofcryptosporidiosisincidenceduring20002005,
accordingtoage(reportingfamilydoctors)

for2000thepopulationsegmentmostaffectedwasthatofpeopleagedover 64(I
1.46%);
themostaffectedsegmentofthepopulationin2001wasthatofpersonsaged1564
years(I1.71%);
for2002itwasobservedalowerincidenceofbetween0.43%forpersonsaged114
yearsand7.23%forchildrenunderoneyear;
in2003thepopulationsegmentmostaffectedwasthatofchildrenaged114years(I
0.42%);

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maximumincidencefor2004wasrecordedbyagecategory1564years(I1.89%);
in2005inBucharest,themostaffectedsegmentofthepopulationwerechildrenaged
underoneyear(I11.18%).
Health monitoring in the human population in terms of cryptosporidiosis in the period
20002005wasalsoachievedbyretrospectiveepidemiologicalsurveyofhospitalphysicians
for specialty reporting. The epidemiological analysis were established following on the
frequencywithwhichnewcasesofdiseaseoccurredinagecategoryduringthesixyearsof
study(table2,scheduleno.3).
Table2

Cryptosporidiosisincidencepercalendaryearandagegroup(reportinghospitalspecialist)
(indexedat100000inhabitants)
Under1
Year
Total
year
114years 1564years Over65years
2000

2001

0.35

0.00

0.00

0.48

0.00

2002

0.10

0.00

0.86

0.00

0.00

2003

0.41

0.00

2.28

0.21

0.00

2004

0.15

0.00

0.48

0.14

0.00

2005

0.10

0.00

0.00

0.14

0.00

Ggraphics3TemporalDistributionofcryptosporidiosiscasesaccordingtothestudygroup
(reportingspecialtyhospital)

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in 2000 were not reported new cases of disease contamination with C. parvum,
NationalCentreforOrganisationandInformationSystemandInformationAssurance
inhealth.
yearwiththehighestincidencewasin2003(I0.41%);
childrenunderoneyearaswellaspeopleover65wererecordedduringtheentire
periodofstudyanincidenceof0.00%;
agegroupmostaffectedwasthatofchildrenaged114years(I0.002.28%);
Comparatively analyzing data from the two sources of reporting, can make the
followingobservations(table3,graph4):
frequency of new cases of cryptosporidiosis in Bucharest for the period under
studyisdifferentdependingonthesourcereporting;
epidemiological data from family practices indicate a higher risk of
contaminationwithhumanpopulationCriptosporidiumsp.;
outpatientspecialtyshowlowervaluesofincidenceduringthesixyearsofstudy
and summary reports (in 2000 were not reported new cases of
cryptosporidiosis).
Table3
Cryptosporidiosisincidenceduring20002005inBucharest,accordingtothesource
reporting

Year
I%cryptosporidiosis
I%cryptosporidiosis(specialty
(familydoctors)
hospital)
2000
0.54

2001

1.55

0.35

2002

1.24

0.10

2003

0.36

0.41

2004

1.81

0.15

2005

1.35

0.10

Graphics4.Comparativemeasurementofthedistributionofcryptosporidiosisincidence
during20002005,accordingtothesourcereporting
Conclusions

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UniversitateadetiineAgricoleiMedicinVeterinarIai
This study the epidemiological investigations have used such descriptive
epidemiologicalinvestigation,aimedatestablishingthefrequencywithwhichnewcasesoccur
in humans cryptosporidiosis in Bucharest, in a period of time. Quantifying epidemiological
indicatortheincidencewasgiventhetemporalcharacteristicofthedisease,measuringthe
agedistribution,reportingdifferentsource.
Statistical processing of the database shows that both the temporal distribution of
diseaseandsettingupthecontaminationriskpopulationisdifferentaccordingtothesource
reporting.Maximumincidenceisdifferentdependingonthesourcereportinghavingvaluesof
0.41%ofambulatoryspecializedreportingand1.81%accordingtodatacollectedfromfamily
practitioners.
Offamilyphysiciansreportingcryptosporidiosisisanintestinalparasitosisthataffects
allagegroups(bothchildrenunderoneyearandadults64yearspast).Alsosmalldifferences
betweenincidencevaluesbasedonage,showsthatthediseasehasanuniformdistributionin
thehumanpopulationwithanaveragefrequencyofoccurrenceofnewcasesofdiseaseduring
thesixyearsisonly1.14%.
Epidemiologicalsurveillanceofcryptosporidiosisishavingasasourceofinformation
andreportingoutpatientspecialistinBucharest,establishedalowfrequencyofoccurrenceof
newcasesofcryptosporidiosisinperiod20002005(I0.100.35%).Regardingthedistribution
of disease according to age categories, children under one year as people over 65 were
recordedthroughoutthestudyperiodanincidenceof0.00%.
In Romania, cryptosporidiosis is a disease of animals included in the Stock
Surveillance Program, Prevention and Control of Animal Diseases, those transmitted from
animals to humans (...) ". Knowing its zoonotic nature, it must be monitored in order to
achieveSurveillanceandPreventionprotocolsforZoonosesofParasiticNature.Tothisend
onecanmakeotherkindlongitudinalepidemiologicalsurveyretrospectivecohort.

BIBLIOGRAPHY

1.
2.
3.
4.
5.

Cacci, S.,M.,DeGiacomoM., Aulicino F.A.,PozioE.Giardia cystsinwastewatertreatment


plantsinItaly.ApplEnviron.Microbiol.,69,33938,2003.
Cox,F.,E.,G.,Zoonoze,EdituratinelorMedicale,2005
Lazr,Lidia,Cocidiozedigestiveumane.Ed.MondocartPres,2002
Thompson,R.,C.,A.,Zoonoze,EdituratinelorMedicale,2005
***A European Network for the detection and Control of Cryptosporidium, Final Report,
februarie2006

915

EPIDEMIOLOGICALRESEARCHONOUTBREAKSOFAVIAN
INFLUENZAINLETEAAND
PLAURFROMTULCEACOUNTY

M.ARSENE,ARSENEMarinela,TERTISMarinela,P.BRIAl,
MAFTEIDaniel,BOICIUCLaurenta
DSVSATULCEA

StudieswereconductedinMarch2010inLeteaandreedvillagesinTulceacounty,and
followedtheclinicalcourseandepidemiologyofavianinfluenzainhouseholdsinthese
twolocalities.Thediseasewasmanifestedbyatrendsuperacutedepression,decreased
appetite,polydipsia,adinamie,headandneckedema,cyanosisandswellingridgesand
beards,conjunctivalcongestionandhypersecretion,cyanosisandbeardsridge.

Avian influenza virus can be isolated from active outbreaks of avian influenza, of
greater or lesser severity, but also from birds with discrete forms of disease or clinically
apparentlyhealthy.Inthefirstcasewespeakofepidemicsandoutbreaksofavianinfluenzain
thesecondcasewespeakonlyofinfectionwithinfluenzavirus.
Amongpoultry,avianinfluenzawasreportedmostfrequentlyinturkey,duck,goose,
partridge, pheasant, chicken and various psitacide. Among many species of birds have been
isolated from avian influenza viruses, it referred primarily waterfowl, birds that inhabit the
shores(seagulls,seaswallows)migratorybirds,especiallywebfooted.
Easy transmission of avian influenza as well as asymptomatic infections, is usually
withinthesamespecies.Inotherwords,whenitisanactiveoutbreakofinfluenza,asourceof
infectionforbirdsareallbirds,andthesourceofinfectionforhumansisstillaman.
Localities of the Delta system practiced extensive growth of poultry. Although this
systemisprohibitedbylawincreaseveterinaryhealthisdifficulttocontrolandimposedaban
growthpoultryinopensystembecausethebenefitsofgrowthshowsthatway.
These flocks reared semisalbaticie and those who are allowed to leave the court
holding daily household contacts of people have permanent or periodic, regular birds,
implicitly wild bird species are incriminated in the epidemiology of the disease (avian
influenza).

MATERIALANDMETHODS

ResearcheswereconductedinMarch2010 inavianinfluenzaoutbreaks inlocalities


Leteaandreed,TulceaCounty,andpresentsepidemiologicalandclinicalaspectsofdiseasein
terms of the Danube Delta. For diagnosis were collected corpses of cloacal and tracheal
samples, serum samples were both serological and virological worked in the Laboratory
VeterinaryandFoodSafetyTulceaandconfirmationwasmade attheInstituteofDiagnostic
Bucharest.

RESULTSANDDISCUSSION

On 03/11/2010 there were the first clinical signs in two households in the village
cycle. Household that was located on the ring canal that connects with the Danube. Letea

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Lucrritiinificevol53seriaMedicinVeterinar
village lies between the arms Chilia and Sulina on hill Letea. In this area have their habitat
manyspeciesofwildbirdsandmigratorywildbirdsthatspendalldevelopmentalstagesofthe
DanubeDeltaterritory.
On 14.03.2010 in the household that all birds died, so it was a rapid evolution of
disease.Birdsofthehouseholdwerethechickens,andtheirgrowthismade,household.
Following implementation of the program to prevent and control avian influenza in
theareawereexaminedclinicallyLetea1621to1561birdsinchickens,57ducks,28turkeys,
24geese,aguinea.Theexaminationsperformedwereidentifiedclinicalsignsofdiseaseand
mortality in three households located near the first outbreak. Clinical examinations were
performedinvillageslocatedinsurveillancezonelocalitiesCARosettiandSfistofca.Inthese
placestherewerereportedcasesofillnessormortality,andhouseholdswerenotonthebank
channel(birdswerenotdirectlyaccesstowater).
Affectedhouseholdsinfiveoutof89hens,atotalof68diedand21werekilledafter
theapplicationprogramtopreventandcontrolavianinfluenza.
On25/03/2010afterclinicalsupervisionwasfoundinahouseholdasthetotalof15
hens that were still held six live birds not clinical signs and two corpses were collected and
senttothelaboratory.Householdthatislocatedabout50mfromthebodyofwaterthatplace
is visited by migratory wild birds. Up to date 26.03.2010 suspicion was confirmed when the
remainingbirdsinthehouseholdhavedied.
Allsixcasesdiagnosedasavianinfluenzainhouseholdscitiesarelocatedonthesame
street,allwithoutthepool.
The program of surveillance affected households were harvested samples were
examined at LSVSA and confirmed at IDAH as positive in the direction A150 of highly
pathogenicH5N1.
Morphologicalandclinicalaspectsfoundinthebodiesofchickenswerethefollowing:
Swellingofface(Fig.3)
Swellinginconnectivetissueoftheneckanddorsalareaoftheneck(Fig.6)
Cyanosisandswellingridgesandbeards(Fig.1)
Hemorrhagictracheitis(Fig.4)
Bronchopneumonia
Liverdegeneration
Bruisingandpetechiaeinthelegs(Fig.2)
Kidneydegeneration
Catarrhalenteritishemorrhagic.
Cerebraledema(Fig.5)
On24/03/2010therewerethefirstcasesofillnessinahouseholdofPlaurwiththe
same clinical signs as the city Letea. Plaur is located downstream on the right arm of Chilia.
HouseholdthatislocatednearthearmChiliaat500mwhichisafloodedareawherethereare
suitable places for wild birds standing. On 25/03/2010 has been confirmed and suspected
avianinfluenzawereatotalof80affectedchickensinwhich28diedand52werekilled.The
diseasewastoofastwithhighmortality.Throughmonitoringlocalbirdshavebeenrecorded
casesofdeathorillnessinbirds.

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Fig.1CyanosisandswellingridgesandbeardsFig.2Bruisingandpetechiaeinthelegs

Fig.3SwellingoffaceatchickenFig.4Hemorrhagictracheitisatchicken

Fig.5CerebraledemaFig.6Swellinginconnectivetissueoftheneck
anddorsalareaoftheneck

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Lucrritiinificevol53seriaMedicinVeterinar

1.
2.

3.
4.

5.

6.
7.

CONCLUSIONS
Existing domestic waterbirds (ducks, geese), high risk areas for avian influenza is
themodeoftransmissionfromwildbirdstopoultry.
An important role in development and disease progression we have species "in
touch" as quasidomestic species to be mixed with water and wild birds while
attendinghumandwellingswherepoultryarekept.
Thediseaseappearedonlyinhouseholdswithsettlementnearshorewaters.
Because cohabitation poultry with migratory birds avian influenza virus could
enterthehouseholds.Thiswaspossiblebecauseofthefarmingsystem,particularly
webfootedinvillagesintheDanubeDelta.
Route of infection was a direct contact with infected migratory birds through
habitat and an indirect (body of water that infected migratory wild birds have
stopped).
Unlike poultry, wild birds typically do not exhibit symptoms of disease, but they
mayconstituteapermanentsourceofinfectioninlargeareasofworld.
Inallcasesclinicalsignswerethesame:headswelling,cyanosisandswellingridges
andbeards,hemorrhagictracheitis,bronchopneumonia,andhemorrhagicenteritis
etc.

BIBLIOGRAFIE
1.

2.

3.
4.

ArseneM.,ArseneMarinela,SavutaGhe.,T.Perianu(2006),Clinicalaspectsinthesummer
birdfluswan(Cygnusolor)ScientificPapers,VolUSAMVVeterinaryMedicine49(8),EdIon
IonescuBradIasi,p.774779;
Arsene M. , Arsene Marinela, Savuta Ghe., T. Perianu (2006), Clinical aspects on bird flu in
chickensScientificPapers,VolUSAMVVeterinaryMedicine49(8)EdIonIonescuBradIasi,p.
780784;
PerianuTudor,(2005)Infectiousanimaldiseases,virusII,EdUniversitasXXIIasi
MafteiD.,SiomcaO.,AvramM,BriaP,TertisM,ArseneM.,(2008)Roleintheevolutionof
avianmigratorybirdsintheterritoryofTulceaCounty,workshop7to8February.2008,Tulcea.

919

ETIOLOGYANDPATHOLOGYINPORCINERESPIRATORYDISEASE
COMPLEX(PRDC)INFINISHINGPIGS

S.BRITREANU ,MihaelaBEZMAN2,S.MARINACHE2,DCOBZARIU1,Gabriela
BAGRINOVSCHI3,MariaOELEA3,
M.V.CMPEANU3,SimonaIVANA1,DoinaDANE1
1
USAMVBucuretiInstitutuldeMedicinComparat
SplaiulIndependenei105,Sector5,Bucureti
2
SCIZOCONMCSRLBucureti
3
USAMVBucuretiFacultateadeMedicinVeterinar
SplaiulIndependenei105,Sector5,Bucureti
doruvet@yahoo.com
1

Respiratoryinfectionsarethemostprevalentinthegrowingpigfarms.Inthispaperitspresented
the resultsofscreeningcarriedout on PRDC factors in 11 farms and their economically impact.
Blood samples collected from 19 finishing pig herds 376 specimens were investigated by
HerdChek Pseudorabies Virus gB Antibody Test Kit, CHEKIT APPApxIV Actinobacillus
pleuropneumoniae Antibody Test Kit, HerdChek Micoplasma Hyopneumoniae Antibody Test Kit,
HerdChekPRRSVAbTestKit,HerdChekSwineInfluenzaVirusAntibodyTestKitH1N1,HerdChek
SwineInfluenzaVirusAntibodyTestKitH3N2,andHerdChekSwineSalmonellaAntibodyTestKit
(IDEXX Laboratories, Inc., USA). For postmortem investigations were collected 20 pigs from two
farms,and38pigsfromonefarmwereMycoplasmahyopenumoniaescoredinslaughterhouse.
The pulmonary lesions registered at the post mortem exam of finishing pigs suggested the
presenceofseveralpathogens,andserologicalinvestigationsrevealedthenext:IgGH1N149%
positive, IgG H3N2 67% positive, IgG PRRSV 12% positive, IgG PRV 39% positive, IgG
M.hyopneumoniae14,6%positive,IgGAPP19%positive,IgGSamlonella49,5%positive.The
investigationsperformedinslaughterhouseshowedthatthelevelofpulmonarylesionsdeclined
daily growth rate with 19 g/day/pig (the economical deficit of 5% pulmonary lesions was
2/pig/productioncycle).

Keywords:porcinerespiratorydiseasecomplex,swinepathology,PRDC

In professional swine herds the swine respiratory disease complex (PRDC) causes
majoreconomiclosses[12,16].AlthoughPRDCisdescribedinallagegroups,thispathologyis
usuallyreportedinfinishingpigswithagebetween14and22weeks[17,13].Theetiologyof
PRDC are involved bacteria, viruses and zoohygiene conditions that in a unfavorable
combination,overcomelocaldefensemechanismsandtriggersspecificrespiratorypathology
[6].
ThemostfrequentpathogenicvirusesassociatedwithPRDCarepigreproductionand
respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Pseudorabies virus (PRV),
porcinecircovirustype2(PCV2)andporcinerespiratorycoronavirus(PRCV)[15,11,15,17,
18]. These viruses can be isolated with one or more pathogenic bacteria. Depending on the
role of bacteria in onset morbid process, they may be primary pathogens (Mycoplasma
hyopneumoniae, Actinobacillus pleuropneumoniae, Bordetella bronchioseptica) or secondary
(Pasteurella multocida, Haemophilus parasuis, Streptococcus suis, Actinomyces pyogenes,
Salmonellacholeraesuis,Actinobacillussuis)[1,5,11,17].

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Lucrritiinificevol53seriaMedicinVeterinar
Evaluation of immune status to the main infectious agents (bacterial and viral) as
primaryorsecondaryrespiratorypathogensisapriorityinpreventivehealthmanagementof
swine farms [1]. List of pathogens active surveillanced by serological tests should be
establishedaccordingtolegalregulationsandcasehistory.
Thispapermainlyaimsdominantetiologicalandpathologicalaspectsassociatedwith
PRDCinRomania.Inthiscontextitanalyzesthedatacollectedfromserologicalinvestigations
ofswinefarmswhereclinicalexaminationandnecropsyrevealedtheexistenceofrespiratory
pathologyinpigsslaughteredordiedduringthefinishingperiod.

MATERIALSANDMETHODS

Pigs (1422 weeks) were collected from 11 farms, and 19 distinct lots. From
September2006toDecember2009werecollected376bloodsamples.Intwofarms(XandXI)
were additionally collected 20 pigs corpses that showed respiratory disease before death.
From a batch of pig (48 pigs) of farm XI have collected data on pulmonary pathology in the
slaughterhouseexamination(pulmonarylesions).
In the farm I samples were collected 25 (Lot 1: 140 150 days finishing pig), in the
farm II were collected 25 samples (Lot 2: 140150 days finishing pig), in the farm III were
collected 25 samples (Lot 3: 110 150 days finishing pig), in the farm IV were collected 40
samples(Lot4:110120daysfinishingpig20samples,Lot5:160180daysfinishingpig20
samples), in the farm V were collected 26 samples (Lot 6: 110125 days finishing pig 13
samples, Lot 7: 160170 days finishing pig 13 samples), in the farm VI were collected 60
samples(Lot8:130daysfinishingpig20samples,Lot9:170daysfinishingpig20samples,
Lot10:185daysfinishingpig20samples),inthefarmVIIwerecollected45samples(Lot11:
110 days finishing pig 20 samples, Lot 12: 130150 days finishing pig 25 samples), in the
farmVIIIwerecollected40samples(Lot13:110120daysfinishingpig20samples,Lot14:
160180daysfinishingpig20samples),inthefarmIXwerecollected20samples(Lot15:140
150daysfinishingpig),inthefarmXwerecollected40samples(Lot16:110120daysfinishing
pig20samples,Lot17:150180daysfinishingpig20samples),inthefarmXIwerecollected
30samples(Lot18:110daysfinishingpig15samples,Lot19:130160daysfinishingpig15
samples).
To assess immune status of pig herds in this study were used the following ELISA
diagnostic kits: HerdChek Pseudorabies Virus gB Antibody Test Kit (IDEXX Laboratories, Inc.,
USA), APPChek ApxIV Actinobacillus pleuropneumoniae (APP) Antibody Test Kit ( IDEXX
Laboratories, Inc., USA); HerdChek Mycoplasma hyopneumoniae Antibody Test Kit (IDEXX
Laboratories, Inc.., USA) Porcine Reproductive and Respiratory Syndrome HerdChek Virus
Antibody Test Kit (IDEXX Laboratories, Inc., USA); HerdChek Swine Influenza Virus Antibody
TestKitH1N1(IDEXXLaboratories,Inc.,USA);HerdChekSwineInfluenzaVirusAntibodyTest
Kit H3N2 (IDEXX Laboratories, Inc., USA); HerdChek Swine Salmonella Antibody Test Kit
(IDEXX Laboratories, Inc., USA). Working protocols used were those specific Serology
Laboratory of the Faculty of Veterinary Medicine Bucharest, in accordance with the
manufacturer'sprotocol(IDEXXLaboratories,Inc.,USA).
Pulmonaryandlymphnodelesionswereassessedandrecordedinconcordancewith
interpretationandclassificationschemerecommendedforthisspecies[14].Pulmonarylesions
characterizedbytheincreaseinvolume,hyperemiaandedemawereclassifiedasacute.
Pulmonarylesionscharacterizedbymoderateedemaandbronchialexudates,butno
increase in volume and hyperaemia were classified as subacute. Atelectic fibrotic lesions,
consolidated lesion with darkred appearance and dilated bronchi filled with inflammatory

921

UniversitateadetiineAgricoleiMedicinVeterinarIai
exudateswereconsideredchronic.Accordingtotheirsizelymphnodeswerescoredasnormal
= 0, moderate enlargement = 1 or marked enlargement = 2. All lungs examined were with
lobulartypelesions:cleardelineationfromlobularlevelofhealthytissuetothesick.

RESULTSANDDISCUSSIONS

Described as a multifactorial disease, PRDC cannot be managed without close


cooperationbetweenthepractitionerandthelaboratory.Diversifyingandimprovingmethods
of antemortem diagnosis for both immunological diagnostic and identification of pathogens
wasbeneficialnotonlyforveterinarylabbutalsoforpractitioner.VeterinaryLaboratorywill
providereliableresultsquickly,andthepractitionerwillbereducedbythedecisionstheytake
economiclosses.
However,postmorteminvestigationscannotberemovedastheyprovide,alongwith
clinical and zootechnical indicators, information on health and possible infection in swine
herds.Inourcasesthepneumonialesionswereheterogeneous.Inaddition,therewereboth
acute and chronic pulmonary lesions. This suggests either codevelopment of at least two
infections or an acute injury healing process. Based on morphological characteristics, lung
lesions can be classified as embolic, bronchopneumonia and interstitial or bronhointerstitial
[4].
In the 20 lungs collected from pig bodies that previously showed signs of respiratory
deathdominantwaslobularbronchopneumoniainthecranialventralregion(16lungs).Four
lungsshowednomacroscopicallyvisiblelesions.
Seromucous, purulent or mucopurulent exudates were found in five pigs (25%) with
acutebronchopneumonia,eightpigs(40%)withsubacutebronchopneumoniaandsevenpigs
(35%) with chronic bronchopneumonia. Seven animals presented fibrotic pleurisy of the
diaphragmlobes.Lymphnodescoreswere:score0in4pigs(20%)(withoutpulmonarygross
lesions),score1in9pigs(45%),andscore2in7pigs(35%).
Tosupporttheswinefarmersisnecessarytoevaluatetheimmunestatusagainstmajor
infectiousagents(suchasbacterialorviral)thatactsprimaryorsecondaryontherespiratory
system.Undoubtedly,theepidemiologicaldata,clinicalsignsandpathologicallesionsprovide
sufficient information to guide diagnosis to development of a porcine respiratory complex,
however many unknown problems it still present in swine industry. Linking the information
previouslydescribedwithserologicalinvestigationsbyELISAinordertoconfirmsuspicionsor
assessmentofpostvaccinationimmunestatusisanactivityusuallypracticed.
Based on epidemiological data, clinical features and lesions attention has focused on
evaluation the immune status for folowing infections: Porcine Reproductive and Respiratory
SyndromeVirus,Aujeszky'sDiseaseVirus,SwineInfluenzaVirus,Mycoplasmahyopneumoniae,
ActinobacilluspleuropneumoniaeandSalmonellaspp.
EvaluationbyELISAofIgGantiH1N1antibodiestothe376samplesshowedthat185of
thepigstested(49%)wereincontactwiththevirus.ELISAH3N2providedpositiveresultsin
252pigstested(67%).Thesedatahighlightthesimultaneouscirculationofseveralstrainsof
influenzainpigherds.
In assessing the immune status against PRRSV were obtained 45 positive samples
(12%),andforAujeszky'sDiseaseVirus39samplespositive(10%).
Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae, the bacteria
considered primary etiologic agents in respiratory pathology, were identified in 55 samples
(14.6%),andrespectivelyin71samples(19%).

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Lucrritiinificevol53seriaMedicinVeterinar
Amongbacteriathatcancauselungdamagewasinvestigatedassecondaryagentsonly
Salmonellasp.Antigenicexposuretosalmonellawasestablishedin186specimens(49.5%).
Lot of pig assessed for respiratory lesions (48 lungs) had average live weight at
slaughterof105120kg.From48lungs19lungswerewithlesions:5lungswithcharacteristic
lesions of swine enzootic pneumonia (Mycoplasma hyopneumoniae), and 9 lungs with
pneumoniaandpleurisies(18.8%);nopigshadpericarditisorlungabscess(Table2).
These data suggest that the farm revealed 11 lesions were pathognomonic of
Mycoplasmahyopneumoniaeon10%oflungsexamined(fiveoutof48lungs),avaluesimilar
withresultofallserologicaltestsperformedforMycoplasmahyopneumoniaeinourstudy.
Each10%lesionofthetotalpulmonaryrespiratoryareacausesalossof37ggain/day.
The pulmonary lesions collected in slaughterhouse suggest a reduction of average daily gain
with 19 g / day / pig, and 1.87 kg at the end of 100 days of fattening. Thus, the cycle of
fatteningwillincreasewithother2days.

(a)

(c)

(d)

923

(b)

(e)

UniversitateadetiineAgricoleiMedicinVeterinarIai

.
(f)

(g)

(h)
Fig.1.RespiratorydiseaseassociatedwithPRDC(a,b)cranioventralacutebronchopneumonia,
(c)chronic cranioventral bronchopneumonia with pulmonary consolidation and focalfibrotic
pleurisyofthediaphragmlobes(d,e)oedematous,hyperaemicandswollenacutelesions(f,g)
mucopurulentexudateintracheaandbronchi(h)enlargedlymphvolume

Table1.SerologicalinvestigationswithELISAresults(14to22weeksage)
Re
Negative
Positive
control
ELISA
samples
No.
%
No. %
No. %
Actinobacilluspleuropneumoniae(APP)AntibodyTes
376
305 81 71 19
0
0
Kit(ELISAAPP)
PseudorabiesVirusgBAntibodyTestKit(ELISAPV)
376
337 90
39 10
0
0
SwineInfluenzaVirusAntibodyTestKitH1N1 (ELISA
376
191 51 185 49
0
0
H1N1)
SwineInfluenzaVirusAntibodyTestKitH3N2 (ELISA
376
124 33 252 67
0
0
H3N2)
SwineSalmonellaAntibodyTestKit(ELISASalm)
376
190 50.5 186 49.5 0
0
PRRSVAntibodyTestKit(ELISAPRRS)
376
331 88
45 12
0
0
MycoplasmahyopneumoniaeAntibodyTestKit
376
321 85.4 55 14.6 0
0
(ELISAMhyo)

Chart1.EtiologyofPRDCinswinefarms

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Lucrritiinificevol53seriaMedicinVeterinar
Table2.Slaughterhouseexamresults:assessinglungdamage
Cantitate
No.swine
%
No.swineslaughtered
48
4800
Respiratorysurfacedamaged
242
%Respiratoryaveragearea/pig
5
No.lungswithoutlesions/animalsslaughtered
29
60
No.lungswithlesions/animalsslaughtered
19
40
Mycoplasmaspecificlesions
5
10
Pleurisies
9
18.8
Pericarditis
0
0
Abscess
0
0

Chart2.Dominantlunglesioncollectedpostmortemexaminationattheslaughterhouse

CONCLUSIONS

Ofthe376samplestested49%weretheH1N1positiveantiIgG,67%werepositiveto
IgGantiH3N2,12%werepositivetoIgGantiPRRSV,10%werepositivetoIgGantiprv,14,6%
were positive to IgG antiMycoplasma hyopneumoniae, 19% were positive to IgG anti
Actinobacilluspleuropneumoniaeand49.5%werepositivetoIgGantiSalmonellasp.
Circulationofinfluenzastrainsinswinefarmssuggestthemonitoringoftheirprogress
through regular serological investigations. It recommends immunoprophylactic actions or
adjustment of already schemes of vaccination implemented in livestock of sows and youth.
Evenifthespecificpreventionprogramsimplementedinfarmsincludedimmunoprophylactic
products from some of the pathogens, adverse effects caused by the actual movement of
virusesorotherpathogenicbacteriaarebeingfeltthroughreducedfeedconversionrateand
averagedailygain.

BIBLIOGRAFIE

1. Baraitareanu S, Baraitareanu AP, Cobzariu D, Balaban A, Campeanu MV, Danes D. Young swine
management for infectious respiratory pathology .Lucrri tiinifice Medicin Veterinar,
Universitatea de tiine Agricole i Medicin Veterinar "Ion Ionescu de la Brad" Iai VOL. 51(10)
Partea2/2p.644647.2008
2. Burns,K.SwineveterinariansresolvetoeliminatethePRRSvirus.JournaloftheAmericanVeterinary
MedicalAssociation.228(9),pp.13151316.2006.

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3. Cadar,D.,Csgola,A.,Dn,A.,Deim,Z.,Spnu,M.,Miclau,V.,Kblkuti,L.,(...),Tuboly,T.Porcine
circovirus type 2 and associated diseases in Romaniashort communication. Acta veterinaria
Hungarica55(1),pp.151156.2007.
4. Caswell J.L., K.J. Williams, Respiratory system. In: M.G. Maxie, Editor, Jubb, Kennedy, and Palmer's
PathologyofDomesticAnimals(5thEdit.),SaundersElsevier,Philadelphiapp.523653.2007
5. Choi Y.K., S.M. Goyal and H.S. Joo, Retrospective analysis of etiologic agents associated with
respiratorydiseasesinpigs,CanadianVeterinaryJournal44,pp.735737.2003.
6. ChristensenG,SorensenV,MousingJ.Diseasesoftherespiratorysystem.In:StrawBE,D`AllaireS,
th
MengelingWL,TaylorDJ,eds.DiseasesofSwine.8 ed.Ames,Iowa:IowaStateUniversityPress;pp.
913941.1999.
7. DinuI.Suinicultur.Tratatdecrestereasuinelor.Ed.CoralSanivet,Bucuresti2002.
8. Elbers, A.R.W., De Jong, M.F., Wellenberg, G.J. Risk factors for clinical signs of PMWS and PDNS in
pigsintheNetherlands:Acasecontrolstudy.TijdschriftvoorDiergeneeskunde131(9),pp.318325.
2006.
9. GrauRoma,L.,Segals,J.Detectionofporcinereproductiveandrespiratorysyndromevirus,porcine
circovirustype2,swineinfluenzavirusandAujeszky'sdiseasevirusincasesofporcineproliferative
andnecrotizingpneumonia(PNP)inSpain.VeterinaryMicrobiology119(24),pp.144151.2007.
10. Hansen M.S., S.E. Pors, H.E. Jensen, V. BilleHansen, M. Bisgaard, E.M. Flachs, O.L. Nielsen, An
InvestigationofthePathologyandPathogensAssociatedwithPorcineRespiratoryDiseaseComplex
in Denmark, Journal of Comparative Pathology, In Press, Corrected Proof, Available online 23
February2010,ISSN00219975.
11. Harms P.A., P.G. Halbur and S.D. Sorden, Three cases of porcine respiratory disease complex
associated withporcine circovirus type 2 infection, Journal of Swine Health and Production 10. pp.
2730.2002.
12. Hoover, T. Porcine Respiratory Disease Complex. Pfizer Animal Health Technical Bulletin. October
1996.
13. KimJ.,H.K.ChungandC.Chae,Associationofporcinecircovirus2withporcinerespiratorydisease
complex,VeterinaryJournal166,pp.251256.2003.
14. Militaru M, Ciobotaru E, Dinescu G, Soare T Ghid practic de anatomie patologic a sistemelor i
aparatelorlaanimaleledomestice.Bucureti,Ed.Elisavaros,2007.
15. PopaV,DAStanuica,EBucur,DCadar,NCatana,DoinaDanes,S.Baraitareanu,DanielaBotus,Irina
Nica, Adela Stan, C Belteghi, F. Pastrama, T Tamas Studies regarding to the presence of Porcine
circovirus type 2 in swine industrial growth farms from Romania Lucrri tiinifice Medicin
VeterinarVol.XXXXI,Timioara784,2008.
16. Srensen V., S.E. Jorsal and J. Mousing, Diseases of the respiratory system. In: B.E. Straw, J.J.
Zimmerman,S.D'AllaireandD.J.Taylor,Editors,DiseasesofSwine(9thEdit.),BlackwellPublishing,
Oxford,pp.149177.2006.
17. Thacker E.L. Immunology of the porcine respiratory disease complex, Veterinary Clinics of North
America.FoodAnimalPractice17,pp.551565.2001.
18. ThackerEL.Lunginflammatoryresponses.VetRes.;37:469486.2006.
19. Truszczyski, M., Pejsak, Z. Postweaning multisystemic wasting syndrome of swine. Medycyna
Weterynaryjna63(5),pp.499502.2007.

926

SIGNSANDLESIONSOFTHESPONTANEOUSACUTEPNEUMONIC
PASTEURELLOSISINPETRABBITS

S.BRITREANU1,D.COBZARIU1,MariaOELEA2,M.V.CMPEANU2,SimonaIVANA1,
DoinaDANE1
1
USAMVBucuretiInstitutuldeMedicinComparat
2
USAMVBucuretiFacultateadeMedicinVeterinar
SplaiulIndependenei105,Sector5,Bucureti,doruvet@yahoo.com

Pasteurellosisisoneofthemostseriousdiseasesofrabbits,withconsiderableeconomiclossin
largeproductionunitsandunfavorableprognosisforsickrabbits.However,Pasteurellaisthought
toliveinthethroatsandnasalpassagesofsomerabbitswithoutcausinganysignsofdisease.Our
case studyisfocusedon petrabbitsthat werediagnosed withacutepneumonic pasteurellosis.
TherabbitscandevelophealthproblemscausedbyPasteurella,especiallyiftheyareunwellor
stressed,butthepetrabbithusbandryismuchdifferentfromthatofrabbitfarm,andtherefore
the risk factor are others. The identification and quantification of risk factors are difficult and
dependent by subjectivity of pet owner, and for a complete disease history we recommend a
detaileddiscussionwithallfamilymembers.Inthisoutbreakthemajorstressfactorwas:phonic
stress, excessive manipulation, transport stress and improper feeding. Clinical and lesion data
collectedinthisstudysupporttheheterogeneityofrabbitPasteurellainfections.

Keywords:rabbitpasteurellosis,rabbitrespiratorypathology

Pasteurellosisorhaemorrhagicsepticaemiaisoneofthemostcommunediseasesof
rabbits[5,6,7,9,11].Pasteurellasp.areubiquitiesinconventionalfarmsofdomesticrabbits,
anditsimplicationsinleporidaepathologywasdiscussedanddetailedinseveralscientifically
papers[2,4,5,7,9].Infectionmostlikelyoccursimmediatelyafterbirth,andtheprevalence
ofpasteurellacarryingrabbitsincreasetoover90%at5monthsofage[2,7].
Thepathologyinrabbitfarmspasteurellainfectedismaintlyrepresentedbyrhinitis,
pneumonia, otitis media, metritis, conjunctivitis, orchitis, subcutaneous abscess and
septicemia [4]. The stress and immunodeficiency play a major role in the onset of auto
infection for the pasteurella carrier rabbits [5]. This required the development of health
management programs in all holdings of domestic rabbits, adapted to the particularities of
eachunitinamannerthatmeetsalltherequirementsofwelfare.Thepasteurellaoutbreaks
can benefit from therapeutically intervention, but for the economical reasons usually all ill
rabbitsareeliminatedfromthefarmherds[2,5,9].TheVeterinarymedicalapproachofpet
rabbitsisdifferent.Theeconomicalvalueisalowmarkinhealthmanagementofrabbitpets,
andthetherapeuticallydecisioncanbeconsiderateiftheownerswantthis.
Unfortunatelythereareseveralcaseswheretheveterinarianinterventionisdelayed,
in context of numerous peracute and acute septicemia infections. For this reasons, the
preventive medicine is an activity that mast be included on the rabbit growth in non
professionalhouses.

MATERIALSANDMETHODS

Inthispaperwefocusontherelationshipbetweenpasteurellapathologyandrabbits
ascompanionanimals.WepresentaclinicalcaseofrabbitpasteurellosisintwoAngorarabbit

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Lucrritiinificevol53seriaMedicinVeterinar
bredwith5weeksofage.Themethodsofinvestigationsusedweredescribedpreviously[8,
12,13].

RESULTSANDDISCUTIONS

Rabbits are particularly sensitive to environmental stressors and handling. For this
reason, in the leporidae health management housing and feeding conditions are priority. In
addition, contention and handling should be limited to that necessary. These objectives are
not respected especially in pet rabbits. In the context of pasteurella portaj, the carrier pet
rabbits stressed by an excessive handling and an improper accommodation (noise, poor
housing, frequent transport, poor diet) have a great risk to develop a grave form of
pasteurellosis by autoinfections. If those stressors are associated with the early removing
from the rabbit nest, the risks are significantly increased. Health problems of pet rabbits
acquirenewfacetsinthecontextoftheheterogeneouscareandfeedingconditions,tailored
toeachownerandmajorinfluencedbythelevelofownereducationininterrelationwiththe
petrabbit.
Thecasehistoryisakeyinanalyzingofeachpathologicaleventinrabbitpasteurella
carriers.However,theidentificationindiseasehistoryofeventsthatconstitutetheriskfactors
is usefully, but not sufficiently in etiological diagnosis. In our cases, unfortunately, the
etiological diagnosis was obtained after death of rabbits. The evolution of disease was
peracute/acute and the therapeutically actions were limited. Usually, rabbit pasteurellosis is
clinicallymanifestedbyrhinitis.Symptomatologyconsistsinsneezing,coughing,nasalserous,
white or purulent discharges and noisy breathing. These secretions can be passed by rabbit
groomingcomportmenttofrontpawsandhead(fig.1),andcancontaminatedthesinuses,eye
andtearductandcausesinusitis,conjunctivitisanddacriocistitis.Sincerabbitsaremandatory
nasal breathing, any obstruction of the nasal passages will result in respiratory compromise
[1].Althoughdescribedinrhinitis,mouthbreathinginrabbitsisratherasignpriortodeath,
underwhichthevitalprognosisisunfavorable.
Peracutesepticemiawithsuddendeathisnotcharacterizedbynasaldischarge;even
more the single element that can be identified is the bleeding diathesis [9]. This type of
pasteurellainfectionisfrequentlydescribedinyoungpetrabbits.
In acute form the pasteurellic pneumonia is associated with fatigue, lethargy,
depression,anorexiaandprogressiveweakness.Inourstudythiswasassociatedinonecase
with rhinitis. Frequently the respiratory pathology is associated with digestive disorders
expressedbycatarrhal(fig.2)orhemorrhagicdiarrhea.Bacteraemiaismostoftenassociated
withthemorevirulentstrainsofP.multocida[10].
Usually,thediagnosticoflowerrespiratorytractdiseaseisbasedonclinicalexamand
Xrays.Oftenthetracheaandlungslisteningpermittheidentificationoftheplacewherethe
airflow is obstructed. Xrays confirm the thoracicdisease, and candifferentiate the origin of
respiratory signs. Thoracic ultrasounds can determine if abscesses or densifications are
present. Blood tests are not useful in determining the etiology, but may be made in any
contexttodeterminethemetabolicprofileandevaluatingthepresenceofconcurrentdisease.
Serologicaltestingofpasteurellacanbeperformed,butresultsaredifficulttointerpret[1].
Inlowerrespiratorytracttheacute/subacuteformsofpasteurellosis,withahistoryof
over5days,areassociatedwithmarkedpulmonaryconsolidation,oftenwithhemorrhageand
fibrinpurulent exudates in affected areas. Pulmonary lesions are frequently accompanied in
subacuteformsbypleuritis,fibrinpurulentpericarditisandrarelywithempyema[8,10].

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Marked pulmonary congestion was described in individuals who died 18 hours
postinfectionexperimentalintravenous[10],butthissituationwasdescribedinyoungrabbit
with natural infections (fig. 4). Symptoms and lung lesion picture are not always correlated.
Thus, experimental infections have been described in animals have remained asymptomatic
for 14 days, but at necropsy showed areas of consolidation or diffuse pulmonary nodules.
However,thereisadirectcorrelationtothedurationoftheevolutionofinfectionanddegree
oflungdamage.Theasymptomaticrabbits,sacrificedat14dayspostinfectionshoweddiffuse
alveolitiswithpneumociticproliferation,leucocyteinfiltrationandobliterationofnormallung
architecture. Acutesubacute forms are histopathological characterized by heterophilic and
macrophage infiltration, and a marked alveolar interstitial infiltrat, but the airways are
relativelyfreeofinflammatoryexudate.
In some cases the cell population that invade lung can be represented by
polymorphonucleated,thiscellularinfiltrationisassociatingwithpleural fibrosis.The rabbits
thatwilldieat7dayspostinfectiondevelopsfibrinpurulentbronchopneumoniawithvarying
degrees of enlargement, while those that die at 9 days postinfection showed a marked
congestionofalveolarcapillariesandpulmonaryedemawithproteinmaterial[10].

Fig.1.Rabbit,Angorabred,4weeks,male.
Purulentrhinitis.Pasteurellosis

Fig.2.Rabbit,Angorabred,4weeks,male.
Enteritis.Pasteurellosis.

Fig.3.Rabbit,Angorabred,4weeks,male.Visceral
Fig.3.Rabbit,Angorabred,4weeks,male.
congestion.Pasteurellosis
Pulmonarycongestion.Pasteurellosis

Clinicalandlesiondatacollectedinthispaper,andtheinformationprovidedinmany
previous works with same subject supporting the heterogeneity of leporidae pasteurella
infections[1,2,4,5,7,9,10].
Specialproblemsindiagnosisarefoundinperacute/acutepasteurellosisofyoungpet
rabbits. The small number of pet rabbits, without or with moderate signs often required

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Lucrritiinificevol53seriaMedicinVeterinar
extensiveinvestigationsforconfirmation,andthatcandelaytherapeuticinterventionandthus
reducethechancesofrecoveringtheanimal.Thelesionsarenotalwayshelpful,oftenthose
havesepticemiacharacter.Inaddition,thepasteurellaisnotthesingleleporidaerespiratory
pathogen. Bordetella bronchiseptica is also involved in respiratory pathology of rabbits,
Klebsiellapneumonia,pneumococcalandvirusinfectionsmayhaverespiratorysymptoms.

CONCLUSIONS

Theyoungpetrabbitwithperacutepasteurellosisdonthaveprodromalsignsbefore
sudden death. In the acute form the signs can consist in dyspnoea, misconduct, rhinorrhea,
sneezing, and death in few days. The pet rabbits identification and quantification of
pasteurellosisriskfactorsisdifficultanddependentofdataprovidedbytheowner.Because
thetransportationtoveterinaryclinics,medicalmanipulationandtherapyarestressfactors,
allyoungpetrabbitswithpossiblesignsofpasteurellosisneedtobeapproachedwithcaution.
When the medical care without pet rabbit transportation is possible, this option can be
consideredtoprotectthepatient.

BIBLIOGRAFIE

1.

2.
3.

4.
5.
6.
7.
8.
9.
10.
11.

12.
13.

Deeb, BJ. Respiratory disease and the Pasteurella complex. Din E. V. Hillyer and K. E.
Quesenberry(eds).Ferrets,Rabbits,andRodents:ClinicalMedicineandSurgery.Philadelphia,
WBSaunders,1997,pp.189201.
DiGiacomoRF:1992,NaturalhistoryofPasteurellamultocidainfectioninrabbits.JApplRabbit
Res15:15151523.
El Tayeb A. B., T.Y. Morishita, E.J. Angrick Evaluation of Pasteurella multocida isolated from
rabbits by capsular typing, somatic serotyping, and restriction endonuclease analysis J Vet
DiagnInvest.2004,16:121125
Flatt,R.E.Bacterialdiseases.DinS.H.Weisbroth,R.E.FlattiA.L.Kraus(coord.),Thebiology
ofthelaboratoryrabbit.NewYork,AcademicPress,1974;pp.194205.
Hillyer, E.V., Quesenberry, K.E. Ferrets, Rabbits, and Rodents Clinical Medicine and Surgery.
W.B.SaundersCo.Philadelphia,2004.
Manning P.J. 1982. Serology of Pasteurella multocida in laboratory rabbits a review.
LaboratoryAnimalScience,32,666671.
ManningPJ,DiGiacomoRF,DeLongD:Pasteurellosisinlaboratoryanimals.Din:Pasteurellaand
pasteurellosis,ed.AdlamC,RutterJM,NewYork,AcademicPress,1989;pp.263302.
MilitaruM,CiobotaruE,DinescuG,SoareTGhidpracticdeanatomiepatologicasistemelor
iaparatelorlaanimaleledomestice.Bucureti,Ed.Elisavaros,2007
Moga Mnzat, R. Boli produse de germeni din genurile Pasteurella i Mannheimia. Din Radu
MogaMnzat(coord.),Boliinfecioasealeanimalelor.Timioara,Ed.Brumar,2001;112139.
Percy D H, Bhasin J L, and Rosendal S Experimental pneumonia in rabbits inoculated with
strainsofPasteurellamultocida.CanJVetRes.1986January;50(1):3641
TakashimaH.,SakaiH.,YanaiT.,MasegiT.(2001):DetectionofantibodiesagainstPasteurella
multocidausingimmunohistochemicalstaininginanoutbreakofrabbitpasteurellosis.Journal
ofVeterinaryMedicalScience,63,171174
ogoe I, Dobrea M, Ivana S, Negoi C. Practicum de bacteriologie special; Bucureti, Ed.
Printech;2005
Vlagioiu, C; Tudor N. Semiologie veterinara si tehnici de examinare. Bucureti, Ed. Sitech,
2008.

930


TREATMENTEFFICACYINUDDERLOCALISATIONOF
CONTAGIOUSECTHYMAINSHEEPANDGOATS

R.BIA1,G.RPUNTEAN2,S.RPUNTEAN2
email:bita.romulusbn@ansvsa.ro
1.DSVSABistriaNsud;2.USAMVClujNapoca

ABSTRACT
InBistritaNasaudCountyareaffectedbycontagiousecthymaapproximately3040%oflactating
herdofsheepandgoatsleadingtosignificanteconomiclossesthroughdecreasedmilkproduction
andweightloss.Diseaseevolvesthroughouttheyear,butisprevalentinsummer,especiallyifthe
pastures are not changed annually. Flocks are mixed, consisting of sheep and goats,
predominantlymostlysheep.Thediseasehasahigherincidenceinthepopulationofgoats,which
are more sensitive. Clinically the dominant locations are on the udder, with the formation of
lesionsontheteats.Diseasedanimalsweretreatedlocallyfor35daysusingdrugsbasedonthe
followingformula:a)methyleneblue+oxytetracyclinepowder+honeyorvaseline,b)methylene
blue+oxytetracyclinepowder+osmatinc)methyleneblue+oxytetracyclinepowder+grease.By
using these treatment formulas good results were achieved, after 5 days the healing rate was
over90%.About5%oftreatedanimalsshowedcomplicationswithdifferentpathogens,resulting
mastitisandeffectivelyrequiringtheremovalofsuchanimals.
Keywords:sheep,goats,contagiousecthyma,udder,treatment.

INTRODUCTION

Contagious ecthyma of sheep and goats is widespread worldwide, causing major


economic losses particularly in lambs and kids by mortality and to youth and adults with
weaknessanddecreasedmilkproduction.Thediseasehashealthimportance(zoonosis)asit
passed from animals to humans, so safety measures must be taken (1, 2, 9, and 15). The
diseaseiscausedbyaspecificvirus(Parapoxvirus)epiteliotrop,whichhasahighresistancein
the external environment, thus ensuring greater importance of the secondary sources of
infectionandtheperpetuationofdiseaseoutbreaks(1,3,14,17).
Clinically,thediseaseismanifestedbytheformationofvesicles,pustules,andfinally
thickcrustsonthelips,nostrils,face,teats,udder,legsandrarelyinthemouth(2,5,8,9,13).
Insuckling,lambsandkids,therearefrequentlesionlocationsinthemouthandaroundthe
mouth,andinlactatingsheepandgoatsthedominantdevelopmentlesionssiteismammary
gland skin, often complicated with mastitis. Less commonly found locations are seen at the
endsoflimbs(thecrownandinterdigitalspace),genital(vulva,foreskin,scrotum,andpenis)
andeyes(1,2,5,7,13).
Ecthyma causes major economic losses through mortality in young animals (lambs
and kids), or complications that arise due to secondary bacterial infections (4, 14, 17).
Morbidityinsomepopulationsmayreach7080%even100%,butmortalityislow,lessthan5
15% (3, 11, 13). Fatalities are more common in lambs and kidswith severe form of disease,
duetoperibuccalinjuries theyrefusesuckingandmaydiefromstarvation(7,8).Theyoung
sheep and goat with oral and peribuccal lesions have difficulties to consume feed, so that
economic losses are due to decrease in weight. The location on the udder is common in

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Lucrritiinificevol53seriaMedicinVeterinar
lactatingperiod;theeconomiclossesareduetoweakeninganddecreasedmilkproduction.To
thisaddscostsoftreatmentsandpreventivenonspecificorspecificmeasures(3,12,and15).

MATERIALANDMETHOD

InBistritaNasaudCountyapproximately3040%oftheflocksofdairysheepandgoat
households are affected by contagious ecthyma. The paper presents results in disease
treatment in sheep and goats dairy farms belonging to three veterinary districts: Bistrita
Bargaului,RodnaandZagra.Notethatusuallytheflocksaremixed,composedofbothsheep
andgoats,sheeparepredominant.Fromeachdistrictoneherdwaschosen(wherethedisease
was diagnosed), and the treatment groups comprises 100 sheep and 50 goats, which were
monitoredduringtreatment.
Sickanimalsweretreatedlocallyfor35days,usingthefollowingdrugformula:
a)methyleneblue+oxytetracyclinepowder+honeyorvaseline;
b)methyleneblue+oxytetracyclinepowder+osmatin;
c)methyleneblue+oxytetracyclinepowder+grease.

Animals were heading back into contention, then a clinical examination was
performed by inspection and palpation, and udder and teats were washed with water to
remove coarse impurities, then medication was topically applied. Treatment was performed
dailyfor35days.Sicksheepandgoatsfromtheflock,werecaughtandhandleddailyduring
milking.Theywereseparatedandtreated,thenwereallowedbackinthefold.Milkfromthese
animalswascollectedinseparatevesselsanddestroyedorusedindogfoodafterboiling.

RESULTS

Onexaminationitwasfoundthatthelesionswerelocatedontheteats,asinoneor
both.Initiallyisnoticedaredarea(macula),whichsoonturnsintovesicleorbleb,whitegray,
which is covered with a brown crust, adhering and with an irregular outline. Lesions were
usually singular and affecting only one teat (Fig. 1), rarely were more and smaller (Fig. 2).
Sometimeslesionshadtheappearanceofthickcrustcoveringalmostentirelytheteat.

Fig.1LesionsontheteatsFig.2Multiplelesionsontheteat

InthefarmofBistritaBargauluidistrict(BargauluiValley)thefollowingformulawas
used for treatment: aqueous solution comprising 100 ml of methylene blue, oxytetracycline
powder 50 g, 500 ml distilled water and honey or neutral vaseline. Before carrying out the
treatmentbeehoneyorgreaseisappliedtoudderfordippingcrusts,thentheyareremoved

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UniversitateadetiineAgricoleiMedicinVeterinarIai
andoxytetracyclineandmethyleneblueaqueoussolutionsweretopicallyapplied.Treatment
wascarriedoutbytopicalapplications,dailyfor35days.
In the farm of Rodna district (Someului Valley) the following formula was used for
treatment: 100 ml of methylene blue, oxytetracycline 100 g, 800 g osmatin. Osmatin was
heateduntilitbecameslightlyliquidandwasmixedwithoxytetracyclineandmethyleneblue,
obtainingasuspensionthathasbeenusedforlocaladministrationfor35days.Neocafspray
wasusedingoats,250mlspraycontainingoxytetracyclineandmethyleneblue,bytopicaland
sprayfor35days.
In the farm of Zagra district (Tibleului Valley) the following formula was used for
treatment: 100 ml of methylene blue, oxytetracycline 50 g and 800 g of grease. Grease was
gentlyheatedtoformauniformmixturewithoxytetracycline.Thefirstphasewasmethylene
bluebrushingandthenthemixtureofgreaseandoxytetracyclinewasapplieddaily,usualyfor
35days.

Contagiousecthymaincidenceishigherinherdsofgoatsthaninsheep.
Imagesoftreatedanimalsareshowninthefiguresbelow(Figs.3and4).

Fig.3Teatduringtreatment
Fig.4Teatsduringtreatment

Numberofanimalstreatedandcuredthenumberandpercentagevaluesobtainedin
thethreefarmsareshowninTable.1.

Theresultoftreatmentobtainedin3daysandafter5days
Table
no.1
After 3daysof
After5daysof
Districtinwichfarmis Thetotalnumberof
treatment
treatment
located
treatedanimals
No.
%
No.
%
CSVBistriaBrgului
150
120
80,0
134
89,33
(BargauluiValley)
CSVRodna
150
123
82,0
136
90,66
(SomeuluiValley)
CSVZagra
150
126
84,0
139
92,66
(ibleuluiValley)
Total
450
369
82,0
409
90,88

Analyzing the data from Table. 1, it appears that after the treatment performed on
sheepandgoatswithcontagiousecthyma(mammaryform)wereobtainedsatisfactoryresults.
After3daysoftreatment,fromthetotalof450animalstreated,in369(82.0%),therewasan

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Lucrritiinificevol53seriaMedicinVeterinar
improvement in the development of lesions and after 5 days the situation improved even
more,sothatwereconsideredascured409animals(90.88%).Differencesintheimprovement
andhealingoflesionsobservedamongthethreeherdsandthedifferentformulaappliedfor
treatment are insignificant, which means that effective treatment was established. We
appreciate that the mixture that included an antiseptic (methylene blue), a broadspectrum
antibiotic (oxytetracycline) and an emollient substance (honey, osmatin, grease or neutral
vaseline),meetthequalitiesofancomplexproductthatfavoredbothhealingandprevention
ofsecondarybacterialcomplications.In45%ofanimalsincludedinthetreatedgroupssome
complications were found with diverse microbial flora, which resulted in suppurative and
necroticlesions,whichledtothecompromiseofudderandactuallyrequiringslaughtering.2
Younglambsandthosebelongingtohouseholds,afterweaningarekeptinseparate
flocks from sheep in lactation, but they have just rarelyserious disease, clinically presenting
peribuccal lesions and mouth problems. When presented serious forms they were treated
locallywithmethyleneblueandapplicationsoftetracyclinesuspensionsorointments.
Contagious ecthyma disease has an endemic trend in sheep and goat farms in the
areaofBistritaNasaud.Illnessaffectsbothyounglambsandfatteningfarms,inwhichclinical
dominates peribuccal and oral forms, but the disease is also present in private backyards in
milkingsheepandgoats,andweappreciatethattheyareaffectedin3040%.Sheepandgoats
heardareusuallymixed,approximately400heads,prevailingsheep.Goatsaremoresensitive
makinginjuriesmoreseriousandfavoredbythegreaterlengthofnipples(1,5,14,and17).
The disease is endemic and outbreaks occurs dominant in summer, during milking, the
appearanceisenhancedbyproducingmorefrequentinjuriesontheteats,andtransmissionas
aresultofmilking,andbyflyinginsects,whichfulfilltheroleofvectors(4,9).

Therapeuticformulasgenerallyseekachievementofanemollienteffect,localasepsis
withbroadspectrumantibioticscombatsecondarybacterialinfections(7,11).Thistreatment
generally does not affect the evolution and pathogenic immune response caused by viral
infection, but prevents bacterial infections that worsen lesions (7, 8). Crusts from the
mammaryglandshouldbekeptflexible,thepurposebeingappliedbydifferentlocalapplied
ointments,iodinatedglycerolsolutions(5,11).

Treatmentconductedinsheepandgoatsinthisstudywasbasedonlocalapplications
of therapeutic formulas that have had their composition with antiseptic effect (methylene
blue, oxytetracycline antibiotic with broadspectrum antibacterial action and an emollient
substancerepresentedbyhoney,neutralgrease,orosmatin).
Methylene blue is recommended because it is moderate and lasting antiseptic,
penetratingthelesionaltissue(wounds,burns,gall,pressuresores,frostbite,glossitisetc.)and
by repeated applications have an analgesic effect, promoting healing. In view of these
propertieswithanantisepticeffectthissubstancewasusedinallthreeformsoftreatment(6,
10).
Oxytetracycline is a broadspectrum antibiotic (tetracycline group), with
bacteriostatic action against many Grampositive and Gramnegative bacteria, contained in
numerousproductsthatareintheformofinjections(solutionsorsuspensions)orointments,
powders,forlocaladministration.Thecombinationsusedincaseswithecthymahadadecisive
impactinpreventingsecondarybacterialinfection,promotinghealing.
Emollient substances are necessary to achieve softening crusts, having protective
effect against moisture, sunlight, cold and prevent the formation of crevasses. Vaseline is a
mixture of saturated hydrocarbons with emollient effect, cheratoplastic and against itching,
buthasalowerpowerofpenetrationandcouldnotbecomerancid.Itcanbeusedassuch(per
se)orinotherointments.Osmatincontainsunsaturatedaliphaticamine,paraffinandvaseline

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UniversitateadetiineAgricoleiMedicinVeterinarIai
andithavebactericidaleffectagainstpathogensthataretransmittedbymilkershands(9,12).
Porkgreasehaveanemollienteffect,butcanbecomerancidveryeasyandtopreventthiswe
canaddpreservative(6,10).Honeyisrecognizedforitsbeneficialeffects.
Other authors recommend the use of iodine solutions, as Betadine, iodine sprays,
povidoneiodine(1,5).ItisconsideredthataniodinesolutioninactivatesGramnegativeand
Grampositive bacteria, fungi, viruses and protozoa. Their application forms a film that
protects tissues from contamination with bacteria, not irritating, ensure full coverage and
good penetration and can be used in many animal species and in different age categories.
Shouldalsoconsideringflyinginsectcontrol(4,9).Whatevertreatmentisaimedatprotecting
tissuesandpreventsecondarybacterialinfections.

CONCLUSIONS

1. In Bistrita Nasaud County are affected by contagious ecthyma approximately 30


40% of backyard lactating herd of sheep and goats (mixed flocks); infection evolving in the
summerandismorecommoningoats.
2. 450 animals were treated, each 150 heads of three different flocks, and in three
differentareas(BistritaBargaului,RodnaandZagra),whichclinicallyshowederuptivelesions
ontheteats;withstadialdevelopmentinwhichhighlyexpressedwasthecruststage.
3.Sickanimalswerelocallytreateddailyfor35dayswithamedicationconsistingof
anantisepticsubstance(methyleneblue),abroadspectrumantibiotic(oxytetracycline)andan
emollientsubstance(osmatin,grease,honey,porklard).
4.Followingtreatmentwasnotedanimprovementoflesionsafter3daysandhealing
after5days,inmorethan90.0%ofanimals;asmallnumberofanimalsshowedcomplications
andmastitisappeared,notsensitivetotreatment,requiringtheirremoval.

REFERENCES

1. Coates W. J., Contagious ecthyma: An unusual distribution of lesions in goats. Can.


Vet.J.,1990,31,209210.
2. HarwingN.,Contagiousecthyma(SoreMouth),SheepHealth,FactSheet,2000,no.1,
p.12.
3. Hopkins M. F., Warren G., Soremouth in Sheep. Annual Meeting of the Virginia
AssociationofLicensedVeterinaryTechnicians,2007.
4. HousawiF.M.T.,AbuElzeinE.M.E.,Contagiousecthymaassociatedwithmyasisin
sheep.Rev.Sci.Tech.Off.Int.Epiz.,2000,19,(3),p.863866.
5. LeiteBrowning,Contagiousecthyma(orf/SoreMouth)inSheepandGoatsExtension
Animal Scientist, Alabama University:2008, http://www.aces.edu/pubs/docs/U/UNP
0063.
6. LicpertaE.,Farmacologieveterinar,EdituraCeres,Bucureti,1980,212213.
7. Luginbuhl JeanMarie, Anderson L. K., Controling Sore Mouth in Meat Goats.
ExtensionAnimalHusbandry.DepartmentofAnimalScience,2009,ANS00601MG:
1. http://www.aces.edu/pubs/docs/U/UNP0063.
8. LyleG.McNeile,ContagiousEcthyma(SoreMouth).1999,TheNavajoSheepProject.
9. NorrisR.,Scabbymouth.Farmanote,DepartmentofAgriculture,VeterinaryServices,
SouthPerth,2005,12.
10. NueleanVeturia,Farmacolgieveterinar,EdituraRizoprint, ClujNapoca,2004,313
314.

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Lucrritiinificevol53seriaMedicinVeterinar
11. PeacockA.,ContagiousEcthyma(ORF)inSheepandGoatsAgriculture,Publication
AP030,2004:http://www.goatworld.com/articles/soremouth.shtml.
12. Pop M., Rpuntean Gh., Vasiu C., Corina Suciu, Efficacy of treatment and
imunoprofilaxie formula in ectima contagiuoas to lambs. Rev.de Cret. Animal.,
1975,3,7478.
13. Raczykowski,C.,ContagiuosEcthyma(Soremouth),1994,
2. http://kinne.net/soremth.htm.
14. RpunteanGh.,BoldizsarE.,Virusologiespecialveterinar,2002,286299.
15. SotoCeaM.,ArenasReyesE.,ThomsBravoA.,EctimaContagioso:UnaEnfermedad
OcupacionalenlaIndustriaOvina.Ciencia&Trabajo,2007,9,25,113116.
16. Stone Niky, Ballart, Scabby Mouth (Orf) A Disease of Sheep and Goats, 2007,
AgricultureNotes,
3. 17.*** Ecthyma Contagieux Caprin. Pathologie de la mamelle: Epidmiologie,
SymptmesetLsions,Diagnostic,Risquespourlhomme.
4. http://etudiant.vet.alfort.fr/pedago/theses/repro/ovicap/femelle/htm/mamelle/ecyh
yma/ecthyacontagieux.htm

936


EFFECTIVENESSOFTREATMENTFORMULAINCONTAGIOUS
ECTHYMAINLAMBSFROMINTENSIVEBREEDINGSYSTEM
R.BIA1,S.RPUNTEAN2,G.RPUNTEAN2
email:bita.romulusbn@ansvsa.ro
1.DSVSABistriaNsud;2.USAMVClujNapoca

ABSTRACT
LivezilefarmfromBistritaNasaudcounty,isgrowingurcanbreedlambsformeatproduction,
byitsownresourcesandthroughacquisitionsfromneighboringcounties.Housingisperformedin
open shelters on metal grates floors. The lambs aged between 34 months showing lesions
developedontheskinofthelipsandfrequentlyextendedtothemucosaofthemouth.150lambs
weretreated,dividedintothreelotsof50head.Localtreatmentconsistedofmanualremovalof
crusts,followedbymethylenebluebrushingandsprayingantibioticspackedinspraybottlesfor5
days.Inmostofthetreatedanimalswasfoundanimprovementindiseasedevelopmentafter3
daysoftreatment,andafter5daystoproducehealinginmorethan90%oftreatedanimals.
The best efficiency expressed as the percentage of healing at after 5 days was found in the
productTerramycin(98.0%),followedbyAlamycin(94.0%)andDonarom(92.0%).Weappreciate
that the product Donarom was better supported by the fact that dexamethasone reduced the
inflammatory phenomena and anesthesine analgesic effect attenuates the pain. Antimicrobial
efficacywasfavoredbyensuringanappropriatediet,consistingofsoftfoodandwateradlibitum
andimprovinghygieneconditions.

Keywords:contagiousecthyma,lambs,treatment

INTRODUCTION
ContagiousEcthymaofsheepandgoatsiswidespreadworldwide,causingsignificant
economicdamage,particularlyinlambsandkidsandintheyouthadultpopulationmortality,
weakeninganddecliningproduction.Diseasehasimportanthealthimportancebeingplacedin
groupzoonosesandeasilytransmittedfromanimalstohumans,sothatstafftoexaminesick
animalsshouldwearprotectiveequipment(1,2,3).
The disease is caused by a specific virus, epiteliotrop (Parapoxvirus), acute and
characterizedclinicallybyavesicularrashwithscabsformingonlips,nostrils,face,eyes,teats,
udder, feet and mouth (2, 3, 4, 6, 9). During weaning young animals are most susceptible,
comparedwithotheragegroups(6).
Contagious Ecthyma cause significant economic damage by mortality, especially in
youth, as a result of complications that may occur consecutively secondary bacterial
infections.Morbiditymayreach7080%even100%,butmortalityislow,lessthan515%(3,
8).Fatalitiescanbefoundinlambsandkidswithsevereformofdiseaseduetolesionsofthe
mouth,theyrefusethesuckingandmaydiebystarvation.Oralandaroundthemouthlesions
inyoung,willreducedweightduetolowerfeedconsumptionleadingtoeconomiclosses.In
thelocationontheudder,mothersrefusetobreastfeedthelambs/kidsbecauseofthepain,
and they may weaken and die, and economic losses are due to both weaken and decrease
weightandreducemilkproduction.Thisisaddedtocostsofnonspecificorspecifictreatments
andpreventionmeasures(7).Itisknownthatdiseasetendstobepermanentinsomefarms,

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Lucrritiinificevol53seriaMedicinVeterinar
due to the fact that the virus has a high resistance to temperature and dryness, which can
surviveindriedscabsforyears.
In Bistrita Nasaud County contagious ecthyma develops in lambs between 34
months, in breeding and fattening farms with oral location or around the mouth and
mammarylocationinadultovineandgoatsfromhouseholds.Thepaperpresentsresultsfrom
treatmentinanintensivefarmingthathasdevelopedendemiccontagiousecthyma.

MATERIALANDMETHOD

Theexperimentwasconductedonthreegroupsofsicklambs,eachbatchconsisting
of 50 subjects, using different formulas for treatment. Lots of sick animals were separated
fromtherestoftheherd.Localtreatmentconsistedofmanualremovalofcrusts,followedby
methylenebluebrushingandantibioticapplicationbyspraying:
Group1wastreatedwithTerramicinespray(Pfizer)150mlbottlescontaining4gof
Oxytetracyclinehydrochlorideandthegreenmarkersubstance;
Group 2wastreatedwithAlamycinspray (Norbrook)140 g vialscontaining5 gof
Oxytetracyclinehydrochlorideandthebluemarkersubstance;
Group3wastreatedwithDonaromspray(Romvac)150mlbottlescontaining10g
ofoxytetracyclineanddexamethasone,crystalviolet,anesthesineandexcipients.
Support staff has been trained on the risk of contamination and have taken safety
measures(useofgloves,gowns,rubberbootsandprovideantisepticsubstances).Toreduce
environmentalcontamination,crustsremovedmanuallyandcollectedinthecontainerswere
burned, and accommodations have been cleaned and repeated disinfection was made with
VirkonandVirocidsolution.

RESULTS

Sheep breeding and fattening farm (urcana race) is in the area of Livezile in the
countyofBistrita,isownedbyaprivatebreeder.Fatteningyoungsheeppopulationisformed
fromitsownsourcesandthroughpurchaseofsheepfromdifferentownersinthecountyof
Bistrita and in neighboring counties (Maramures, Satu Mare, Cluj, Salaj, Mures, Suceava).
Growthishoused(opensheds),overthemetalgratingsonatotalof10lines,eachlinehaving
twosections(Fig.1and2).Feedingisdonewithalfalfa,naturalhayandconcentratesandthe
wateringisautomaticwithconstantleveltroughs.

Fig.1&2Aspectsoftheaccommodationsectionsofyounganimals

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UniversitateadetiineAgricoleiMedicinVeterinarIai
In the flock of young sheep for fattening, at the age of 34 months is evolving
contagious ecthyma; illness is most common in the months from July to August, the
occurrence of red by poor hygienic conditions. Transmission shall directly benefit from the
closecontactbetweenanimalsduetothegrowthsystem.

Clinically ill animals show more frequently lesions around the mouth and oral sites,
rarelyothersites.Vesicularrashappearsonthelips,corners,nosetipofthesnoutandrarely
onthecheeks,eyelids,earsorproliferativelesionsappearinthemouth.Evolutionarystages
veryquicklysucceededeachother,oftenthediseasewasseeninthescabsstage.Following
the opening of vesicles occur bleeding lesions, painful, becoming scabs, leading to thick
formations,lightgraybrown,irregular,adherenttounderlyingtissues(Fig.3and4).

Fig.3&4Youngsheepwithecthyma:crustlocatedaroundthemouthandnasal
Mostanimalshavelesionslocatedaroundthemouth,lipsbeingcomprisedentirelyor
onlysomeareasmorefrequentlylipcorners.Sometimesextendingstageisobserved,starting
from the nose and catching the mouth. In cases with more severe forms, swollen lips and
muzzlewereprocessedinalargenecroticwound,whichbleedeasilyifdetached(Fig.5and6).
Lesionswereverypainful,preventinganimalsconsumefeed.

Fig.5&6Contentandapplicationofmedicationbyspraying
Aftermanualremovalofthecruststhemethylenebluebrushingsweremade,taking
into consideration the whole area from which the crusts have separated, after which
proceeded to spray the antibiotic to the affected areas. Spraying was done from a short

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Lucrritiinificevol53seriaMedicinVeterinar
distance(1015cm)forafewsecondsuntilitcoveredthewholeaffectedarea.Treatmentwas
givendailyfor5days.

Fig.7&8Animalswithlesionsaroundthemouthinthetreatmentphase
Feeding sick animals was made with soft and liquid feed, and improved hygiene.
AccommodationwassubjectedtorepeateddisinfectionusingVirkonandVirocidsolutionsand
cleanbeddingwasprovided.
Treatedanimalsweredailyobservedandassessmentsbeingmadeofefficacyatafter
3 days and 5 days. The assessing focuses on the healing, regression of lesions and restored
ability to feed properly. The results obtained for each of the products used are listed in
Table.1.
Treatmentoutcomeinthethreegroups
Table1
No.

Groups

Results

Antibiotics
(spray)

After 3days
No.
%
39
78,0
35
70,0
32
64,0

1
2
3

After5days
No.
%
49
98,0
47
94,0
46
92,0

Group1
Terramycin(Pfizer)
Group2
Alamycin(Norbrook)
Group3
Donarom(Romvac)

Analyzing the table data can be seen that treatments have proved effective in all
threeappliedformulas.Inmostofthetreatedanimalswasfoundanimprovementindisease
development after 3 days of treatment and after 5 days the cure occurs in over 90.0% of
treatedanimals.Thosewhowerenotcuredwerestilltreated.Bestefficiency,expressedasthe
percentageofhealingafter5dayswasfoundintheproductTerramycin(98.0%),followedby
Alamycin(95.0%)andDonarom(92.0%).WeappreciatethatDonaromwasbettersupported
by the fact that dexamethasone reduced the inflammatory phenomena and anesthesine
attenuatesthepainbyanalgesiceffect.

DISCUSSIONS
To treat infectious ecthyma many and varied therapeutic formulas were tested
(brushingswithiodinesolution,iodizedglycerine,potassiumpermanganate,methyleneblue,

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UniversitateadetiineAgricoleiMedicinVeterinarIai
sprayingwithsulfaandantibioticsetc.). (3, 6,7,11).Inthetreatmentofaround mouthand
nostrils forms, generally good results are obtained, but in oral form, which usually occurs in
lambsandkids,thetherapeuticefficacyislower(6).
The principle is accepted that in cases of benign forms of disease no treatment is
necessary, the animals heal in about 23 weeks, achieving a satisfactory immunity to new
infections(2,3,4).Insevereforms,whichmayoccurinlambsandyounganimals,treatment
mustbedonetopreventsecondarybacterialinfections, whichcansometimescauseserious
complications.Forthispurpose,mostoftenisbeingmadealocaltreatment(spray,ointments)
withbroadspectrumantibioticsincombinationwithdifferentantiseptics(3,4).
Pop M, et al., (1975) performed treatments by using one of the following formula:
local applications of lotagen, lotagen in saline solution (1/1), bromocet, 2% methylene blue
solution, iodized glycerine, ramcemic chloramphenicol ointment 10%, 5% terramicine
ointment.Inallperibuccalformcasesbeforemedicationthecrustsscrapewasmade(6).

Methylene blue is recommended because it is moderate and lasting antiseptic,
penetratingthelesionaltissue(wounds,burns,gall,pressuresores,frostbite,glossitisetc.)on
which, by repeated applications have an analgesic effect, promoting healing. Given these
properties, this substance was used for its antiseptic effect in all three forms of treatment,
precedingadministrationoftheantibioticspray.

Antibioticsinasprayformhastheadvantageofeasyapplicationandsprayedfroma
convenient distance provides a uniform distribution on the affected area. Small size of
particles splashed ensure penetration into the smallest crevices, without any further
maneuvers,benefitinginsmoothandrapidwoundhealing.Sprayingshouldbedonewiththe
bottleuprightatadistanceof1015cmandalengthofatleast5secondsuntiltheproduct
evenlycovertheentireareaaffected.

All three sprays (Terramycin, Donarom and Alamycin) that have oxytetracycline as
activesubstanceincombinationwithmarkercoloringsubstanceandsomeexcipients,have
bactericide effect knowing that oxytetracycline hydrochloride is a broadspectrum antibiotic
that act as bacteriostatic on many Gram positive and Gramnegative bacteria, the rickettsia,
and spirochaetes. After spraying a protective film is formed having a protective effect over
lesiontissue,bacterialcomplicationsareavoidedandhealingisfavored.

Topreventdiseasewillavoidfeedingcoarsefeed,whichcancausethenoseormouth
lesions(5)andwillcombatflyingstinginginsects(3,5,7,and10).Antimicrobialefficacymay
be enhanced by ensuring adequate diet consisting of soft food and water ad libitum, and
improvinghygieneconditions(11).

CONCLUSIONS


1.InLivezilefarmfromBistritaNasaudcounty,youngTurcanabreedsheeparereared
for meat production. In young animals aged between 34 months is evolving oral and
peribuccalformsofcontagiousecthyma.

2.Atotalof150lambs,dividedintothreebatchesof50headswereinitiallytreated
topicallydoingmanualremovalofcrusts,followedbymethylenebluebrushingandspraying
antibioticsfor5days.

3. Efficacy of products used resulted in gradual healing of lesions after 3 days of
treatmentandcompleterecoveryafter5days,thebestperformancewasfoundtoTerramycin
spray(98%),followedbyAlamycin(94%)andDonarom(92%).

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Lucrritiinificevol53seriaMedicinVeterinar

4. Antimicrobial efficacy was enhanced by ensuring adequate diet consisting of soft
foods,wateradlibitumandbetteraccommodation.

REFERENCES
1.
2.

BttnerM.,Ecthymaofsheepandgoats.Tierarztl.Prax.,1985,13,(2),163169.
HarwingN.,Contagiousecthyma(SoreMouth).SheepHealth,AnimalScience,2000,FactSheet,
no.1.
3. HiggsA.,NorrisR.T.,BaldockF.C.,CampbellN.J.,KohS.,RichardsR.B.,Contagiousecthymain
thelivesheepexportindustry.Austral.Vet.J.,2008,74,(3),215220.
4. LuginbuhlJeanMarie,AndersonL.K.,ControlingSoreMouthinMeatGoats.ExtensionAnimal
Husbandry.DepartmentofAnimalScience,ANS00601MG.
5. Norris R., Scabby mouth. Farmanote, Department of Agriculture, Veterinary Services, South
Perth,2005,12.
6. Pop, M., Rpuntean, Gh., Vasiu, C., Corina Suciu, Eficacitatea unor formule de tratament i
imunoprofilaxienectimacontagioaslamiei.RevistadeCretereaAnimalelor,1975,3,7478.
7. Peacock,A.,SoreMouth,ContagiousEcthymaalsoknowasCE
1. http://www.goatworld.com/articles/soremouth.shtml.
8. Raczykowski,C.,ContagiuosEcthyma(Soremouth),1994,http://kinne.net/soremth.htm.
9. Rpuntean Gh., Boldizsar E., Virusologie special veterinar, Editura Academic Pres, Cluj
Napoca,2002,286294.
10. Stone, N., Scabby Mouth (ORF) A Disease of Sheep and Goats. Agriculture, State of Victoria,
DepartmentofPrimaryIndustries,2007,12.
11. VasiuC.,Virusuri,virozeiboliprionicelaanimale,EdituraMega,ClujNapoca,2009,237244.

942


CLINICALANDANATOMOPATHOLOGICALRESEARCHINPORCINE
CIRCOVIRUSTYPE2INFECTIONINYOUNGSWINE

N.CTANA,V.HERMAN,IONICA,FODOR,V.PETRUSE
FacultateadeMedicinVeterinarTimioara
epirovet@yahoo.com

Abstract
Porcinecircovirosisisadiseaseofviraletiology,withspecialsignificanceintheintensivefarming
ofpigs.Theinvestigationswereconductedduring20072009inthreeswinefarms,intheWestern
part of country, where it was pursued the presence of PCV2 infection by clinical and
anatomopathologicalexaminations.Duringtheresearch,intheherdsofthesethreefarmsitwas
reported only the post weaning multisystemic wasting syndrome (PMWS), the other syndromes
notbeingreported.Observationsofclinicaltrialsconfirmthatbasedonclinicalexaminationmay
be detected the 6 fundamental symptoms which may create the suspicion of PMWS syndrome.
We found these symptoms described above, except the presence of jaundice, in the herds from
thethreefarms.Grosslesionsencounteredmostfrequentlywere:enlargedinguinallymphnodes
and necrotichemorrhagical tonsillitis, catarrhal and hemorrhagic bronchopneumonia,
hyperplasticsplenitis,enlargedmesentericlymphnodesandpallidityofmucosaeandtissues.

Keywords:swine,PCV2,symptoms,anatomopathologicallesions.

Swine pathology in intensive farming has changed significantly due to the emergence of
nosologicalentities,especiallywithviraletiology,thatiscurrentlyadominantcomponentof
infectiouspathology.
Porcinecircovirosisis adiseaseofviraletiology,withspecialsignificance intheintensive
farmingofpigs.Firstdescribed inCanadain1991,thatdiseasehas spreadworldwide, being
alsoreportedandstudiedbyamultidisciplinaryteaminRomania.Inmanycountries,giventhe
economic importance of the disease and some unexplained aspects of epidemiology,
pathogenesis, anatomoclinical signs, diagnosis and prevention of this disease, several
collectiveshavedonelargeanddiversestudiesinthisarea(1,4,6).
The research of this work were made in order to obtain epidemiological and
anatomoclinical data, in three swine farms in Western part of the country where it was
suspectedthepresenceofinfectionwithPCV2,respectivellyPMWSsyndromeevolution.

MATERIALSANDMETHODS

Theinvestigationswereconductedduring20072009inthreeswinefarms,intheWestern
part of country, where it was pursued the presence of PCV2 infection by clinical and
anatomopathologicalexaminations.
Farm A of pigs breeding, in Arad county, has organized activities in three areas: maternity,
youngswineandfatteningpigs,andthebreedeffectiveisthehybridPIC,LargeWhitebreed
andKahibhybrid.Inthefarmispracticedonlytheartificialmounting.Inthebeginningofthe
researchtheherdstructurewasasfollows:1442sows,363youngsows,12boars,1719infant
piglets,6315youngpigsand5477fatpigs.

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Lucrritiinificevol53seriaMedicinVeterinar
Farm B, in Bihor county, is also organized in three sectors: maternity, young swine and
fatteningpigs,andthebreedeffectiveisthehybridPIC,LargeWhiteandDurocbreeds,from
theimport.
From the beginning of the experiment at this farm herd structure was as follows: 1200
sows,405youngsows,15boars,1520pigletsinfant,5800youngpigsand6200fatpigs.
FarmC,situatedinCarasSeverincounty,isafatteningfarmforyoungswinedesignedto
beslaughtered.Severaltimesayear,forthatpurposetheyareimportedeffectivesofyoung
pigsagedof2,53months.Averagetheimportedeffectivesarearound3000headsperseries.
ResearchonthepresenceofPCV2infectionwasmadeinthesefarms,becausetheexisting
swineherds,PMWSsyndromewassuspected.
Clinicalexamination.Clinicalexaminationswereperformedonagegroups,seekingtothe
presenceofsyndromescausedbyPCV2virus.Inthethreefarms,intermsofclinicalsigns,only
PMWSsyndromewassuspected.
Anatomopathological examination. Anatomopathological examinations were performed
only on young swine corpses in which PMWS syndrome was suspected. From these corpses
weresampledorgans(lungs,spleen,kidneysandinguinallymphnodes).Thesesampleswere
intendedforlaboratorytests.
Histopathologycalexamination.Itwasperformedontheorganssampledinthenecropsy
ofcorpses,beingusedtheconventionalmethodology,byfixinginabsolutealcohol,including
intotheparaffin,cuttingandsectionscoloringbytrichromicHEA(HematoxilineEosineMetil
Blue)stain.Thisexaminationwasconductedinordertohighlightsomecharacteristiclesionsof
PCV2infections(4).
Cytologicalexamination.Thisexaminationwasperformedonfingerprintssmearsfromthe
inguinallymphnodes.FortheevidenceofchangesincytologyMayGrundwaldGiemsastain
wasused(5).

RESULTSANDDISCUSSIONS

Research conducted in the three farms confirmed the presence of PCV2 infection, which
hasevolvedduringthatperiodasPMWSsyndrome.Atthesametimetheseresultsconfirmed
the observations of other researchers who consider that to certainty specify the diagnosis
there are required more laboratory tests and by corroborating the obtained results may be
confirmedthepresenceofthedisease.
Resultsofclinicalexamination.Clinicalexaminationwas performedonagegroupsbeing
recordedalldetectedsymptoms.Duringtheresearch,intheherdsofthesethreefarmsitwas
reported only the post weaning multisystemic wasting syndrome (PMWS), the other
syndromesnotbeingreported.
Inthesickpigsfirstsignsbegantoappearaftertheageof6weeks.Thedominantsymptom
wasprogressiveweakeningoftheaffectedpigs.Alongwiththatsymptomtheyalsohavebeen
reported:dyspnea,diarrhea,pallidityofskinandmucousmembranesandenlargedsuperficial
lymphnodes,especiallyoftheinguinallymphnodes.Inmanyanimalsthesesymptomshave
beenmaskedbysymptomsofsomeassociateddiseasesusuallywithrespiratorytracking.
Observationsofclinicaltrialsconfirmthatbasedonclinicalexaminationmaybedetected
the6fundamentalsymptomswhichmaycreatethesuspicionofPMWSsyndrome.Wefound
these symptoms described above, except the presence of jaundice, in the herds from the
three farms. In farm A the clinical examination confirmed that there were the most clinical
casesofPMWSsyndromethatdevelopedconstantly,ineveryseriesofpigletsaftersixweeks
ofage,butrecordsofmorbidityandmortalitywerenotkeptclearlyinthefarm.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
In thefarm Band farmC the numberof sick animalswas lower but the development of
symptomswasidenticaltothatoffarmA.
These findings are similar to existing data in the literature on the prevalence of PMWS
syndromewhichismorefrequentcomparedwithothersyndromesandthemorbidityofthis
syndromeisvariable(2,6).
ClinicalobservationshaveconfirmedthepresenceofsocalledassociateddiseasesPCVAD
(Porcine Circovirus Associated Disease) which in the three farms were as follows:
pasteurellosis, contagious pleuropneumonia, enzootic pneumonia and dysentery with
Brachyspira.
Results of anatomopathological examination. Anatomopathological examinations
conductedonthecorpsessuspectedofPMWShaverevealedcharacteristicgrosslesionsmore
or less associated with lesions caused by associated infections. Gross lesions encountered
most frequently were: enlarged inguinal lymph nodes and necrotichemorrhagical tonsillitis,
catarrhal and hemorrhagic bronchopneumonia, hyperplastic splenitis, enlarged mesenteric
lymph nodes and pallidity of mucosae and tissues. In the three farms there were not found
characteristiclesionsofdermatitisnephritissyndromeofpigstothenecropsyofcorpses.
In other organs that liver, kidney, stomach, small intestine and large intestine, the
observed lesions were specific to other diseases, usually bacterial infections or dystrophic
injuriesconsecutiveofsome nutritionaldiseasesor mycotoxicosis. Inmorecorpses,perhaps
withlongerclinicalevolution,specificlesionsofPCVADdiseaseswerefound,suchas:fibrinous
pleuritis, fibrinous or hemorrhagic bronchopneumonia, poliserositis and necrotic
hemorrhagicalcolitis.
The results of anatomopathological exam conducted in the three farms are linked to
existingliteraturedataregardingthefrequencyandthesignificanceofcharacteristiclesionsof
thissyndromeandofthelesionscausedbyPCVAD(3,4,6).
Resultsofhistologicalandcytologicalexamination.Characteristichistologicallesionswere
found in the lymphnodes and spleen and lesions of viral andbacterial associated infections
havebeenrevealedinotherorgans.
Ininguinallymphnodesbyhistologicalexaminationwerehighlightedthefollowinglesions:
renderedlymphocyticdepletion,histiocytesmassiveinfiltrationandthepresenceofgiantcells
(fig.1).Thesecellsconfirmthepresenceofgranulomatoustypeinflammation,respectivelythe
granulomatouslymphoreticulitischaracteristictoPCV2infection.
In the spleenwas found also a marked lymphocyticdepletion until the disappearance of
lymphoidfolliclesandthepresenceofgiantcellscharacteristictogranulomatoussplenitis.In
the case of that organ also the found lesions are characteristic to PCV2 infection, having a
specialmeaningtheinflammationofgranulomatoustype.
Cytologicalexaminationperformedonfingerprintssmearsofinguinallymphnodesasthe
methodology recommended by LUPPI and all. (2008) highlighted a small number of
lymphocytes and a large number of histiocytes. Multinucleated giant cells have also been
highlighted. This exam has the advantage of speed on the diagnosis of PMWS syndrome, it
couldbeperformedinanylaboratoryandtheresultsprovidedhelptospecifythediagnosis,
nottoreplacethehistologicalandserologicalexaminationandthePCRtest(5).
According to the results obtained by LUPPI and all (2008), depending on the number of
giant cells and on the infiltration with histiocytes it can appreciated the evolution of PCV2
infection.Theseauthorsconsiderthatthechangesfound,depending on theirintensity,may
beofgrade1,grade2andgrade3(5).

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Lucrritiinificevol53seriaMedicinVeterinar
Theobtainedresultsintheresearchcarriedoutbythisexaminationrevealedgranulomatous
inflammation of lymph nodes but has not allowed us to appreciate the intensity of existing
cytologicalchangesinlymphnodes,respectivelythemomentofinfection.

Fig.1Giantcellsininguinallymphnodes

CONCLUSIONS

Inthethreefarms,PCV2infectionwasintroducedbytheboughtanduncontrolledswines
thatwereviruscarrying.
Clinical examination confirmed the development of PMWS syndrome with dominant
symptoms,lessjaundice,inthethreefarms.
Anatomopathologicalexaminationconstantlyrevealedenlargedinguinallymphnodesand
thepresenceofgrosslesionsproducedbyPCVAD.

REFERENCES

1. Cadar,D.Analizaepidemiologicievaluareamodificrilormorfofuncionalealesistemuluiimun
ncircoviroza(PCV2)suin,USAMVClujNapoca,Tezdedoctorat,USAMVClujNapoca,2008.
2. Carman, S., Mcewen, B., Delay, J., Van Dreumel, T., Luis, P., Cai, H., Fairles, J. Porcine
circovirus2associateddiseaseinswineinOntario(2004to2005).In:CanVetJ.,2006,47,(8),p.
761762.
3. Carr,J.,Odea,M.,Mclachlan,S.,Wilcox,G.Thesuperficialinguinallymphodeofthepigand
PCV2.Proceedingsofthe20thIPVSCongress,Durban,SouthAfrica,2226June2008,39.
4. Chae,C.Areviewofporcine circovirus2associatedsyndromesand diseases, VetJ. May,2005,
169,(3),32636.
5. Luppi Andrea, Bonilauri, P., Di Lecce, Rosanna, Paoletti, Francesca, Bosseti, M., Cordioli, P.
Clinical and Pathological in an outbreak of PMWS and diagnostic methods comparison.
Proceedingsofthe20thIPVSCongress,Durban,SouthAfrica,2226.VI.2008,49.
6. Segals, J., Allan, G., Domingo, M. Viral Diseases, section II, Porcine Circovirus Diseases. n:
DiseasesofSwine,9thEdition,Edit.STRAWB.,ZIMMERMANJ.,D'ALLAIRES.,TAYLORD.,Wiley
Blackwell,Hardcover,2006,299305.

946

SEROLOGICALANDPCRRESEARCHDIAGNOSISOFTHEINFECTION
WITHPORCINECIRCOVIRUSTYPE2INYOUNGSWINE

N.CTANA ,VIRGILIAPOPA2,V.HERMAN1,IONICAFODOR1
1
FacultateadeMedicinVeterinarTimioara
2
S.N.InstitutulPasteurS.A.
epirovet@yahoo.com

Abstract
In european countries PCV2 infection evolves epidemic, with the starting point of western
countries,from where itpermanently expanded to central and eastern Europe.To confirm PCV2
infectionintheexistingswineherdsofthethreefarmsweremadethefollowinglaboratorytests:
immunoassay test, Polymerase chain reaction.By PCR reaction viral DNA was revealed,
respectivelyORF2gene,insamplesoflymphnodesandspleen,beingconfirmedthepresenceof
PCV2 virus, by a reliable diagnosis method.Analyzing the results obtained in the three farms,
regardingtheseroprevalencebyagegroupsandtypeofinfectionwecannotethatPCV2infection
was present in all production groups from farms with the exception of infant piglets, these
infectionsbeingclassifiedinall3categoriesaccordingtotheevolutionmoment..
Keywords:PCV2infection,granulomatousinflammation,seroprevalence.

IneuropeancountriesPCV2infectionevolvesepidemic,withthestartingpointofwestern
countries,fromwhereitpermanentlyexpandedtocentralandeasternEurope.Thus,thereare
europeancountrieswherethediseasedevelopssevere,withlargeeconomiclosses,countries
where the disease is not a major problem or is less monitored as Romania and countries
wherethediseasehasnotarecognizedstatus(2,3).
Inthiscontextithasbeenincludedtheresearchconductedinamultidisciplinaryresearch
programofC.E.E.X.type,coordinatedbySNPasteurInstituteSA,theDepartmentofInfectious
DiseasesofFMVTimioarabeingforeignpartner.
The research covered the work were made in order to confirm the presence of PCV2
infectionbylaboratorytestinginthreeswinefarmsfromthewesternpartofthecountry.

MATERIALSANDMETHODS

To confirm PCV2 infection in the existing swineherds ofthe threefarms were made the
followinglaboratorytests.
Immunoassaytest.Avariantofcatchwasusedtodetermineantibodytitres(IgMandIgG
type)specifictoPCV2.TothispurposeitwasusedthecommercialkitIngezimCircovirusIgG/
IgM,madebyIngenasaMadridSpain,whoalsosuppliedthesoftwareforinterpretation.
Polymerasechainreaction.ItwasdonetodetectORF2genethatispresentintheviralDNA
(5).TodetectthatwereusedgeneprimersORF2andamplicons967bp.Thistechniquewas
performedintheLaboratoryofMolecularBiologyofSNPasteurInstituteS.ABucharest.The
reactionwascarriedoutonsamplesoforganstakenfromcorpseswithnecropsy(2).

RESULTSANDDISCUSSIONS

Results of polymerases chain reaction. By this reaction viral DNA was revealed,
respectivelyORF2gene,insamplesoflymphnodesandspleen,beingconfirmedthepresence

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Lucrritiinificevol53seriaMedicinVeterinar
of PCV2 virus, by a reliable diagnosis method. This reaction is recommended to confirm
diagnosis of PCV2 infection as virusological examination, respectively the isolation and
cultivationofthisvirus,cannotbeimplementedinalllaboratories(1,4).
Serological examination conducted by the reaction of immunoassay, catch variant
highlighted the presence of specific antibodies in serum collected from animals in the three
farms.ThevaluesofthesetitresexpressedinOD(opticaldensity)wereprocessedthroughits
programfortheinterpretationoftheusedcommercialkit.Theprogramfortheinterpretation
groupedtheobtainedresultsinpositivesamplesandnegativesamplesbasedonODvaluesof
theinvestigatedseracomparedwithODvaluesofcontrolsera.
Thisprogramallowsinthepositivesera,dependingontheopticaldensityforIgMandIgG,
theclassifyingofinfectionsinthreecategories,asfollows:activeinfection,recentinfections,
oldinfections.
Table1
Theserologicaltestresults

FarmA
FarmB
FarmB
Category
Results
No.
%
No.
%
No.
%
Negative
5
9,6
13
32,5

31,5
Youngpigs
Pozitive
6
11,5
12
30

68,4
Negative
8
15,3
1
2,5
6

Sows
Pozitive
16
30,7
4
10
13
Negative

2
5

Fatpig
Pozitive
13
25
8
20

Negative
2
3,85

Boars
Pozitive
2
3,85

Negative
15
28,8
16
40
6
31,5
Pozitive
37
71,1
24
60
13
68,4
Active
2
3,8
6
15
13
68,4
Centralization infection
results
Recent
8
15,4
8
20

infection
Old
27
52
10
25

Infection

PCV2infectionwasconfirmedserologicallyinallagegroupsunderthistest.
InfarmA,themostpositivesamples(37.7%)werefromsows followedby fat pigs(25%)
andyoungpigs(11.5%).
InfarmBthemostserologicallypositivesampleswerefromyoungpigs(30%),followedby
fatpigs(20%)andsows(10%).
InfarmC,intheimportedyoungswine,68.4%ofsampleswerepositive.
Dynamicsofpositivesamples,whosetitreswereexpressedinODisreproducedintable1.
The interpretation program of the commercial kit also established the results of the
infectiontype,respectivelythethreecategories(active,recentandold).
In farm A, 71.1% of samples were positive, 52% being old infections and 15.4% recent
infections.
InfarmB,60%ofsampleswerepositive,ofwhich25%witholdinfection,20%withrecent
infectionand15%withactiveinfection.
InfarmC,68.4%ofsampleswerepositive,allbeingactiveinfection.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Analyzing the results obtained in the three farms, regarding the seroprevalence by age
groups and type of infection we can note that PCV2 infection was present in all production
groupsfromfarmswiththeexceptionofinfantpiglets,theseinfectionsbeingclassifiedinall3
categoriesaccordingtotheevolutionmoment.
Although initially most researchers arguedthat serological examination is not sufficiently
decisive, currently by using enzyme immunoassay test with kits and performantes programs
for the interpretation of results, it can be determined with certainty so PCV2 infection
seroprevalenceandtypeofinfection.
Theseresultscompletedtheresultsofotherexamsandtheyareinterrelatedwithexisting
dataintheliterature(3,4).
CONCLUSIONS

Characteristichistologicallesions(lymphocyticdepletion,infiltrationwithhistiocytes,giant
cells)wereconsistentlypresentininguinallymphnodes.
Inflammation of granulomatous type, represented by multinucleated giant cells, was
confirmed by cytological examination, this examination being recommended as a guidance
andrapidtestforPMWSdiagnosis.
Serological examination conducted by the capture enzyme immunoassay test confirmed
thepresenceofPCV2infectionandallowedtheclassificationofinfectionsinthreecategories:
active,newandold.
By using performing kits the enzyme immunoassay test can be used in epidemio
surrveillanceofPCV2infectioninswinefarms.

REFERENCES

1.

2.

3.

4.

Kwang, S.L., Kim, H.B., Han, S.J. Evaluation of a nested polymerase chain reaction assay to
differentiate between two genotypes of Porcine circovirus2, Journal of Veterinary Diagnostic
Investigation,2008,Vol.20,Issue3,283288.
PopaVirgilia,Stnuic,D.A.,Bucur,E.,Cadar,D.,Ctan,N.,DaneDoina,Britreanu,S.,
BotuDaniela,NicaIrina,StanAdela,Belteghi,C.,Pastram,F.,Tma,T.Studiesregardingto
the presence of porcine circovirus type 2 in swine industrial growth frams from Romania,
LucrritiinificeMedicinVeterinar,2008,XLI,Timioara,784792.
Segals, J., Allan, G., Domingo, M. Viral Diseases, sectionII, Porcine Circovirus Diseases. n:
DiseasesofSwine,9thEdition,Edit.STRAWB.,ZIMMERMANJ.,D'ALLAIRES.,TAYLORD.,Wiley
Blackwell,Hardcover,2006,.299305.
Sibila, M., Calsamiglia, M., Segales, J., Blanchard, P., Badiella, L., Le Dimna, M., Jestin, A.,
Domingo, M. Use of a polymerase chain reaction assay and an ELISA to monitor porcine
circovirus type 2 infection in pigs from farms with and without postweaning multisystemic
wastingsyndrome,AmJVetRes.,2004,65,(l),8892.

949

RESEARCHCONCERNINGTHEANTIBACTERIALACTIVITYOF
HONEYAGAINSTCOAGULASENEGATIVESTAPHYLOCOCCI
WITHUSEINVETERINARYMEDICINE

C.CEAUI,I.OGOE,
ANCAMARIAGALI,L.TUDOR,I.L.ILIE,
*FacultyofVeterinaryMedicineBucharest

Abstracts
Honey is considered to have a great potential to treat infected wounds, but for the
momentthedetailsintheresearchfieldarenotsoconcludingandstillnotenoughas
number. The apparition and development of antibioticresistant strains of coagulase
negativestaphylococciisconsideredaverycomplicatedandimportantprobleminthe
medicalfield.
The objective of this study was the to determine the optimal dilution of 3 types of
honey,for25clinicalcaseswithisolatesofcoagulasenegativestaphylococci.Inorderto
determine the active dilution, it was used a technique of agar incorporation, with
dilutionstepsof5%.Theplateswereinoculatedwith25mlofculture,fromtheisolated
coagulasenegative staphylococci. Honey presented an inhibitory effect at dilutions
from4,2to1,8%foracaciahoney,3,7to1,2%forlindenhoneyand3,9to1,1%for
polifloralhoney.Themainconclusionwasthatthesetypesofhoneymayhavebeneficial
activitieswhentreatingdifferenttypesofwounds,especiallywhencoagulasenegative
staphylococciarepresent.
Keywords:coagulasenegativestaphylococci,honey,infection,wounds.

Regarded as saprophytic commensals of low pathogenicity that normally inhabit


humanskinandmucosalmembranes,coagulasenegativestaphylococciwerenotconsidered
but until now opportunist pathogens, being now among the five most frequently isolated
causativeagentsofhospitalacquiredinfection(2,5,7),frequentlyassociatedwiththeuseof
temporaryandpermanentinvasivemedicaldevices(intravenouscatheters,dialysiscatheters,
urethral stents, endotracheal tubes, etc.). The most commonly isolated coagulasenegative
staphylococcus is the S. epidermidis, but there were reports including other species too (1,
10).Theapparitionofamethicillinresistantstrainofthesecoagulasenegativestaphylococci
and also of the other species which reveal a multiple antibiotic resistance, has made this
problem a very important and difficult one (4, 8, 9). Until now, coagulasenegative
staphylococciwerenottestedsufficiently,sothisstudyhadtheobjectiveofinvestigatingtheir
susceptibilitytohoney.Notallthehoneytypespresentanantimicrobialactivitywhichcould
influencepreciselythecoagulasenegativestaphylococciactivity,thereforehoneywithmedian
levelsofactivitywasused.

MATERIALSANDMETHODS

Thethreenaturalhoneysusedwereselectedtobeclosetothemedianantibacterial
activity for each type of activity and tested against Staphylococcus aureus: acacia honey,
linden honey and polifloral honey. The isolated cultures of coagulasenegative staphylococci

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Lucrritiinificevol53seriaMedicinVeterinar
were obtained from the veterinary clinic, in number of 25. The cultures were isolated from
peritonealfluid,cerebrospinalfluidandbreastaspirate.Theisolateswereidentifiedusingthe
classic biochemical and morphological techniques. The media used for this investigation
included:TSB,nutrientagarandbloodagar(withdefibrinatedsheepblood).
Beforetheinitiationofthetests,eachisolatewasintroducedtocultureinTSB,and
after incubation at 37oC for 16 hours, the agar incorporation technique was used. Nutrient
agarwasmadeupatdoublestrength,measuredoutinto25mLaliquotsandautoclaved.To
preparetheplatesitwasmeltedandtemperedina50oCwaterbathuntilpoured.Thehoney
samples were prepared in sterile deionized water, and poured into duplicate Petri dishes,
formingamixturewiththeagar.
A dilution series with honey concentrations in the range 520% (v/v) final honey
concentration,in5%increments,wasusedforthesusceptibilityassaysforthenaturalhoneys.
Theinoculatedplateswereincubatedat37oCfor16h,andtheresultswererecordedoneach
plate.

RESULTSANDDISCUSSION

In Table 1, it can be seen that the growth of all 25 coagulasenegative


staphylococcus isolates was inhibited by acacia, linden and polifloral honey in different
degrees and at different concentrations (dilutions). Honey presented an inhibitory effect at
dilutionsfrom4,2to1,8%foracaciahoney,3,7to1,2%forlindenhoneyand3,9to1,1%for
polifloralhoney.Acaciahoneypresentedvaluesof4.0,4.1and4.2%(samplesno.20,21and
22)ofdilutionswhichinhibitedtheS.epidermidisgrowth,while,lindenhoneyhadvaluesof
3.5,3.6and3.7%(samplesno.15,14and9)andpolifloralhoney3.5and3.7%(samplesno.9
and 21), for the same species. In what concerns S. haemolyticus, the highest values were
registeredforpolifloralhoney(3,8%,sampleno.24),whilelindenhoneyhadavalueof3,7%
(sampleno.16)andacaciahoneyonly2.9%(sampleno.1).

Table1Theaveragevaluesforthedilutions(%)ofdifferenttypesofhoneyonthe
speciesofStaphylococcusspp.isolatedfromthe25clinicalcases
Typesofhoney
No.
Species
Acacia
Linden
Polifloral
2
Staphylococcusepidermidis
1,8
2,8
2,0
5
S.epidermidis
2,0
3,1
2,9
9
S.epidermidis
1,8
3,6
3,5
10
S.epidermidis
2,2
2,0
2,4
11
S.epidermidis
2,6
1,5
1,5
12
S.epidermidis
3,6
2,7
3,0
14
S.epidermidis
2,8
3,7
1,7
15
S.epidermidis
3,4
3,5
1,1
20
S.epidermidis
4,0
2,9
2,2
21
S.epidermidis
4,1
1,2
3,7
22
S.epidermidis
4,2
3,1
1,8
25
S.epidermidis
3,9
2,8
2,8
1
Staphylococcushaemolyticus
2,9
1,9
3,2
13
S.haemolyticus
3,1
3,3
2,4

951

UniversitateadetiineAgricoleiMedicinVeterinarIai
16
23
24
3
4
7
6
8
19
17
18

S.haemolyticus
S.haemolyticus
S.haemolyticus
Staphylococcussimulans
S.simulans
S.simulans
Staphylococcuscapitis
S.capitis
S.capitis
Staphylococcuswarneri
S.warneri

2,7
1,9
2,0
3,5
4,0
3,8
3,6
3,7
4,1
4,2
3,3

3,7
1,8
2,9
2,2
3,0
2,8
3,1
2,2
1,9
1,2
1,6

1,6
2,7
3,8
2,8
3,1
1,1
1,8
3,4
2,5
3,9
3,7

Sampleno.4,presentedthehighestvaluesforS.simulans,4.0%foracaciahoney,
3.0 % for linden honey and 3.1 % for polifloral honey. S. capitis was inhibited at 3.1 %
concentrationforlindenhoney(sampleno.6),at3.4%forpolifloralhoney(sampleno.8)and
4.1%foracaciahoney(sampleno.19).S.warneriwasinhibitedat4.2%foracaciahoney,3.9
%forpolifloralhoneyandonly1.6%forlindenhoney(samplesno.17and18).

Acacia

Linden

S. warneri

S. warneri

S. capitis

S. capitis

S. capitis

S. simulans

S. simulans

S. simulans

S. haemolyticus

S. haemolyticus

S. haemolyticus

S. haemolyticus

S. haemolyticus

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S.epidermidis

S. epidermidis

5
4,75
4,5
4,25
4
3,75
3,5
3,25
3
2,75
2,5
2,25
2
1,75
1,5
1,25
1
0,75
0,5
0,25
0

Polifloral

Fig.1Theaveragevaluesforthedilutions(%)ofdifferenttypesofhoney
onthespeciesofStaphylococcusspp.isolatedfromthe25clinicalcases

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Lucrritiinificevol53seriaMedicinVeterinar
CONCLUSIONS

Themainconclusionafterperformingthisstudy,isthatacaciahoneypresentsthe
highestantimicrobialactivityconcerningcoagulasenegativestaphylococci,withvaluesofthe
concentrationof4,2%,andinthefuture,thismaybeusedwithsuccesswhenclinicalcases
appearinmedicalinstitutionswithveterinaryprofile.

REFERENCES

1.

Tunney MM, Gorman SP, Patrick S. Infection associated with medical devices. Rev Med
Microbiol1996;7:195205.
2. KloosWE,BannermanTL.Updateonclinicalsignificanceofcoagulasenegativestaphylococci.
ClinMicrobiolRev1994;7:11740.
3. Vandenesch F, Eykyn SJ, Etienne J. Infections caused by newlydescribed coagulase negative
staphylococci.RevMedMicrobiol1995;6:94100.
4. Molan PC, Betts JA. Clinical usage of honey as a wound dressing: an update. J Wound Care
2004;13:3536.
5. Cooper RA, Molan PC, Harding KG. Antibacterial activity of honey against strains of
Staphylococcusaureusfrominfectedwounds.JRSocMed1999;92:2835.
6. Cooper RA, Molan PC, Harding KG. Honey and gram positive cocci of clinical significance in
wounds.JApplMicrobiol2002;93:85763.
7. Cooper RA, Halas E, Molan PC. The efficacy of honey in inhibiting strains of Pseudomonas
aeruginosafrominfectedburns.JBurnCareRehabil2002;23:36670.
8. Molan PC.Theantibacterialactivityofhoney.2.Variationinthepotencyoftheantibacterial
activity.BeeWorld1992;73:5976.
9. Allen KL, Molan PC, Reid GM. A survey of the antibacterial activity of some New Zealand
honeys.JPharmPharmacol1991;43:81722.
10. Molan PC. Reintroducing honey in the management of wounds and ulcerstheory and
practice.OstomyWoundManage2002;48:2840.

953

RESEARCHESREGARDINGTHEEFFICIENCYOFPLANTSESSENTIAL
OILSEXTRACTSONSOMEBACTERIALSTRAINSISOLATEDFROM
MASTITISCOWMILK

F.CHIRIL,N.FI,S.RAPUNTEAN,G.NAD,O.NEGREA
UniversityofAgriculturalScienceandVeterinaryMedicine,ClujNapoca,
FacultyofVeterinaryMedicine,email:nfit@usamvcluj.ro

Abstract:BetweenJanuary2009May2010intheLaboratoryofMicrobiologyoftheFacultyof
VeterinaryMedicineinClujNapoca,17samplescollectedfrom16dairycowsandbuffaloeswith
acute and recurrent clinicalmastitis have been examined by classic bacteriological examination
andmycologicalexamination.Fromtheexaminedsampleswereisolatedthefollowingbacterial
species:12strainsofStaphylococcusspp.,onestrainofStreptococcusspp.,onestrainofBacillus
spp., one strain of Klebsiella spp., a strain of Candida spp. and from buffalo milk one strain of
Streptococcusagalactiae.
Sensitivity of these strains was tested by aromatograma on MuellerHinton medium for
bacteriaandSabouraudmediumforCandidatothefollowingessentialoils:pine,mint,lavender,
St John's wort, basil and thyme, as a possible alternative to current antibiotics. Aromatograma
diffusimethrictechniquewasperformed,eachessentialoilwasapplieddiluted1/10,20ml/well.
Wereusedascontrolsthefollowinginternationalreferencestrains:StaphylococcusaureusATCC
6538P,BacilluscereusATCC14579,andCandidaalbicansATCC90028.Ofthe12Staphylococcus
strains tested, 10 strains (83.33%) were sensitive to pine oil, 9 strains (75%) for oil of thyme, 7
strains (58.33%) for peppermint oil and three strains (25%) for lavender oil. Control strain of
StaphylococcusaureusATCC6538Pwasverysensitivetothymeoilandmoderatesensitivetopine
oil,beingresistantfortheothertestedoils.

Keywords:clinicalmastitis,largeruminants,essentialoilsfromplants,bacteria,yeasts.

INTRODUCTION

Widespread use of antibiotics in animal breeding, particularly intensively, lead to


selectionofresistantstrainsoforganismswithincreasinglyhigherresistancetomostofthese
products(2,3,4,8;9,10,11).Theirretentioninthebody,theireliminationinmilk,whichisa
staplefoodforchildrenandtheelderly,leadtorestrictionsinusingthisproduct,insignificant
economiclossesforthefarmer,thepublichealthrisksandlimittheuseofantibiotics.

For this reason, the world is seeking alternatives to using antibiotics, one of which
being represented by aromatherapy. Antibacterial and antiinflammatory properties of
essential oils are known for some time but they are still little used in veterinary medicine,
althoughtheymayrepresentafuturealternative(7,12,13,14).
AromatherapyusedinanimalhealthisreglementedwithAnnex2ofRegulationEEC
2377/90 showing that 19 essential oils can be administered to all food producing species
withouttheneessitytosetmaximumresiduelimits.Thisinformationledustoinvitrotestof
the effect of plant essential oils on some species of microorganisms isolated from clinical
mastitisinlargeruminants(cows,buffalo).

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Lucrritiinificevol53seriaMedicinVeterinar
MATERIALSANDMETHODS

Our investigations were performed on 17 microbial strains isolated from milk from
cows with acute and recurrent clinical mastitis in Mures and Cluj counties and three
international reference strains used as controls. Isolation of microbial strains was done by
classicbacteriologicalandmycologicalexam,50mlofeachsamplewasinoculatedinbrothon
bloodagarandSabouraudmedium. Tubeswereincubatedat37Cfor24hours,prolonging
the incubation to 4872 hours if Sabouraud environment, with daily examination of cultural
characters. Genus identification was made based on morphological characters on Gram
stained smears, cultural character, complemented by selective or differential biochemical
propertiestests.Microbialstrainsisolatedwerethensowedinsoftagartubesandincubated
24hoursat37C.
International reference strains, kept in soft agar in the laboratory's own collection
and other strains of interest isolated from cases of mastitis were resowed in broth for 24
hoursbeforethearomatogramawasmadeandincubatedat37C.
Aromatograma was made by a technique similar to that used in antibiogram
diffusimetricalmethod.BacterialstrainsweresowedonMuellerHintonmediumandyeastsof
thegenusCandidaonSabouraudmedium.IneachPetridishwithadiameterof5mm,aradial
patternwasmade,forthesixessentialoilsusedfortesting.24hoursculturesofthebacterial
andyeaststrainswerediluted1/1000intubeswithsterilenutrientbrothandinoculated by
floodingthePetridishesinwhichpreviouslywellswerepracticed.Aftercoatingthesurfaceof
theculturemediaevenly,excesscultureofwellsandsurfacePetridishwas removed witha
sterile Pasteur pipette, the dishes were then left in a laminar flow hood to dry for 1015
minutes.

AfterdryingthesurfaceenvironmentofPetridishessowedwiththeteststrain,each
wellwasfiledby20loftestoil,diluted1/10,thentheywereincubated24hoursat37C.
Inthetestwereusedsixessentialoils,madeintheplateinthefollowingorder:pine,
peppermint,lavender,StJohn'swort,basilandthyme.Readingandinterpretationofresults
was performed the next day, based on the presence or absence of microbial culture lysis
areas.

RESULTSANDDISCUSSIONS

Of the 17 milk samples collected from cows with clinical mastitis the following
microorganismswereisolated:12strainsofStaphylococcusspp.,twostrainsofStreptococcus
spp., and one strain of Bacillus spp., Klebsiella spp. and Candida spp. In nutrient broth both
GrampositiveandGramnegativebacteriacausedanntenseturbiditywithalowdeposit,easy
homogenized. On blood agar, staphylococci formed Stype colonies 23 mm in diameter,
surrounded by a zone of 12 mm of or and haemolytic while streptococci presented
smallcoloniesofupto1mmindiametersurroundedbyareducedzoneofhaemolysistype.
Morphological characters exam on smears stained by Gram method, staphylococci were
presented as Gram positive cocci, grouped as irregular clusters of cocci while streptococci
formGrampositiveovoidshaped,groupinthechainlengthsvariabledependingonthestrain
andspecies.
Effect of six essential oils from plants on the strains isolated from mastitis milk
samplesandthethreeinternationalreferencestrainsusedascontrolsarepresentedinTable
1.

955

UniversitateadetiineAgricoleiMedicinVeterinarIai

Testedstrain

Table1:Effectofsixessentialoilsfromplantsonpathogenicmicroflora
isolatedfromclinicalmastitisincows
Pine
Pine
Lavander
StJohns
Basil
Thyme
essential
essential
essential
wort
essential essential
oil
oil
oil
essential
oil
oil
oil
13mm
15CR
10CR
R
R
11mm
8mm
8mm
6mm
R
R
12mm

115Staph.
0753M+
Staph.
0753RStaph.
13mm
11mm
8mm
R
R
13mm
2675Staph
20mm
15mm
8mm
R
R
11mm
8626Staph
22mm
20mm
7CR
R
8mm
R
8674Staph
17mm
17mm
13mm
R
R
23mm
2060Staph.
15mm
12mm
10mm
R
10mm
13mm
Staph.3
15mm
R
6mm
R
R
12mm
Staph.5
13mm
7mm
7mm
R
10mm
15mm
1231Staph.
10IP
R
10IP
R
10IP
8mm
3056Staph.
13mm
15mm
11mm
R
R
12mm
8556Staph.
13mm
10IP
10mm
R
R
10mm
3057Str.
R
R
R
R
R
R
64LStr.
13mm
17mm
9mm
R
R
18mm
Bacillusspp.
R
R
R
R
R
12mm
Klebsiellaspp.
R
R
R
R
R
R
Candidaspp.
R
R
R
R
R
8IP
S.aureusATCC
9mm
R
R
R
R
22mm
6538P
B.cereusATCC
11mm
12mm
11mm
R
R
14mm
14579
C.albicans
R
R
R
R
R
10IP
ATCC90028
Legend:CR=resistantcoloniespresentintheinhibitionarea;
IP=partialinhibition;
R=totalresistance.
StrainofKlebsiellaspp.andBacillusspp.weredevelopedaslargecolonies46mmin
diameter, smooth, free of haemolytic activity. Candida spp. strain was developed as small
colonies of 12 mm after 24 hours of incubation which subsequently grew to 45 mm size,
mattewhite.Onmorphologicalcharactersexam,KlebasiellawereobservedasGramnegative
bacilli isolated or grouped diplo, the genus Bacillus form of rods with rounded ends, Gram
positive,isolatedorasshortchainswhileyeastofthegenusCandidawereasovalformations
characteristicaspectofkeg,isolatedpilesasmanycellsareburgeoning.
Aromatograma analysis results performed on the 17 microbial strains isolated from
clinical mastitis in milking cows and three international reference strains used as controls
showedthatfrom12strainsofStaphylococcusspp.,10strains(83.33%)weresensitivetopine
oil,9strains(75%)tothymeoil,7strains(58.33%)tomintoiland(25%)tolavenderoil.Forthe
other strains tested, belonging to other genres can not yet draw meaningful conclusions
regardingtheireffectbecauseofthesmallnumberofstrainstested.

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Lucrritiinificevol53seriaMedicinVeterinar
It was noted however that in vitro effect of essential oils on pathogen strains
isolatedfromclinicalmastitisincowsissimilartoantibiotics.Somestrainsthataresensitiveto
one or more oils and others are moderately sensitive or has a total resistance to all the
essentialoilsused.

The results we obtained by testing in vitro are consistent with those obtained by
otherauthors(1,6,7,15,16)andmayconstituteanalternativetocurrentantibiotictherapy.
Butnotethat,tohavethedesiredefficiencyinthisapproachismandatorytestingofessential
oils by aromatograma sensitivity of isolates from cases of mastitis. Figures 1, 2, and 3 are
presenting the effect of essential oils from three different types of microorganisms:
Staphylococcus,StreptococcusandBacillus.

Fig.1Staphylococcusspp.

Fig.2Streptococcusspp.

Fig.3.Bacilluscereus

Thefirstthreegenera,Grampositivesgrouparesensitivetopineoil,mintandthyme
whileKlebsiellastrainhasatotalresistancetothesixessentialoilstested.

CONCLUSIONS
1. Bacteriological examination allowed us to isolate the following types of
microorganisms in 17 samples of milk from cows with mastitis: Staphylococcus,
Streptococcus,Bacillus,KlebsiellaandCandida.
2. In vitro tests by aromatograma, indicate that strains of Staphylococcus spp. are
sensitivetoessentialoilsin4of6situationsinthefollowingpercentages:10strains
(83.33%) to essential oil of pine, 9 strains (75%) to essential oil of thyme, 7 strains
(58.33%)toessentialoilofmintand3strains(25%)toessentialoiloflavender.
3. Thefirstthreeessentialoilscanbeanalternativetocurrenttherapyofstaphylococcal
mastitis with antibiotics, but only after in vitro susceptibility testing isolates by
aromatograma.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
4.
5.

IncaseofbacterialstrainsbelongingtoStaphylococcusandBacillus,thesensitivityis
variablefromonestraintoanother.
StrainofKlebsiellaspp.,isolatedfrommastitisincowswasresistanttoallsixessential
oilsusedinaromatogramatest.

BIBLIOGRAPHY

1.
2.

3.
4.
5.

6.

7.

8.

9.

10.

11.
1.
12.
13.
14.
15.

BekechiC.,AtikBekkaraF.,AbdelouahidD.(2008)Compositionetactiviteantibacteriennedes
huilesessentiellesdOriganumglandulosumdAlgerie,Phytotherapie,vol.6,No.3,153159.
BenHassenS.,MessadiL.,BenHassenA.(2003).Identificationetcaractrisationdesespces
de Staphylococcus isoles de lait de vaches atteintes ou non de mammite, Ann. Med. Vet.
2003,147,4147.
Bidaudetal.:EtiologiedesmammitesbovinesenFranceentre2005et2007.JourneBovine
Nantaise,2007,121122.
ChaducL.,etLabrePh.(2002),Traitementdepathologiesdominanteschezlesruminantspar
leshuilesessentiellesEssai"Aromavet",AlterAgriNo.54,jouillet/aout,2325.
Chantaigner B., Messad S., Bony J., Tillard E. (2007). Prvalence des principales bactries
responsablesdesmammitesalaRunionetrsistancesantibiotiquesdesbactriesidentifies,
Renc.Rech.Ruminants,2007,14,222.
KaloustianJ.,ChevalierJ.,MikailC.,MartinoM.,AbouL.,VergnesM.(2008)Etudedesixhuiles
essentielles: composition chimique et activite antibacterienne, Phytotherapie, vol. 6, No. 3,
160164.
C.Lefevre,M.Kammerer,M.LeGuenic,P.Roussel,C.Alby,O.Linclau,G.Cartaud,D.Tainturier,
M.Larat,N.Bareille,Letraitementdesmammitescliniquesdelavachelaitirepardeshuiles
essentielles,InnovationAgronomique2008,4,7983.
PavelMariana(2009)Contribuiilastudiulbotanic,fitochimicifarmacologicalunorspeciide
Thymus(Cimbrior)dinfloraspontanaRomniei,UniversitateadeMediciniFarmacie"Iuliu
Haieganu"ClujNapoca,FacultateadeFarmacie,Tezdedoctorat.
S. Pop, F. Chiril, N. Fi, S. Rpuntean, G. Nad (2009) Mamitele clinice la vac:
Microorganisme implicate i sensibilitatea lor la antibiotice i antimicotice Clinical mastitis in
cows,themicroorganisminvolvedandtheirsensitivitytoantibioticsandantimycotics,Lucrri
tiinificevol.52(11)MedicinVeterinarParteaI,USAMV"IonIonescudeBrad"Iai,Editura
"IonIonescudeBrad"Iai.
SandholmM.,KaartinenL.,PyoralaS.(1990).BovinemastitisWhydoesantibiotictherapynot
alwaysworc?Anoverview.JournalofVeterinaryPharmacologyandTherapeutic1990,13,248
260.
UngureanuC.,MinciunV.,1983.Afeciunileglandeimamarelavaci,Ed.CeresBucureti.
12.http://www.interbiobretagne.asso.fr/upload/File/Recherche/Elevage/Resultats%20_2006/e
xpe06L4.pdf
http://www.biopaysdelaloire.fr/documents_blocs/161.pdf
http://www.adage35.org/wpcontent/uploads/2009/11/ArticleLADHuilesessentielles.pdf
http://www.airpharmalabo.com/docs/etude3.pdf
http://www.google.ro/#q=Aromatogramme&hl=fr&start=30&sa=N&fp=bc1772dacef022f1

958


RESEARCHONTHEFREQUENCYOFBACILLUSCEREUS
INMILKASRAWMATERIAL

C.CIOTU2,E.V.INDILAR1
1
FacultateadeMedicinVeterinarIai
2
DireciaSanitarVeterinaripentruSiguranaAlimentelorSuceava

Abstract
The contamination of milk as raw material for processing purposes with Bacillus cereus is
important under sanitary as well as technological aspects. Studies performed by the authors
showedanaveragefrequencyofcontaminationofthewholerawmilkwithBacilluscereus5,02%.
Other species by Bacillus genus that was identified from the samples: B. cuagulans 5,02%, B.
mycoides3,86%,B.firmus2,32%,B.circulans1,44%,B.licheniforms1,16%,B.thuringensis0,77%
andB.subtilis0,77%.
Keywords:rawmilk,Bacilluscereus,microbiologicalquality.

Bacillusgenusincludesagreatnumberofspecies,22ofthembeingbetterknown.(2,
11). This genus is made of Grampositive bacilli, aerobic or optional anaerobic, with
development temperatures between 30 and 750C, optimal temperature of 250370C. All the
speciesaresporogeneous,someofthemaremobileandsomehavecapsules.(2,3,4,11)
Bacillus genus is a heterogeneous genus, aspect that reflects the large variety of
environmentsinwhichthesemicroorganismslive(2,3,4,10).Thisheterogeneityandthefact
thatG+CfromtheDNAvariesbetween32%69%suggeststhatsomespeciesincludedinthis
genuscouldformdifferenttaxa(7,12,13).
Bacillus genus includes both saprophytes and pathogenic bacteria for humans,
animalsandinsects. Thisgenusincludesbacilluscereus,abletoproducefood poisoningand
occasionallyotherinfections(3,11,12).Likealltaxaincludedinthisgenus,bacilluscereusis
widespreadinnature,usuallyinsoil,dust,plantmaterials,humanandanimalfaeces.
Overall the food and milk contamination is made by unsanitary conditions of
production,foodprocessingandstorage(3,8,9,14).
BacilluscereusisabacteriummorphologicallysimilarwithBacillusanthracis,towhich
is antigenic related, but forms shorter chains and the ends appear rounder. It is
noncapsulogenic,mobilewithnumerousperitrichcillia,withtheovalsporuminthemiddleor
subterminal,whichdoesn`tchangethecellshape,aerobicoroptionalanaerobic,haemolytic,
licithinolytic,catalasepositive,oxidationnegative(1,2,3,4,9,11,12).
Growth and development temperatures varies, depending on the strains, and for
othersisbetween150500C(4,8,11,12).Optimaltemperatureforgrowthandmultiplicationis
280350C when the generation time is shortest (1827 minutes). pH limits in which B. cereus
canmultiplyare4,9and9,3andtheminimalawvalueis0,95.B.cereusgrowsandmultiplies
insalinemediawithNaClconcentrationlessthan7.5%anditisinhibitedbynisinandascorbic
acid. Most B. cereus strains have a protease and licithinase shown on egg yolk medium,
formingawhiteyellowprecipitate(3,8,12,15).
The biochemical features of B. cereus are: glucose fermentation, casein and starch
hydrolysis,gelatinliquefaction,reductionofnitratesinnitrits,useofamoniumcitrateasonly
carbonsource.

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Lucrritiinificevol53seriaMedicinVeterinar
Bacillus cereus can live both in liquid and solid media, but for growth, isolation,
identificationandquantificationarespecialmediathattendtothebacterialpropertiesnotto
ferment mannitol, insensitive to polymyxin, to produce lecithynase and haemolysin (2, 3, 9,
10,12).
Afteralatencyperiodinfood,B.cereusgerminatesandmultiply,inducingenzymatic
changesofthesubstrate,andproducesvomitinganddiarrheacausingexotoxinswhichcross
thecellmembrane(3,6,10,11,13).
The presence of the bacterium in food has sanitary importance when their number
exceeds105/gorml105/gorml.

MATERIALANDMETHOD

The study analyzed 259 milk samples collected by the processor on two collection
routes.Ofthetotalsamples,128milksampleswerefromroute1and131sampleswerefrom
route2.
Investigations were aimed at determining the number of Bacillus cereus / ml milk,
and the number of other bacteria of the genus Bacillus / ml milk found in the examined
samples.

Procedure
Thepreparation of samples and dilutions of raw milk was done according to SR EN
ISO 8261/2005, and the method of analysis was done according to EN ISO 7932/2005,
usingthetechniqueofcountingcoloniesat30C.
Initialinoculumofproductanddecimaldilutionswereinoculated1mloftheoriginal
product and 0.1 ml of decimal dilutions on the surface substrate MYP (mannitol agar and
phenolred),withextraeggyolkandpolymyxinBin2Petridishes(140mm)andthefollowing
dilutions in 2 Petri dishes (90 mm). The Petri dishes were incubated at 30 C for 24 hours.
After the incubation period the dishes that had between 15 and 150 colonies on two
successivedilutionswerekept.Foridentificationandconfirmationthecoloniessuspectedof
beingBacilluscereuswereselected.Theywerepinkandalmostalwayssurroundedbyazone
ofprecipitation,indicatingtheproductionoflechitinase.CertainstrainsofB.cereusproduce
littleornolechitinaseatall.Thesecoloniesweresubjectedtobiochemicalconfirmationtest.

IsolationandselectionofcoloniesforconfirmationofB.cereus
From each selected Petri dish 5 suspected B. cereus colonies were subjected to
confirmationtest.Wheresomesampleshavemanycoloniesanditwasnotpossibletoselect
wellisolatedsuspectcolonies,thereplicationof5coloniesonselectivesubstratecontaining
MYPwasconducted,toobtainisolatedcolonies.
Incubationwasdoneat30Cfor1824hours.Fromeachdishwellisolatedcolonies
wereselectedandsubjectedtoconfirmationtestonthefollowingsubstrates:
- glucose agarmedium forfermenting glucose. Thecoloring inyellow of
theseedingindicatedapositivereaction;
- medium for VogesProskauer reaction with potassium hydroxide
solution, Naphtol and creatin crystals, which highlighted the
productionofacetylmethylcarbinol,observedbecauseofthepinkeosin
colorofthemedium,meantthepositivetestforB.cereus;
- nitratemediumforhighlightingnitriteinthepresenceofreactivesof52
ANSA, sulfamidic acid and zinc powder. The reducing of the nitrate to

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UniversitateadetiineAgricoleiMedicinVeterinarIai
nitritemarkedbythechangeofcolorfromyellowtoredinthepresence
ofthereactivesmeantthepositivetestforB.cereus.

Methodofcalculation
The number of Bacillus cereus, confirmed in at least 80% of colonies selected from
the dishes which contained between 15150 colonies on two successive dilutions was
calculatedwiththeformula,accordingtoISO7932/2005:
a
N=
(n1+0,1n2)xd
inwhich:
asumofB.cereuscoloniesfromalltheselecteddishes;
n1numberofdishesselectedonfirstdilution;
n2numberofdishesselectedonseconddilution;
ddilutionratelevelcorrespondingtothefirstdilution.

If from the selected dishes with 15150 colonies on the 2 successive dilutions were
confirmedlessthan80%ofselectedcolonies,thecalculationofthenumberofB.cereuswas
madepercentage,dependingonthenumberofconfirmedcolonies.Iftheselecteddisheshad
lessthan15B.cereuscolonies,theaverageofthenumberofcoloniesfromthe2disheswas
made.
ExpressionoftheresultonthenumberofB.cereuspermlorpergramproductwas
made by a number between 1.0 and 9.9, multiplied by 10x, where x is the corresponding
powerof10.
Identification of Bacillus cereus species from the cultures obtained on the selective
isolationmediumMYP,bythemethodaccordingtoISO7932/97wasmadewiththeminiAPI
microbiologicalanalyzerusingAPI50CHgallery,withsuspensionmediumAPI50CHB/E.

Preparationoftheinoculation
The characteristic colonies from the selective medium MYP was made a culture on
nutritive agar to obtain a sufficient number of colonies for the preparation of the bacterial
suspension. The bacterial suspension was prepared on API 50 CHB/E medium for the
identification on Bacillus bacteria, which allows the observation of the fermentation of 49
carbohydratesfromthemicrotubesoftheAPI50CHgallery.
Galleryinoculationwasdoneby pipette,fillingthemicrotubes carefullytothelevel
indicatedbyfull,avoidingtheappearanceoftoofull.Addingparaffinoilinmicrotuburiletests
allowbettervisualizationofcolor.GalleriesIncubationwasat30Cfor2448hours.
ThereadingofAPI50CHgallerieswasdonein2stages:oneat24hoursandsecondat
48 hours.The biochemical processes occurring during incubation in glucose catabolism,
producing organic acids which change the pH indicator (phenol red in the environment), to
yellow.Thischangeindicatesapositiveresult.Alsotestedinmicrofuge25aesculincolorturns
fromredtoblack,indicatingapositiveresult.
For reading the galleries the reading program was typed. Analytical module called
readerrecordsthecolorreactionfromeachmicrotubeandtransmitstheinformationtothe
computerwhereitisinterpretedwiththeidentificationsoftware.

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Lucrritiinificevol53seriaMedicinVeterinar
RESULTSANDDISCUSSION

ThestudyforisolationandidentificationofBacillusspeciesfromthemilksamplesof
route1showsthefactthattherewere24positivesamples,representing18.5%,fromwhich
aftertheconfirmationtest5sampleswerepositiveforBacilluscereus(3.91%),and19samples
(14.84%)provedtobeotherspeciesofBacillus.
Onroute2,aftertheanalysisofthe131milksampleswefound29positivesamples
forspeciesofBacillus,representing22.14%.Fromthepositivesamples,8confirmedtohaveB.
cereusrepresenting6.11%,and21milksamples(16.03%)hadotherspeciesofBacillus.
From the total of 2596 milk samples investigated on routes 1 and 2, resulted 53
positive samples (20.46%) for Bacillus spp, from which in 13 samples was confirmed the
presence of B. cereus (5.02%), and in the other 40 samples (15.44%) was confirmed the
presenceofotherspeciesofBacillus.

Table1TheincidenceoftheBacilluscereusinrawmilkcollectedfromtworoutes

FrequencyofBacillusspp inmilksamples

Positivesamples
Positive
Normalsamples
Positive
Route Nr.of
samplesforB. forotherspeciesof
(negatives)
samplesfor
samples
Bacillus
cereus
Bacillusspp
A
R
A
R
A
R
A
R
IV.D.
128
104
81,25
24
18,75
5
3,91
19
14,84
II
131
102
77,86
29
22,14
8
6,11
21
16,03
C.M.
Total
259
206
79,54
53
20,46
13
5,02
40
15,44
Aabsolutefrequency;Rrelativefrequency.

In the second work stage we identified the other species of Bacillus found in the
investigatedmilksamples.TheresultsarepresentedinTable2.

Table2TheidentificationofthespeciesofBacilluscereusinrawmilk
collectedfromtworoutes

Frequencyofidentifiedspecies
Species
RouteI=128samples Route II=131samples Total=259samples
A
R
A
R
A
R
Bacilluscereus
5
3,91
8
6,11
13
5,02
Bacilluscuagulans
6
4,68
7
5,34
13
5,02
Bacillusmicoides
5
3,91
5
3,82
10
3,86
Bacillusfirmus
3
2,34
3
2,29
6
2,32
Bacilluscirculans
2
1,56
2
1,3
4
1,44
Bacilluslicheniformis
1
0,78
2
1,53
3
1,16
Bacillusthuringiensis
1
0,78
1
0,76
2
0,77
Bacillussubtilis
1
0,78
1
0,76
2
0,77
Total
24
18,74
29
22,11
53
20,46
Aabsolutefrequency;Rrelativefrequency.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Analyzing the data from Table 2 reveals that, in addition to B. cereus were still
identifiedsevenspeciesofthegenusBacillusidentificationwithadecreasingrate,asfollows:
B. cuagulans 5.02%, B micoides 3.86%, B. firmus 2.32%, B. circulans 1.44%, B. licheniformis
1.16%,B.subtilisandB.thuringensiseach0.77%.
Dataanalysisshowssomeaspectsthatrequirefurtherdiscussion.Inthiscontextitis
notedthatthemilkcollectedonthetwocollectionroutesiscontaminatedwithseveralspecies
ofbacteriaofthegenusBacillus.
Fromthetotalnumberof259milksamplesinvestigated,206samples(79.54%)were
free of bacteria of the genus Bacillus, and a number of 53 samples (20.46%) were
contaminatedwithvariousspeciesofbacteriaofthegenusBacillus.Thisindicatesdeficiencies
regarding compliance with hygiene rules to harvesting, handling and cooling the milk
immediatelyaftermilking.
OfallspeciesofBacillusfoundininvestigatedmilksamples,Bacilluscereusisinvolved
in producing food poisoning by consumption of milk.Isolation percentage of 5.02% of
investigatedsampleswithvalues<10CFU/mlofmilkisamajorrisktopublichealth.

CONCLUSIONS

In the socioeconomic conditions in the mountainous area from which the milk
samples were collected and the investigation of the presence of B. cereus involved in
producingoffoodpoisoningshowsthat:
CollectedmilkiscontaminatedwithbacteriaofthegenusBacillusinapercentageof
20.46%,ofwhichwithBacilluscereusonly5.02%and15.44%differencewithotherspeciesof
thegenusBacillus;
The situation resulting from the investigations made on milk samples from the two
collection routesimposes taking measures for improving the hygiene conditions by
implementingrules ofgoodpracticeinproduction, andgood practiceespeciallyinobtaining
andconditioningofthemilkindairyfarms.

BIBLIOGRAPHY

1. BRZOID.,(1985)Microbiologiaproduseloralimentare,EdituraCeres, Bucureti.
2. BRZOI D., APOSTU S., (2002) Microbiologia produselor alimentare, Editura Risoprint, Cluj
Napoca.
3. BRZOID.,MEICAS.,NEGUM.,(1999) Toxiinfeciilealimentare,EdituraDiaconCoresi,Bucureti.
4. CARPCRAREM.,(2000)Microbiologieveterinarvol.IiII,EdituraUSAMV,Iai.
5. CIOTUC.,(2006) Cercetriprivindcalitatealapteluimaterieiaunorsortimentedelaptepraf.
Tezdedoctorat.FMV,Iai.
6. CRISTIANSSON A., BERTILSSON J., SVENSSON B., (1999) B. cereus spores in raw milk: factors
affectingthecontaminationofmilkduringgrazingperiod.JournalofDairyScience,82,305311.
7. DROBNIEWSCHIF.,A.,(1993)Bacilluscereusandrelatedspecies,ClinicalMicrobiologycs,Revievs,
oct.137.
8. FLORITEANU VIORELCEZAR, (2006) Caracterizarea bacteriologic a unot tulpini de Bacillus
cereusizolatedinalimente.Tezdedoctorat,FMV,Iai.
9. KORNR.,(1989)IncidenaspecieiB.cereusnunelealimentedeorigineiproprietiletulpinilor
izolate.Tezdedoctorat,FMV,Bucureti.
10 MUREANU I., (2000) Cercetri privind incidena n unele alimente de origine animal i
particularitile morfologice ale speciei Bacillus cereus, potenial patogen pentru om, n zona
centralaTransilvaniei.Tezdedoctorat,USAMV,Bucureti.
11 RDUCNESCUH.,VALERIABICAPOPA,(1986) Bacteriologieveterinar,EdituraCeres,Bucureti.

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Lucrritiinificevol53seriaMedicinVeterinar
12 RPUNTEANGH.,RPUNTEANS.,(2005)Bacteriologiespecialveterinar,EdituraAcademicPres,
ClujNapoca.
13 SCHOENIJ.L.,WONGA.C.,(2005) Bacilluscereusfoodpoisoninganditstoxins.JournalofFood
Protection,68,636647.
14 INDILAR E., BONDOC I., (1996) Toxiinfeciile alimentare produse de Bacilus cereus. Lucrri
tiinifice,UAMVIai,seriaMedicinVeterinar,3839,125130.
15 OGOE I., TUDOR L., OGOE D., (2004) Investigaii privind patogenitateatulpinilor de B. cereus
izolatdinlaptelepraf.Lucrritiinifice,USAMVBucureti,SeriaC,4647,419425.

964

OBSERVATIONSREGARDINGTHEHISTOLOGICALSTRUCTUREOF
THEFOOTMUSCLEOFTHEFOODSNAILH.pomatia

AndreeaFlaviaCIRLAN,E.SINDILAR,
UniversityofAgriculturalSciencesandVeterinaryMedicine
IonIonescudelaBradIasi,FacultyofVeterinaryMedicine,MihailSadoveanuAlleyno.8,
700489Iasiemail:flavia_cirlan@yahoo.com

ABSTRACT
Vineyard snails (H. pomatia) meat has always been highly valued for its dietetic and nutritive
properties. Given the fact that very few studies have been made on this snail, the aim of the
presentresearchwastostudythehistologicalstructureofthevineyardsnailsfootmuscle,which
isthemainpartusedforconsumption.Themuscleshavebeensampledfrom15snailsfoundina
garden in the city. After removing the shell and the organs, we isolated the muscle. This was
processedbythecommonparaffintechniqueandthenforthehematoxylineeosin(HE)andthe
Alcianbluestaining.Weexaminedthemmicroscopicallyandobservedthehistologicalstructure
ofthefootmuscle.

KeyWords:Helixpomatia,foodsnail,muscle,histologicalstructure

INTRODUCTION

Food snails H. pomatia meat has always been highly valued for its dietetic and
nutritiveproperties.Lately,inourcountry,manypeoplebegantoorganizefarmsinwhichthey
raisethisspecie.Evenafabricforsnailproductshasbeenmadeandmostoftheseproducts
areexported.Giventhesefacts,wedecidedthatthefoodsnailHelixpomatiashouldbemore
studied and we began by revealing the histological structure of the main part used for
consumption:thefootmuscle.

MATERIALANDMETHODS

Theresearchtookplaceinfourconsecutivesteps:
Step1TwentyadultsnailsfromthespecieHelixpomatiawerecollectedfromagardenand
thenintroducedintoabowlwithethanolfortheanesthesia.
Step2Weremovedtheshellandthenwedissectedthesnailsbody.Wemadeanincision
fromthemouthandcutthesnailbodyandthenweremovedtheorgans.
Step3Wedividedthemuscleintothreeparts(antherior,middleandpostherior)whichwere
usedforthehistologicalanalysis.Inordertodothis,thesampleswerefixedinformaldehyde
10%, dehydrated in 3 alcohol baths, cleared in acetone, rinsed in benzen, embedded in
paraffinansectionedat7mthickness.Paraffinsectionsweredewaxedinxylene,hydratedin
4alcoholbathsandinonedistilledwaterbath.
Step4Finally,thesectionsweretreatedforhematoxylineeosin(HE)stainingandforAlcian
Bluestainingandthenmicroscopicallyanalyzed.

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Lucrritiinificevol53seriaMedicinVeterinar
RESULTSANDDISCUSSIONS
Thehistologicalexamofthefoot,themobileorganofthesnail,revealeditsmuscular
structure. The muscle is composed of smooth muscle cells intermingled with large spaces
representedbyhaemocoeliccapillarysinuses(Fig.1).

The smooth muscle cells are surrounded by collagen fibrils in the subepithelial
stratum of integument. The muscle fibres are dispersed beneath the epithelium and have a
longitudinal,transverseorradialorientationwithrespecttothemajorbodyaxis.

The Alcian Blue staining revealed the presence of the colagenic connective tissue
underthesurfaceepithelium(Fig.2).

Fig.1.Smoothmusclefibresandhaemocoeliccapillarysinuses.Col.HEAx100

Fig.2.Connectivetissueandcapillarysinusesinthestructureofthefoot.Col.ABx200

Using the Alcian blue staining and the microscope with polarized light we could
observetheshiningcollagenfibrils(Fig.3).

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Fig.3.Thecollagenfibrilsinthestructureofthefootmuscle,observedwiththemicroscope
withpolarizedlight.Col.ABx200

Nearthesurfaceepitheliumwecoulddistinguishmucussecretingandmucusstoring
cells.Theyaresurroundedbysmoothmusclecellsandbyconnectivetissue(Fig.4).
Intheventralzone,thetegumentiscoveredwithabundantmucus.Unlikethedorsal
zone,wecouldnoticeheretheuniformityoftheepithelialstructureandofthemucus.Thisis
probably connected with the existence and the form of the shell in the dorsal zone, as
concludedbysomeauthors:BariatiandComazzi(2001)andTomarandMarkos(2003).

Fig. 4. Many mucus cells, muscular tissue and connective tissue under the surface
epithelium.Col.HEAx100.

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Lucrritiinificevol53seriaMedicinVeterinar
CONCLUSIONS
1. Inthestructureofthesnailsfootaresmoothmusculartissue,connectivetissue
andhaemocoeliccapillaryvessels.
2. Using the microscope with polarized light we could notice the numerous
collagenefibrilsinthesnailsfoot.
3. In the ventral zone of the foot we could observe an abundency of the mucus
secretingandmucusstoringcells,thetegumentbeingcoveredwithathicklayer
ofmucus.

REFERENCES

1. Bairati A., Comazzi M. (2001) An ultrastructural study of connective Tissue in


molluscintegument:Gastropoda.TissueandCell;333:426438
2. RamsayJ.A.(1939)Anervemusclepreparationfromthesnail.
3. Royuela M., Fraile B., Arenas Mi., Paniagua R. (2000) Characterization of several
invertebratemusclecelltypes:aComparisonwithvertebratemuscles.Microsc.Res.
Tech.;48:107115
4. TonarZ.,MarkosA.(2004)Microscopyandmorphometryofintegumentofthefoot
ofPulmonateGastropodsArionRufusandHelixpomatia.ActaVet.Brno;73:38

968

OBSERVATIONSREGARDINGTHEHISTOLOGICALSTRUCTUREOF
THEGENITALSYSTEMOFTHEGARDENSNAILH.POMATIA

AndreeaFlaviaCIRLAN,E.SINDILAR,
UniversityofAgriculturalSciencesandVeterinaryMedicine
IonIonescudelaBradIasi,FacultyofVeterinaryMedicine,MihailSadoveanuAlleyno.8,
700489Iasiemail:flavia_cirlan@yahoo.com
ABSTRACT
Theaimofourstudywastorevealthehistologicalstructureofthegenitalsystemofthe
garden snail Helix pomatia. We used in this research 15 snails from which we have
isolatedthe3partsofitsgenitalsystem:theovotestis(thehermaphroditegland),the
female part and the male part. These were processed by the common paraffin
techniqueandthenfortheperiodicacidSchiff(PAS),thehematoxylineeosin(HE)and
theAlcianbluestaining.Weexaminedthemmicroscopicallyandobservedthecomplex
histologicalstructureofthefollowing:theovotestis,thespermchannel,thepenis,the
oviductandthealbumengland.

KeyWords:Helixpomatia,snails,histologicalstructure,genitalsystem

INTRODUCTION

Helix pomatia is a hermaphroditic snail with a complex genital system, composed


mainlyofthreeparts:theovotestis,themalepartandthefemalepart.Giventhefactthatin
our country very few studies have been made on this snail, we decided to carry some
researchesonitshistologicalstructure,mainlyonitscomplexreproductivesystemstructure.

MATERIALANDMETHODS

Theresearchtookplaceinfourconsecutivesteps:
Step1TwentyadultsnailsfromthespecieHelixpomatiawerecollectedfromagardenand
thenintroducedintoabowlwithethanolfortheanesthesia.
Step2Weremovedtheshellandthenwedissectedthesnailsbody.Wemadeanincision
fromthemouthandcutthesnailbody.Thenweremovedthegenitalsystem.
Step 3 We divided the genital system into its composing parts which were used for the
histological analysis. In order to do this, the samples were fixed in formaldehyde 10%,
dehydratedin3alcoholbaths,clearedinacetone,rinsedinbenzen,embeddedinparaffinan
sectionedat7mthickness.Paraffinsectionsweredewaxedinxylene,hydratedin4alcohol
bathsandinonedistilledwaterbath.
Step 4 Finally, the sections were treated for periodic acid Schiff (PAS) staining, for
hematoxylineeosin (HE) staining and for Alcian Blue staining and then microscopically
analyzed.

RESULTSANDDISCUSSIONS

Helix pomatia is a hermaphroditic snail, which has three important parts in his
reproductivesystem:theovotestis(thehermaphroditegland),thefemalepartandthemale
part.

969

Lucrritiinificevol53seriaMedicinVeterinar
Theovotestisconsistsoffourlobes,eachofthemrepresentedbyalargenumberof
follicles intermingled with loose connective tissue. Inside the follicles we could notice: male
germcellsthatoccupythelumenofthefollicles,femalegermcellsobservedonthewallsof
thefollicles,folliclecellsandSertolicells(Figure1).
Themaleelementswererepresentedbythespermatogonia,whichwerelarge,round
cells with a narrow basophilic cytoplasm around the nucleus. We could also notice primary
spermatocytes, similar to the spermatogonia, but with an eosinophilic cytoplasm, secondary
spermatocytesandelongatedspermatids.
Examining the hermaphroditic channle, we revealed in its structure a
pseudostratificatedcylindricalepitheliumandlooseconnectivetissue.

Fig.1.Theovotestislobesintermingledwithlooseconnectivetissue.Thelumenisoccupiedby
thespermatogoniaandinthewallsaretheoocytes.Col.HEAx40

The sperm channel, through which the sperm is transported to the penis, is a
functionalductwithanarrowlumenoutlinedbyfoldedepithelialcrestssurroundedbyafew
muscleandconnectivefibres.
The penis is a muscular organ with circular, longitudinal, radial and oblique muscle
fibres.Itisoutlinedbyapseudostratificatedcylindricalciliatedepithelium(Fig.2).

Fig.2.Thecircular,longitudinal,obliqueandradialmusclefibresinthestructureofthepenis.
Col.PASx40

970

UniversitateadetiineAgricoleiMedicinVeterinarIai

Theoviductisamuscularductwithsimpleprismaticepithelium.Wenoticedheretwotypesof
cells:ciliatedandsecretorycells(Fig.3).Theepitheliumformsvariouslongitudinalfolds.

Fig.3.Thesimpleprismaticepitheliumoftheoviduct,withbigsecretorycells.
Col.ABx100.

The albumen gland is involved in the formation of the envelopes surrounding the
fertilized oocytes and it opens in the oviduct. In the parenchymal glandular mass we could
observealbumensecretorycellsandlabyrinthiccells(Fig.4).

Fig.4.Intheparenchymalmassofthealbumengland,mostofthecellsarebigsecretorycells
withanovoidform.Col.HEAx40

971

Lucrritiinificevol53seriaMedicinVeterinar
CONCLUSIONS
1. The hermaphrodite gland consist of 4 lobes, each of which is represented by a large
numberoffolliclescontainingmalegermcells,femalegermcells,follicularcellsandSertoli
cells.
2. Thepenisisamuscularorgananditsfibresarecircular,longitudinal,radialandoblique.
3. Inthestructureoftheoviductaretwotypesofcells:cilliatedcellsandbig,secretorycells.
4. Inthestructureofthealbumengland,involvedintheformationoftheeggs,wecouldalso
observetwotypesofcells:secretoryandlabyrinthiccells.

REFERENCES
1.BudI.,OroianElvira(2004)Melcii.Cretere,nmulireivalorificare.Ed.Ceres,Bucureti
2. Castello V.M., Brown D.I. (2008) Microscopic anatomy of the male reproductive system in
Echinolittorinaperuviana.Int.J.Morphol;26
3.CatalanN.M.Y.,FernandezS.N.,WinikB.C.(2002)Oviductalstructureandprovisionofeggenvelops
intheapplesnailPomaceaCanaliculata(Gastropoda,Prosobranchia,Ampullariidae).Biocell;26(1):91
100
4.ChaseR.,DarbisonE.(2008)Differentialsurvivalofallospermbylocation within the female storage
organofthesnailCornuAspersum.Can.J.Zool.86:12441251
5. Luchtel Dl., DeyrupOlsen I. (2001) Body wall. Form and function. In The biology of terrestrial
molluscs.CABIPublishing,Wallingford;147178

972

THECONCORDANCEBETWEENTHETOTALGERMSNUMBER
(TGN)FROMTHESHELLANDTHETOTALGERMSNUMBERFROM
THEFOOTOFTHEFOODSNAILHELIXPOMATIA

AndreeaFlaviaCIRLAN,ESINDILAR,
UniversityofAgriculturalSciencesandVeterinaryMedicineIonIonescudelaBradIasi,The
FacultyofVeterinaryMedicine,MihailSadoveanuAlleyno.8,700489Iasiemail:
flavia_cirlan@yahoo.com

ABSTRACT
Letely, in our country, the foodsnail Helix pomatia is very appreciated. Many people began to
raise this specie in organized farms. The snails are processed or exported. Our purpose was to
identifytheTGNfromtheshellandfromthefootofthissnailandtoseeiftherearebacteria
involvedinthehumandigestivepathology.Themethodsweusedare:theserialdecimaldilutions
method,theinoculationonseveralmediaandtheGramstain.ThevaluesoftheCFU/cm2
obtainedfromtheshellsurfaceweresignificantlyhigherthanthevaluesoftheCFU/cm2obtained
from the foot surface. This fact may be related to the quantity and quality of the mucous
produces by the snails glands. On the Sabouraud medium, two species of fungi have grown:
Cladosporium spp and Chrysosporium spp. No specie has grown on the selective media for
pathogenicbacteriathatwehaveused.TheidentificationoftheGramnegativebacilliwasmade
usingtheAPIgalleries.Furtheridentificationsaregoingtobemade.

Keywords:totalgermsnumber,foodsnail,Helixpomatia

INTRODUCTION

Food snails H. pomatia meat has always been highly valued for its dietetic and nutritive
properties.Lately, in our country, many people began to organize farms in which they raise
thisspecie.Thesnailsmeatisusuallyexported.Still,inourcountry,themicrobiologyofthe
foodsnailshasnotbeenproperlystudied.Consequently,theaimsofthepresentpaperare:
todetermine thetotalgerms number from theshell;to determinethetotalgerms number
fromthefoot;toseeifthereisaconcordancebetweenthesecounts;toisolatesomebacteria
involvedinthehumandigestivepathology.

MATERIALANDMETHODS

Theresearchtookplaceinthefollowingsteps:
Step1TenadultsnailsfromthespecieHelixpomatiawerecollectedfromagardenatthe
endofseptember(thebeginingoftheirhibernationperiod).
Step 2 The shell and the foot were examined separately, using the following technique:
moistened cottonwool swabs have been used for the whole surface sampling. After
swabbing, the head of the swab was broken off into diluant (Brain Heart Infusion broth),
which is shaken to release the organism. Serial decimal dilutions were then prepared. The
dilutions1/10and1/100wereincubatedat37C,for24hours.
Step 3 Plates with the following media were then inoculated with 10 l of dilution: Agar
with sheep blood, MacConkey with Crystal Violet, XLD Agar, Brilliant Green Agar, Yersinia
Selectivemedium,BileEsculinmediumforisolationofListeriaandSabouraudmediumforthe
isolationfofungi.

973

Lucrritiinificevol53seriaMedicinVeterinar
The media were incubated at 37C for 24 hours and at the room temperature for other 24
hours.TheSabouraudmediumhasbeenstudiedfor7days.
Step4ThevalueswereexpressedinlogCFU/cm2 oftheshellsurfaceandlogCFU/cm2 of
thefootsurface.

RESULTSANDDISCUSSIONS

Thevaluesobtainedfromthedilution1/10arepresentedinthetables1and2.

Snails
number

1
2
3
4
5
6
7
8
9
10

Snails
number

1
2
3
4
5
6
7
8
9
10

Media CFU/cm2
Brain
Blood
MacConkey
Bile
Heart
Agar
with
Esculin
Agar
Infusion
Crystal
(BHI)
(BAP)
Violet
(BEA)
+
6.6
0
0
+
37
37
0
+
12.9
0
0
+
30.8
29.6
0
+
13.6
8.6
0
+
23.4
11.6
0
+
18.5
11.1
0
+
25.3
7.4
0
+
24.8
13.5
0
+
11.1
2.22
0

Yersinia
selective
medium
(CIN)
0
0
0
0
0
0
0
0
0
0

Brilliant
Green
Agar

XLD
Agar

Sabouraud

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

Brilliant
Green
Agar

XLD
Agar

Sabouraud

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

Table1.ThenumberofCFU/cm2isolatedfromtheshellsurface.
Media CFU/cm2
Bile
Brain
Blood
MacConkey
Agar
with
Esculin
Heart
Infusion
Crystal
Agar
(BHI)
(BAP)
Violet
(BEA)
+
0.8
0
0
+
16
4.4
0
+
4
1.2
0
+
4
0
0
+
6
0
0
+
4
0
0
+
4
0
0
+
4.4
0
0
+
60
30
0
+
6.8
0
0

Yersinia
selective
medium
(CIN)
0
0
0
0
0
0
0
0
0
0

Table2.ThenumberofCFU/cm2isolatedfromthefootsurface.

Afteranincubationattheroomtemperaturefor48hours,wecouldnoticethatthe
CFUdoubledtheirnumberinmostcases,whichprovesthattheisolatedbacteriagrowwellat
temperaturesbelow37C.

On the nonselective, but very rich medium Blood Agar, we obtained both a large
numberanddensityofthecoloniesisolatedfromtheshell.TheCFU/cm2wasbetween54and

974

UniversitateadetiineAgricoleiMedicinVeterinarIai
confluent cultures. The number of CFU/cm2 isolated from the shell on Blood Agar was
between8and210.

Morphology on Blood
Agar
Very
small,

haemolyticalcolony

Morphology
MacConkeyAgar

Small, white colony,


with
a
narrow
haemolyticalzone
Yelloworange, non
haemolyticalcolony

Big,
yellowbrown
colony
Citrinyellowcolony

Viscous, salmon pink


colony
Bigcolonywithdouble
haemolysis
Colony with a narow
haemolytical
zone,
with a browngreen
pigment and irregular
edges
Grey,nothaemolytical
colonies, with a
mucousaspect
Small,
whitegrey
colonies
Grey,
mucous
colonies,fromsmallto
verybigones
White, fluffy colony,
withabrownbase
Black, fluffy colony,
appearedafter7days

Lactose

on

Morphology on Gram
staining
Grampositive bacilli,
arranged in V or Y
forms
Grampositive cocci,
arrangedinclusters
Grampositive
diplococci and cocci
arrangedinclusters
Gramnegative,
polymorphousbacilli
Grampositive bacilli,
arranged in V and Y
formsandinpallisades
Gramnegative,
polymorphousbacilli
Grampositive,
sporulatedbacilli
Gramnegative,
polymorphousbacilli

Lactosenegative

Gramnegative, bipolar
stainedbacilli

Lactosenegative

Gram variable, thick


coccobacilli

Identification

Coagulasenegative

Coagulasenegative

Pseudomonas
mendocina
Rataibacterspp.

Pseudomonas
putrefaciens

Pseudomonas
pseudoalcaligenes

Pseudomonas
pseudomallei

Lactosenegative

Pseudomonasputida

Cladosporumspp

Chrysosporiumspp.

Table3.ThecoloniesaspectsonBloodAgarandonMacConkeyAgar

OnthelowselectivemediumMacConkeyAgar,thenumberobtainedwasbetween0
andsemiconfluentcultures,whileonthemediumandhighselectivemediafortheisolation
ofthehumanpathogenbacteria,nocolonywasobserved.

After 5 days, on the Sabouraudgentamicincloramphenicol agar plates we could


noticeatonlyonesnail,theappearanceoftwocolonies(ablack,fluffyoneandawhite,fluffy
one)ontheculturemadefromtheshellandofonewhite,fluffycolony,whichhadabrown
baseafter7daysandwhichwasintensivelyadherenttothemedium.

975

Lucrritiinificevol53seriaMedicinVeterinar

ThedimensionsofthecoloniesontheBloodAgarvariesfrompunctiformcoloniesto
big,greycolonieswithamucuosaspect.

Some of the colonies present hemolyze, other colonies present a small area of
hemolyzeandotherspresentordoublehemolyze.

Somecoloniesarepigmentedincitrinyellow,inbrowngreen,salmonpinkorred.

On the MacConkey Agar, most of the colonies are lactose negative after 24 hours.
After 48 hours, some of them have a yellow aspect which proves their slow capacity for
lactosefermentation.

WeusedtheGramstainforallthetypesofcolonies.Theresultsareintable3.

TheidentificationoftheGramnegativebacillihasbeenmadeonAPI gallerieswith
automaticreading.

CONCLUSIONS

1. The number of CFU/cm2 is significantly smaller on the foot surfacecompared to that on


theshellsurface.
2. Nobacteriaimplicatedinthehumandigestivepathologywereisolated.
3. ThenumberofCFU/cm2 issignifanctlysmalleronthefoot surfacecomparedtothaton
theshellsurface,withtheexceptionofonesnail:thesnailno.9.
4. ThesmallnumberofCFU/cm2 at9ofthe10studiedsnailscandberelatedtothequantity
andthequalityofthemucousproducedbythesnailsglands.

BIBLIOGRAPHY

1. Bondoc I., indilar E.V. (2002) Controlul sanitar veterinar al calitii i salubritii
animale.Vol.1.Ed.IonIonescudelaBrad,Iai
2. ComanI.,MareM.(2000)Micologiemedicalaplicat.Ed.Junimea,Iai
3. KiebreToeM.B.,BorgesE.,MaurinF.,RichardY.,KodjoA.(2003)Etudedelaflore
bactrienne arobie Gram ngatif de lescargot dlevage (Helix aspersa). Revue
MdicineVtrinaire;154:605610
4. SvecP.,DevrieseL.A.,SedlacekI.,BaeleM.,VancaneytM.,HaesebrouckF.,SwingsJ.,
Doskar J. (2002) Characterization of yellowpigmented and motile Enterococci
isolatedfromintestinesofthegardensnail
5. Helixaspersa.JournalofAppliedMicrobiology;92:951957

976

MAINANTIBIOTICSUSEDINTHETREATMENTOFUDDER
INFECTIONSINHUMANCONSUMPTIONMILKSUPPLIERANIMAL
SPECIESFROMSOUTHEASTERNSIBIUCOUNTY

M.CRSTEA1,R.TRIF2,S.MORARIU2,FloricaMORARIU3
DSVSASibiu,CaleauriiMari,12;cirstea.mariussb@ansvsa.ro
2
FacultyofVeterinaryMedicineTimioara,CaleaAradului119;
3
FacultyofAnimalSciencesandBiotechnologies,CaleaAradului,119.

Abstract
Antibioticsarewidelyusedintreatmentofanyinfectiousdisorder,includingmastitisalso.Their
improperusecouldseriouslyharmtheconsumerofmeat,milk,eggsandotherderivedproducts
obtainedfromtreatedanimals.Thispaperemphasizedtheuseofantibioticsinfivelocalitiesfrom
SibiuCountyinthreespeciesofanimals(cows,buffaloesandewes)whichprovidemilkforhuman
consumption. The most used antibiotic was a combination of Penicillin and Streptomycin
(31.84%),followedbyEnrofloxacyn(20.72%)andOxytetracycline(18.77%).
Keywords:antibiotics,milk,SibiuCounty.

Antibiotics are vital drugs, used as last strategy in infection treatments. But their
efficacyisthreatenedbytheimproperandexaggerateduseofthem,notonlyinmedicinebut
alsoinagriculture.Inveterinarypractice,antibioticsareusedmostlyininfectiontreatmentor
prevention(2).
Thepresenceofantibioticsinmilkisdue,inmostcases,totheimproperuseofvarious
drugsagainstmastitisorotherinfectiousdiseases(1).
The antibiotic contaminated milk is considered compromise by the Human Health
Authorities and represents a potential danger for consumer health. The presence of drug
residuesinfoodovertheadmittedmaximumlevelisconsideredharmfultohumanhealthby
manyinternationalpublicauthorities(4).
Also, some pathogens (i.e. Staphylococcus aureus) can produce several toxins which
exacerbatetheirpathogeny.Someof theseexotoxinscouldselectivedestroythepolymorph
nuclear cells and monocytes. These leucotoxins belongs to a family with two components,
being composed by two proteins: S (sloweluted) and F (fasteluted), which are acting
synergistically to produce veritable holes into the membrane of phagocyte cells. Panton
Valentineleucocydine(LukSPV+LukFPV),haemolysine(HlgA+HlgBandHlgC+HlgB)and
recentlydescribedLukM(LukM+LukF`PV)andLukE/D(LukE+LukD)(3;5;7;8;10).

MATERIALSANDMETHODS

During2009fivelocalitiesfromsoutheasternSibiuCounty(ArpaudeJos,Porumbacu
de Jos, Avrig, Turnu Rou and Tlmaciu) were monitored concerning the antibiotic use in
variousaffections,butmainlyformastitisofeconomicinterestanimals.
Only animals which produce milk for human consumption were selected from
consultationregisters.

977

Lucrritiinificevol53seriaMedicinVeterinar
RESULTSANDDISCUSSIONS

Onlycows,buffaloesandeweswereselectedforthisstudy.
Because the lack of treatment can cause serious injuries both to the health of the
animalandtothemilkproduction,thedrugtreatmentisthefirstoption.Theantibioticsused
inthefivelocalitiesofSibiuCountyarelistedintheTable1.

Table1.
Theantibioticsusedintreatmentofmastitisinanimalsfromthefiveinvestigatedlocalities.
Crt.
No.
1.
2.
3.
4.
5.
6.
7.
8.

Antibiotic
Amoxicillintrihydrate
Ampicillintrihydrate
Penicillin+ Streptomycin(PenStrept)
Enrofloxacyn
Erythromycinthiocyanate
Gentamicinsulfate
Oxytetracycline
Tylosintartrate

No.of cases
(%)
414(16.85%)
24(0.97%)
782(31.84%)
509(20.72%)
63(2.56%)
174(7.09%)
461(18.77%)
29(1.18%)

Waitingtimefor
milk
3days
3days
5days
5days
7days
7days
3days
4days

Outof the2456cases ofmastitisinall 3monitored speciesitcanbeobservedthat3


antibiotics or their combinations were intensely used, with more or les satisfactory results:
PenStrept, Enrofloxacyn andOxytetracycline. These 3 antibiotics represented more then 2/3
from all used antibiotics (71.33%). The most used combination was PenicillinStreptomycin
(31.84%)inalmost1/3ofcases,dueprobablytoitslowestcostcomparingwithbenefits.
It is obvious that the mastitis can not be totally eliminated from a herd, but it is also
obviousthattheirevolutioncouldbecontrolledirrespectiveofbreedingsystem.Thus,in2006,
Jones (6) considers that therapeutically approach in these situations must be the same of a
surgeonbeforestartingthesurgicaltreatment.
Thei.m.injectionoftheantibiotichada90%efficacy,whilethelocaltreatmenthealed
only50%ofcases.Inthesametime thecombinationbetweensystemicandlocaltreatment
curedallcases,eventhoseconsideredcompromised(9).
The prevalence of antibiotics used in the treatment of mastitis in all five investigated
localitiesisshowninFigure1.
1.18
18.77

Am o tri

16.85

Am pi tri
0.97

PenStrept

7.09

Enro

2.56

Eri
31.84

Genta
Oxi

20.72

Tilo

Figure1.Theprevalenceofantibioticsusedinmastitistreatment.

978

UniversitateadetiineAgricoleiMedicinVeterinarIai

CONCLUSIONS

The most used antibiotic was the combination PenicillinStreptomycin (31.84%),


followedbyEnrofloxacyn(20.72%)andOxytetracycline(18.77%).

REFERENCES

Albright, J.L., Ormiston, E.E., Brodie, B.O., Witter, L.D., 1991 Penicillin in milk following
intramuscular and intramammary administration of penicillin in normal and mastitic cows.
J.A.V.M.A.,138,7073.
2. Ambedkar S. S., Deshpandek B. S., Jadhav P., Shewale J.G., 1991 Role of side chain in the
hydrolysisofpenicillinbylactamase.AssociationofOfficialAnalyticalChemists.
3. Choorit,W.,Kaneko,J.,Muramoto,K.,Kamio,Y.,1995.Existenceofanewproteincomponentwith
the same function as the LukF component of leukocidin or gammahemolysin and its gene in
StaphylococcusaureusP83.FEBSLett.,357,260264.
4. Diliello, L.R., 1982. Methods in Food and Dairy Microbiology. AVI Publishing Company, Westport,
CT.
5. Gravet,A.,Colin,D.A.,Keller,D.,Giradot,R.,Monteil,H.,Prevost,G.,1998.Characterizationofa
novelstructuralmember,LukELukD, ofthebicomponentstaphylococcalleucotoxinsfamily. FEBS
Lett.436,202208.
6. Jones,G.M.,2006.Understandingthebasicsofmastitis.VirginiaCooperativeExtension.Publication
No.404233.VirginiaStateUniversity,USA,pp:17.
7. Kaneko, J., Muramoto, K., Kamio, Y., 1997. Gene of LukFPVlike componentof PantonValentine
leukocidininStaphylococcusaureusP83islinkedwithlukM.Biosci.Biotechnol.Biochem.,61,541
544.
8. Rainard,P.,Corrales,J.C.,BelenBarrio,M.,Cochard,T.,Poutrel,B.,2003.Leucotoxicactivitiesof
Staphylococcus aureus strains isolated from cows, ewes, and goats with mastitis: importance of
LukM/LukFPVleukotoxin.Clin.Diagn.Lab.Immunol.,10,2,272277.
9. Sayed,M.,AbdelRady,A.,2008.AcuteclinicallymastiticanimalsinvillagesofAssiutGovernance:
diagnosisandtreatment.Vet.World,1,9,261264.
10. Woodin,A.M.,1960.PurificationofthetwocomponentsofleukocidinfromStaphylococcusaureus.
Biochem.J.,75,158165.

1.

979

PREVALENCEOFMASTITISINCOWS,SHEEPANDBUFFALOESIN
FIVELOCALITIESFROMSIBIUCOUNTY
M.CRSTEA1,R.TRIF2,S.MORARIU2,FloricaMORARIU3
DSVSASibiu,CaleauriiMari,12;cirstea.mariussb@ansvsa.ro
2
FacultyofVeterinaryMedicineTimioara,CaleaAradului119;
3
FacultyofAnimalSciencesandBiotechnologies,CaleaAradului,119.

Abstract
Mastitis is a widespread inflamation of udder all over the world. This paper describes the
prevalence of mastitis in cows, sheep, and buffaloes from five localities of southwestern Sibiu
County. Mean mastitis prevalence in cows was 3.84%, 0.84% in buffaloes, and 5.44% in sheep,
respectively.
Keywords:SibiuCounty,mastitis,prevalence.

Mastitis is the udder inflammation. The diagnosis is accomplished by some criteria,


which implies both the clinical part (they are recognized after inspection or palpation) and
paraclinicalexams(milkdetectionofbacteriaorsomaticcells).
Cows mastitis represents a serious problem, because of its devastating economic
effects.Annually,approximately2billiondollarslossesarerecordedinUSA,andoverthan500
billiondollarsarerecordedinIndia(1;8;12).
Theaimofthisstudywastoinvestigatetheprevalenceofmastitisinsomelocalities
fromsoutheasternSibiuCounty.

MATERIALSANDMETHODS

Duringthe2009yearfivelocalitiesfromSibiuCounty(ArpaudeJos,Porumbacude
Jos,Avrig,TurnuRouandTlmaciu)weremonitoredconcerningtheprevalenceofclinicaland
subclinicalmastitis(Figure1).
There were investigated 47744 animals from households as follows: 4549 cows,
41658ewes,and1537buffaloes,respectively.

Figure1.GeographicaldistributioninsidetheCountyofthefiveinvestigatedlocalities.

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Lucrritiinificevol53seriaMedicinVeterinar
Milk samples were collected in sterile recipients from all affected quarters, after a
thoroughlydisinfectionofthetitsandonlyafterthefirstdropswerediscarded.Sampleswere
immediately transported to bacteriology laboratory of DSVSA Sibiu where they were
processed. The suspected colonies were macroscopically, morphologically and biochemical
identified,onthebasisofselectedliterature(2;9;13).

RESULTSANDDISCUSSIONS

Table1showsthedataconcerningtheprevalenceofclinicalandsubclinicalmastitis
inthefivemonitoredlocalitiesfromSibiuCounty.

Table1.TheprevalenceofcowmastitisinfivelocalitiesfromSibiuCountyin2009.
Cows
Buffaloes
Ewes
Crtno.
Locality
Total Mastitis (%) Total Mastitis (%) Total Mastitis (%)
1.
2.

ArpaudeJos
763
PorumbacudeJos 1564

29(3.8%)
47(3%)

282
665

1(0.35%)
7(1%)

4550
12695

273(6%)
698(5.5%)

3.
Avrig
1100
55(5%)
309
3(1%)
14685
734(5%)
4.
TurnuRou
486
19(3.9%)
281
2(0.7%)
2070 126(6.1%)
5.
Talmaciu
636
25(3.9%)

7658 437(5.7%)
TOTAL
4549 175(3.84)
1537
13(0.84)
41658 2268(5.44)

Investigatingthedistributionofmastitisitcanbeobservedthatthisismostprevalent
in sheep. Localities with the highest prevalence were: Avrig for cows and buffaloes,
PorumbacudeJosforbuffaloes(togetherwithAvrig)andTurnuRouforsheep.
Nextgraphshowsthemastitisprevalenceonspeciesinthefivelocalities,basedon
thedatafromTable1.
12
10

5.5

3.8

0.35

3
1

Arpau

Porumbacu
Cows

6.1

5.7

3.9

3.9

0.7

0
Avrig
Ewes

Turnu
Rou Tlmaciu
Buffaloes

Figure2.Theprevalenceofmastitisincows,sheepandbuffaloesinthefivelocalitiesofSibiu
County.
A similar study carried out in 2008 in Egypt revealed an increased prevalence of
mastitisincows(22.5%),sixtimesgreaterthanregisteredinthefivelocalitiesinvestigatedin
SibiuCounty.Inthesametime,theprevalencewaslowerinsheep(2.63%)andgoats(4.63%)
thaninthemonitoredlocalities(10).

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UniversitateadetiineAgricoleiMedicinVeterinarIai
This fact could be due to the improper hygienic conditions of cows benefit of,
comparing with the cattle from Romania. Talking about sheep, the related prevalence of
mastitisexpressesthesameinconvenientinthebreedingsystemsfrombothcountries.
Indeed,theprevalenceofclinicalandsubclinicalmastitisisdifferentfromcountryto
country,sinceitisduetodifferencesbetweenbreedingtechnologies,management,and,not
thelast,intheapproachingmethodsinthisconcern.
So,incows,theprevalencewashighinUSA(27.3%),Kenya(28.7%)andEthiopia(38.2
%60.8%),whileinsheep,thisrangedbetween12%and37%inUSA,Israel,Greece,England,
Wales, and Spain, respectively (3; 5; 6; 7; 11; 14). Concerning buffaloes mastitis, a study
conductedin2009inIndiareported3.1%prevalence,namelyoutof1000examinedquarters,
only31wereaffectedbyaclinicalorsubclinicalmastitis(4).

CONCLUSIONS

The prevalence of mastitis was greater in sheep (5.44%) and lower in buffaloes
(0.84%)inthefiveinvestigatedlocalitiesfromSibiuCounty.
ThehighestprevalenceofmastitisincowswasnoticedinAvrig(5%).

REFERENCES

1.
2.
3.
4.
5.
6.

7.

8.
9.
10.
11.
12.
13.
14.

AbdelRady, A. Sayed, M., 2009. Epidemiological studies on subclinical mastitis in dairy cows in
AssiutGovernorate.Vet.World,2,10,373380.
Carter, G.R., Cole, J.R.J., 1990. Diagnostic procedures in veterinary bacteriology and mycology.
Essentialsofveterinarybacteriologyandmycology.5thEd.AcademicPress,USA.
Dego, O.K., Tareke, F., 2003. Bovine mastitis in selected areas of southern Ethiopia. Trop. Anim.
HealthProd.,35,197205.
Kavitha, K.L., Rajesh, K., Suresh, K., Satheesh, K., Syama Sundar, N., 2009. Buffalo mastitis risk
factors.BuffaloBull.,28,3,134137.
Las Heras, A., Dominguez, L., FernandezGarayzabal, J.F., 1999. Prevalence and aetiology of
subclinicalmastitisindairyewesoftheMadridregion.SmallRum.Res.,32,2129.
Leitner,G.,Chaffer,M., Zamir,S.,Mor,T.,Glickman,A.,Winkler,M., Weisblit,L., Saran,A.,2001.
Udder disease etiology, milk somatic cell counts and NAGase activity in Israeli Assaf sheep
throughoutlactation.SmallRum.Res.,39,107112.
McDougall, S., Pankey, W., Delaney, C., Barlow, J., Mardough, P.A., Scruton, D., 2002. Prevalence
andincidenceofsubclinicalmastitisingoatsanddairyewesinVermont,USA.SmallRum.Res.,46,
115121.
Miller,R.H.; Pape, M.J.; Fulton,L.A.and Schutz, M.M.,1993.Therelationship ofmilkand somatic
cellcounttomilkyieldsforHolsteinheifersafterfirstcalving.J.DairySci.76,728733.
Quinn, P.J., Carter, M.E., Markey, B.K., Carter, G.R. 1994. Clinical veterinary microbiology. Mosby
Ed.,U.K.
Sayed,M.,AbdelRady,A.,2008.AcuteclinicallymastiticanimalsinvillagesofAssiutGovernance:
diagnosisandtreatment.Vet.World,1,9,261264.
Stefanakis,A.,Boscos,C.,Alexopoulos,C.,Samartzi,F.,1995.Frequencyofsubclinicalmastitisand
observationsonsomaticcellcountsinewes'milkinnorthernGreece.Anim.Sci.,61,6976.
Varshney,J.P.,Naresh,R.,2004.Evaluationofhomeopathiccomplexintheclinicalmanagementof
udderdiseasesofriverinebuffaloes.Homeopathy,93:1720.
Waage,S.,Mork,T.,Roros,A.,Hanshamar,A.,Odegaard,S.A.,1999.Bacteriaassociatedwithdairy
heifers.J.DairySci.,82,712716.
Watkins,G.H.,Burriel,A.R.,Jones,J.E.T.,1991.Afieldinvestigationofsubclinicalmastitisinsheepin
southernEngland.Brit.Vet.J.,147,413420.

982


RESEARCHRELATEDTOTHEREFINEMENENTOFTHEPOULTRYAS
PRODUCTSWITHOUTPROTECTIVEMEMBRANE

LilicaCOCLEA ,AngelaGAVRILA2,D.SIMEANU2,CristinaSIMEANU2
1
SCTabcoCampofrioSATulcea,2USAMVIai

Abstract
The present work has the purpose of testing the quality of the raw chicken meat and that ofa
productwithoutprotectivemembranemanufacturedintheframeworkofSCTabcoCampofrio
SATulcea.Theanalysiscarriedouthasbeenintherangeoforganoleptic,physical,chemicaland
microbiologicaltests.Followingthesetestswehavenotedthattherawmaterialsutilizedwereof
goodquality,thusleadingtofinalproductswellvaluedbyconsumers,especiallysincealllinksin
the manufacturing technology have been followed. For the two analyzed products high protein
contenthasbeendetected;atthesametime,fatcontentwaslow.Microbiologically,thestudied
products did not endanger the consumers health. In the reported context, the products
manufacturedbytheabovementionedcompanyhaveenjoyedagoodappreciationofconsumers
from all categories. For the future, the company where the research was carried out plans to
diversify more the chicken meat product range, so as to cover the growing consumer
requirements.Inthesametimethenaturalflavorsrangewillbeextendedforthemanufactured
products,soastosatisfyconsumersrequirementsinthisrespectaswell.

Keyword:pastrami,,products,organoleptic,physical,chemical

Inthesupplypopulationwithfood,withhighbiologicalvalue,aparticularlyimportantrole
rests to the animal origin products, and among these, meat and meat derivatives are
prevalent. Thus, poultry occupies a special place, whereas it features characteristics that
makesitnotonlyverynutritious,butdietaryaswell.
Concernsforsuperiorrefinementofpoultryasproductsareagrowingconcerninducedby
amoreopenmarketforsuchproducts.
Theareainwhichresearchhasbeencarriedout,NorthDobrogearespectively,theproblem
addressed is very topical, since here is recorded a lesser consumption of chicken meat and
products,duevariousreasonslike:
- lackoftraditioninpoultryconsumption
- limitedrangeofchickenmeatproductsinthegrocerystoresandhighpriceoftheexisting
ones,notmeanttostimulatepublicinterestintheirconsumption
- lackofmeatpoultryfarms,ensuringasupplyofprocessingunitswithrhythmicmaterial
andtodeterminethelowestproductioncostforfinishedproducts
To those shown we believe that our research results will lead to an increase in chicken
meatprocessingintheareaandwillprovideincentivesforbusinesstoinvestinpoultry.

MATERIALSANDMETHODS

This work has analyzed the product Chicken legs smoked pastrami in regards with
organoleptic,physical,chemicalandmicrobiologicalcharacteristics,whichallowedustomake
aproperassessmentoftheproductsquality,inconformitywiththequalityoffoodrequired
bycurrentRomanianlegislationaswellasbyEUregulations.

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Lucrritiinificevol53seriaMedicinVeterinar
Organoleptic characteristics were related to: product appearance, color, its consistency
andsmell,respectingtheprovisionsoftheSTAS7031/83.
pHvalue
Theacidityisassessedobjectively(byelectrometricmeasurement,usingapHmeterora
ionometer).
EasilyhydrolysableNitrogen
Nitrogenfromaminogroupsisreleasedbyhydrolysiswithaweakbaseandtogetherwith
the free ammonia, is driven by steam distillation in an acidic solution quantitatively and
qualitativelyknown.Excessacidisdeterminedbytitrationwithanequivalentalkalinesolution.
HydrolysableNitrogenderivesbothfromaminogroupsandfreeammonia.Freeammonia
isonlyapartoftheeasilyhydrolysablenitrogen,soitshouldnotbeconfound.
Hydrogensulfide
Hydrogensulfideinthesampletobeanalyzedformsinreactionwiththeleadacetatethe
leadsulfide(darkbrowncompound).
Watertestwasdonebytheovendryingmethod.Principleofthemethodconsistsindrying
thesampletobeanalyzedintheovenatatemperatureof+105oC.
Mineralstest
Todeterminetheash,calcinationsmethodintheelectricalfurnacewasused.Principleof
themethodconsistsincalcinationsofthemeatsampleat+550oCinanoven.
ProteintestwascarriedonusingKjeldhalmethod.
The principle of the method: combinations of organic nitrogen, by heating with
concentratedsulfuricacid,inthepresenceofacatalyst,areconvertedintoammoniumsulfate.
Byaddingastrongbase(NaOH33%),ammoniaisreleased,andthroughdistillation,captured
intoafixedamountofsulfuricacidwithknownnormality.Theexcessacidwastitratedwithan
alkaline solution of same normality, and the difference was the total amount of N fixed.
Chemicalreactionsthatoccurare:

N(protein)+H2SO4(NH4)2SO4

(NH4)2SO4+2NaOH=H2SO4+2NH4OH

2NH4OH+H2SO4=(NH4)2SO4+H2O
Fat test was carried out using Soxhlet method. Principle of the method consists in fat
extractionwithorganicsolvent.
Microbiology
N.T.G.M.A.testisbasedontheinclusionofanamountofmeattobetestedinasuitable
nutrient environment, poured in Petri plates. Following incubation, each group of germs or
eachmicroorganismwilldevelopacolony.
For this determination the following culture environments are used: yeast extract agar
glucosegelatin(Frazier);agartryptoneyeastextractglucose(Platecountagar)
Principleofthemethod.PresenceofSalmonellaisdetectedbyseedingthesamplein an
preenriching nonselective liquid environment, incubation at 37 oC, then seeding in two
enrichingselectiveliquidenvironmentsusingculturesfromthepreenrichingenvironmenton
selective solid media, which after incubation at 37 oC are checked for the presence of
Salmonellasuspectedcoloniesandconfirmedbyidentificationtest.
The main experimental data were statistically processed, calculating: arithmetic average,
variance, average standard deviation, variation coefficient, and the homogeneity of the
experimentalvariancesweretestedbyFishertest(SanduGh.,1995).

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UniversitateadetiineAgricoleiMedicinVeterinarIai
RESULTSANDDISCUSSIONS

Organolepticcharacteristics
For the fabrication of the sort Chicken legs smoked pastrami are used chicken thighs,
trimmed, weighing between 350450 g. The raw material is chilled, but it comes from both
fresh and frozen meat. Organoleptic characteristics of the raw chicken legs used in the
manufacturingofthesortChickenlegssmokedpastramiarepresentedintable1.

Table1.Organolepticcharacteristicsoftherawchickenmeat
STAS703183
Peculiarity
Presentation
A1:1997pct.4
Themeatsurfacewasdry,withshiny Meatsurfacehasadryfilm.
andelastictendons.
Fat,color,consistencyandtasteare
Fatcompositionwasnormal.
normal.
Meat
Synovialfluidwasclear
Tendonsareshiny,elasticandstrong.
appearance
Articulatedsurfaceissmoothand
glossy.
Synovialfluidisclear.

Color

Texture
(elasticity)

Smell

Themeathadapinktoredcolor,
moistwithoutbeingsticky,andthe
muscularjuicewasdifficulttoobtain
bypressing.

Onthesurfacethemeatispinktored;
onthecrosscutitisglossy,slightly
moist,withoutbeingsticky.
Musclejuiceisdifficulttoobtainandis
clear.
Whenpressingafingergrooveswere Themeatisfirmandelastic;itdoesnt
keepgroovesafterpressingbyfingers;
notformedonthesurface,having
itiscompactinthecrosscut.
anelastictexture.
Fragrant,bothonthesurfaceand
Onthesurface,incrosscutandinside
deep,withoutshowingabnormal
thecarcassthesmellispleasant,
smells.
aromatic,typicalforthefreshmeat,
withoutabnormalsmells.

We note in the table above tat the chicken legs, used as raw material, match the
requirementsofSTAS703183A1:1997pct.4,thusbeingfitforhumanconsumption.
Physicalcharacteristics
MeasurmentofthepHvalueontherawmaterialsusedforthefabricationoftheChicken
legssmokedpastramiwasmadeusingthepHmeasuringapparatus.
In table 2 the pH values are shown, acquired for both fresh meat and frozen and stored
meat as well. The values recorded were normal; thus, for the fresh meat the values ranged
between6.136.28,whileforthefrozenmeatthevaluesrecordedareitheinterval6.336.26,
thesebeingtypicaltothepoultrymeatstoredforsometime.
Regarding the variance coefficient, this highlights the homogeneity of the studied
characteristic(V%<10).
Values obtained by laboratory analysis on the chicken legs to determine the easily
hydrolysablenitrogencontentarerecordedintable3.Thesevalueswere20.52mgNH3/100g
meatforthechilledmeatandgrewandup to23.31mg NH3/100gmeatforthefrozenraw
material.

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Lucrritiinificevol53seriaMedicinVeterinar
Table2.pHvalueoftherawchickenmeat(n=25)
Typeofraw
meat

24h
x sx
V%

Storage
24h

7days
x sx
V%

V%

x sx

3months
V%

x sx

6months
x sx

V%

Refrigerated
chicken
6,130,11 4,12 6,280,14 4,25

meat
Frozen
chicken

6,330,09 4,91 6,190,07 3,87 6,260,08 4,19


meat

*Infabricationitwasusedonly3monthsstoredfrozenmeat

The recorded values are lower than the maximum admissive stated by the STAS 9065
7/2007.
The variance coefficients recorded values bellow 10%, proving a very good uniformity of
thestudiedcharacteristic.

Table3.Easilyhydrolysablenitrogencontentoftherawmeat(n=25)
Rawmeatstate
Statisticalestimators

STAS90657/2007
Refrigeratedmeat

Frozenmeat

x sx

NH3mg/100gproduct

20,520,24

23,310,32

V%

3.87

4.08

25

Hydrogensulfide.Hydrogensulfidewasnotrevealedintotherawmaterialunderreview,
demonstratingonceagainthedegreeoffreshnessoftherawmaterial.
Chemicalcharacteristics
Watercontent.Refrigeratedrawmaterialhadawatercontentof69.6%,whilethefrozen
one recorded a water content of 67.8%. In comparison with the literature consulted, which
providedawatercontentvalueof67.5%(VacaruOpriI.icol.,2004;2005),wenotethatour
resultsareslightlyhigherfortherefrigeratedrawmaterial.
In calculating the variance coefficient, values were small (V%<10), which shows the
homogeneityofthestudiedcharacteristic.
Drymattercontent.Laboratoryanalysis todeterminethedrymattercontentof theraw
chicken legs revealed comparable results with the specialized literature data (32,5 27,9%),
(VacaruOpriI.icol.,2004,2005).
Calculatedvariancecoefficientsarebellow10%,whichdemonstratestheuniformityofthe
rawmaterialbatchesanalyzed.
Protein content. For protein content the following results were obtained: 22.7% for the
refrigeratedmeatand22.96%forthefrozenone.Thesevaluesdontdiffertotheonesinthe
specializedliterature.
Variancecoefficientsdidnotexceed10%,correlatedwithaverygoodhomogeneityofthe
characteristicstudied.
Fat content. On the fat content parameter laboratory results indicated values in a range
between5.53%6.16%,Thevalueswehaveobtainedarecomparablewiththosefoundinthe
literatureconsulted.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Calculatedvariancecoefficientsbellow10%certifythatthebatchesarehomogenous.
Mineralcontent.Whendeterminingthemineralcontent,theiramountintherawmaterial
rangedbetween1.99%to2.12%,exceedingthe1.3%asisreflectedindatafromtheliterature
consulted(VacaruOpriI.icol.,2005,etc).
Values set for the variance coefficients were very small, (V%<10), which shows a high
degreeofhomogeneityforthischaracteristic.
The overview of the chicken legs raw materials chemical characteristics are presented in
table4.

Table4.Chemicalcharacteristicsoftherawmaterialmeat(n=25)
Rawmeatstate
Chemical
Statistical
characteristics
estimators
Refrigeratedmeat
Frozenmeat
x sx
69,60,71
67,80,86
Watercontent(%)
V%
6.18
6.37
x sx
30,40,32
32,20,39
Drymattercontent
(%)
V%
5,29
4,37
x sx
22,70,18
22,960,16
Proteincontent(%)
V%
6,27
4,08
x sx
5,530,06
6,160,08
Fatcontent(%)
V%
5,39
5,41
x sx
1,990,04
2,120,05
Mineralscontent(%)
V%
3,84
3,49

Microbiologicalcharacteristicsoftherawmaterial
Followinglaboratorytests,noaerobemezofilegermswerefoundinthemeatdepth,while
onthesurfacetheirpresencewasverylow(table5).
Thevaluesobtained,of10.11germs/gmeat,foundonthesurfaceoftherefrigeratedmeat
samples and 11.58 germs/g frozen meat are considered normal and bringing insignificant
changes to the used raw material, given that the maximum allowed germ density on a
microscopefieldis30(ProfessionalStandardsCollectionforthepoultrymeat,1997).
Table5.Totalnumberofaerobemezofilegermsintheanalyzedmeat(n=25)
Rawmeatstate
Specification
Refrigeratedmeat Frozenmeat
NTGMA/gmeatindepth
absent
absent
NTGMA/gmeatonsurface
10,110,25
11,580,28
V%
4,35
6,42

Calculatedvariancecoefficientswerebellow10%
Organoleptic, physical, chemical and microbiological characteristics of the studied sort:
Chickenlegssmokedpastrami
Organolepticcharacteristics
Inaccordancewiththeinternalfabricationnorms,thefinishedproducthastheappearance
of chicken thighs well shaped, with no fringes, with well developed muscles, orangebrown
colored,weighing~300g.Tasteandsmellwerepleasant,boiledandsmokedlike,specificto
theflavorsandspicesused.Meattendernesswashigh,whilejuicinesswastheoneparticular
tothechickenlegs.

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Lucrritiinificevol53seriaMedicinVeterinar
In analyzing the final product organoleptic characteristics compliance with the above
requirementswasfound,thesortbeingcharacterizedbyapleasanttasteandsmell,without
foreign odors, its surface was uniformly dry, showing small breaks of the skin, but without
affectingthecommercialvalueoftheproduct.
Physicalcharacteristics
pHvalue.DeterminationofthepHvaluewasperformedonthefreshproduct,productat
midtermwarranty,after10days,andontheproductattheendofitswarranty,20daysafter.
OnemaynotefromtheacquiredresultsthegradualdecreaseofthepHvaluefrom6.58,
value on the fresh product, to 6.28 for the product at mid warranty term, after 10 days
storage,forattheendofwarranty,after20daysstorage,pHvaluetoreach6.06
Thevaluesobtained,consideredinthelightofthevariancecoefficientsvalues,indicatea
verygoodhomogeneityofthestudiedcharacteristic(V%<10).
Easily hydrolysable nitrogen. Easily hydrolysable nitrogen tests on the product Chicken
legssmokedpastramirevealedvaluesof20.48 mgNH3/100gproductonthefreshoneand
28.96mgNH3/100gproductafter20daysstorage.
Giventhatthelawimposesamaximumof45mgNH3/100gproduct,wecansaythatour
productiscomplyingwiththeoutstandingregulations.
Smallvaluesofthecalculatedvariancecoefficient(V%<10)demonstratesthehomogeneity
ofthecharacteristicwatched.
Hydrogensulfide.Laboratoryanalysisdidnotrevealedthepresenceofhydrogensulfidein
thesamplesstudied.
Saltcontent.Thisproductrangeisrequiredtohaveamaximumsaltcontentof3.5%,but,
asallchickenmeatbasedproducts,saltcontentwasalowone.Inourcase,saltcontentvaried
between 1.8%, value recorded on the fresh product, to 2.1% value at the end of warranty
term.
Batches homogeneity is confirmed by the small values obtained when calculating the
variancecoefficients.
Nitritescontent.Forthetimebeingthemaximumallowablenitritescontentonthemeat
based products is 10 mg NaNO2/100 g product. This limitation implies testing all meat
productstoverifythischaracteristic.
Wehaveobtainedvaluesrangingfrom4.52to4.63mgNaNO2/100gproduct,thesebeing
wellbellowthemaximumadmissiblegivenbythelawinforce.
Viewedintermsofvariancecoefficients,thebatchesweretheanalyzedproductsoriginate
fromarehomogenous.
Table6presentsthelaboratoryanalysisresultsforthephysicalcharacteristicsofthesort
Chickenlegssmokedpastrami.
Chemicalcharacteristics
Todeterminethechemicalcharacteristicswehaveappealedtodeterminationsofwater,
proteinandfatcontentofthefinishedproduct.
Watercontent.Freshproductwatercontentwas77.12%,butdecreasedduringtheperiod
ofvalidity,sothatafter20daysreachedavalueof74.48%.
The variance coefficients recorded small values, bellow 10%, demonstrating the
consistencyofproductbatcheswherethesamplesanalyzedweretakenfrom.
Proteincontent.Analyzesrevealed17.76%proteincontentinthefreshproduct,18.19%in
theproductafter10daysofstorageand19.01%attheproductafter20daysstorage.Wecan
say that our product meets legislative requirements, given the values above 15% recorded,
whichisminimumrequiredbylawforproteincontent.
Thevariancecoefficientsvalueswerebellow10%.

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Table6.PhysicalcharacteristicsofthesortChickenlegssmokedpastrami(n=25)
Productafter
Productafter
Statistical
Fresh
Physicalcharacteristics
10days
20days
estimators
product
storage
storage
x sx
6,580,08
6,280,06
6,060,08
pHvaluedynamics
V%
5,08
4,67
5,13
Easilyhydrolysable
x sx
20,480,11
24,140,17
28,960,23
nitrogencontent(NH3
V%
2,56
3,08
4,24
mg/100gproduct)
Hydrogensulfide
Negative
reaction
x sx
1,80,03
1,90,08
2,10,10
Saltcontent(%)
V%
3,29
3,97
4,06
Nitritescontent
x sx
4,520,11
4,60,10
4,630,12
(NaNO2mg/100g
V%
4,08
4,61
4,27
product)

Fatcontent.Valuesresultingfromfatcontentdeterminationsrangedbetween3.38%and
3.62%. Although its not a limitative factor, for this product range it is taken into account in
fabrication.
Variancecoefficientsremainedbellow10%.
n continuare n tab7. sunt prezentate rezultatele determinrilor caracteristicilor chimice
alesortimentuluianalizat.
Next, in table 7, we are presenting the results of chemical characteristics test for the
analyzedproduct.
MicrobiologicalcharacteristicsofthesortChickenlegssmokedpastrami
DeterminationstoshowanycontaminationwithSalmonellaandListeriashownusthatthe
analyzedproductsareconsideredappropriate,nothavingcontaminationthatmayrenderthe
productunfitforhumanconsumption.

Table7.ChemicalcharacteristicsofthestudiedsortChickenlegssmokedpastrami(n=25)
chemical
Statistical
Fresh
Productafter10
Productafter20
characteristics
estimators
product
daysstorage
daysstorage
x sx
77,120,73
76,270,70
74,480,76
Watercontent(%)
V%
5,39
6,08
6,47
x sx
17,760,12
18,190,11
19,010,13
Proteincontent
(%)
V%
4,38
5,27
4,35
Fatcontent(%)

x sx

3,380,11

3,560,16

3,620,17

V%

6,27

4,71

4,61

CONCLUSIONS

From our findings clearly emerges that in our research was used a high quality raw
material, which led to obtain finished goods valued by consumers, especially since all
fabricationtechnologicalstepshavebeenfollowed.

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Lucrritiinificevol53seriaMedicinVeterinar
All finished products analyzed had high protein content, while the fat content was low.
Microbiologically,thestudiedproductsdidnotendangerthehealthofconsumers.
In the context reported, the products manufactured by us enjoyed a very good
appreciationfromallcategoriesofconsumers.
Forthefuture,thecompanywheretheresearchwasperformedaimstofurtherdiversify
itsrangeofchickenmeatproducts,soastocovertheeverincreasingrequirementsof
consumers.Atthesametime,willexpandtherangeofnaturalflavorsfortheproducts
manufacturedsoastomeetthediversifiedrequirementsofconsumersinthisregard.

BIBLIOGRAPHY

***Regulamentul(CE)nr.852/2004alParlamentuluiEuropeanialConsiliuluicuprivirelaigiena
produseloralimentare.
2. ***Regulamentul(CE)nr.853/2004alParlamentuluiEuropeanialConsiliuluidin29aprilie2004.
3. ***Regulamentul(CE)nr.854/2004alParlamentuluiEuropeanialConsiliuluidin29aprilie2004.
4. ***Regulamentul(CE)nr.882/2004alParlamentuluiEuropeanialConsiliuluidin29aprilie2004.
5. ***Publisher:OPOCE;Regulamentul(CE)nr.2073/2005alComisieidin15noiembrie2005.
6. ***Regulamentul(CE)nr.2074/2005alComisieidin5decembrie2005.
7. ***560/1271/339/210/2006Ordinalministruluiagriculturii,pduriloridezvoltriirurale,al
ministruluisntiipublice,alpreedinteluiAutoritiiNaionalepentruProteciaConsumatorilor
ialpreedinteluiAutoritiiSanitareVeterinareipentruSiguranaAlimentelorpentruaprobarea
Normelorcuprivirelacomercializareaproduselordincarne.
8. VacaruOpriI.(coordonator),2000TratatdeAvicultur,vol.I.EdituraCeres,Bucureti.
9. VacaruOpriI.(coordonator),2002TratatdeAvicultur,vol.II.EdituraCeres,Bucureti.
10. VacaruOpriI.(coordonator),2004TratatdeAvicultur,vol.III.EdituraCeres,Bucureti.
11. SanduGh.,1995Metodeexperimentalenzootehnie.EdituraCoralSonivet,Bucureti.
1.

990

RESEARCHRELATEDTOTHEREFINEMENENTOFTHEPOULTRYAS
PRODUCTSWITHPROTECTIVEMEMBRANE

LilicaCOCLEA1,AngelaGAVRILA2,D.SIMEANU2,CristinaSIMEANU2
1
SCTabcoCampofrioSATulcea,2USAMVIai

Abstract
Thepresentworkhasthepurposeoftestingthequalityoftherawchickenmeatandthatofone
membrane product manufactured in the framework of SC TabcoCampofrio SA Tulcea. The
analysis carried out has been in the range of organoleptic, physical, chemical and microbiology
tests.Followingthesetestswehavenotedthattherawmaterialsutilizedwereofgoodquality,
thus leading to final products well valued by consumers, especially since all links in the
manufacturingtechnologyhavebeenfollowed.Fortheanalyzedproducthighproteincontenthas
beendetected;atthesametime,fatcontentwaslow.Microbiologically,thestudiedproductdid
not endanger the consumers health. In the reported context, the product manufactured by the
abovementionedcompanyhasenjoyedagoodappreciationofconsumersfromallcategories.For
thefuture,thecompanywheretheresearchwascarriedoutplanstodiversifymorethechicken
meat product range, so as to cover the growing consumer requirements. In the same time the
naturalflavorsrangewillbeextendedforthemanufacturedproducts,soastosatisfyconsumers
requirementsinthisrespectaswell.
Keywords:Viennasausage,products,organoleptic,physical,chemical.

Inthesupplypopulationwithfood,withhighbiologicalvalue,aparticularlyimportantrole
rests to the animal origin products, and among these, meat and meat derivatives are
prevalent. Thus, poultry occupies a special place, whereas it features characteristics that
makesitnotonlyverynutritious,butdietaryaswell.
Concernsforsuperiorrefinementofpoultryasproductsareagrowingconcerninducedby
amoreopenmarketforsuchproducts.
Theareainwhichresearchhasbeencarriedout,NorthDobrogearespectively,theproblem
addressed is very topical, since here is recorded a lesser consumption of chicken meat and
products,duevariousreasonslike:
- lackoftraditioninpoultryconsumption
- limitedrangeofchickenmeatproductsinthegrocerystoresandhighpriceoftheexisting
ones,notmeanttostimulatepublicinterestintheirconsumption
- lackofmeatpoultryfarms,ensuringasupplyofprocessingunitswithrhythmicmaterial
andtodeterminethelowestproductioncostforfinishedproducts
To those shown we believe that our research results will lead to an increase in chicken
meatprocessingintheareaandwillprovideincentivesforbusinesstoinvestinpoultry.

MATERIALSANDMETHODS

ThisworkhasanalyzedtheproductChickenViennasausageinregardswithorganoleptic,
physical, chemical and microbiological characteristics, which allowed us to make a proper
assessmentoftheproductsquality,inconformitywiththequalityoffoodrequiredbycurrent
RomanianlegislationaswellasbyEUregulations.
Organoleptic characteristics were related to: product appearance, color, its consistency
andsmell,respectingtheprovisionsoftheSTAS7031/83.

991

Lucrritiinificevol53seriaMedicinVeterinar
pHvalue
Theacidityisassessedobjectively(byelectrometricmeasurement,usingapHmeterora
ionometer).
EasilyhydrolysableNitrogen
Nitrogenfromaminogroupsisreleasedbyhydrolysiswithaweakbaseandtogetherwith
the free ammonia, is driven by steam distillation in an acidic solution quantitatively and
qualitativelyknown.Excessacidisdeterminedbytitrationwithanequivalentalkalinesolution.
HydrolysableNitrogenderivesbothfromaminogroupsandfreeammonia.Freeammonia
isonlyapartoftheeasilyhydrolysablenitrogen,soitshouldnotbeconfound.
Hydrogensulfide
Hydrogensulfideinthesampletobeanalyzedformsinreactionwiththeleadacetatethe
leadsulfide(darkbrowncompound).
Watertestwasdonebytheovendryingmethod.Principleofthemethodconsistsindrying
thesampletobeanalyzedintheovenatatemperatureof+105oC.
Mineralstest
Todeterminetheash,calcinationsmethodintheelectricalfurnacewasused.Principleof
themethodconsistsincalcinationsofthemeatsampleat+550oCinanoven.
ProteintestwascarriedonusingKjeldhalmethod.
The principle of the method: combinations of organic nitrogen, by heating with
concentratedsulfuricacid,inthepresenceofacatalyst,areconvertedintoammoniumsulfate.
Byaddingastrongbase(NaOH33%),ammoniaisreleased,andthroughdistillation,captured
intoafixedamountofsulfuricacidwithknownnormality.Theexcessacidwastitratedwithan
alkaline solution of same normality, and the difference was the total amount of N fixed.
Chemicalreactionsthatoccurare:

N(protein)+H2SO4(NH4)2SO4

(NH4)2SO4+2NaOH=H2SO4+2NH4OH

2NH4OH+H2SO4=(NH4)2SO4+H2O
Fat test was carried out using Soxhlet method. Principle of the method consists in fat
extractionwithorganicsolvent.
Microbiology
N.T.G.M.A.testisbasedontheinclusionofanamountofmeattobetestedinasuitable
nutrient environment, poured in Petri plates. Following incubation, each group of germs or
eachmicroorganismwilldevelopacolony.
For this determination the following culture environments are used: yeast extract agar
glucosegelatin(Frazier);agartryptoneyeastextractglucose(Platecountagar)
Principleofthemethod.PresenceofSalmonellaisdetectedbyseedingthesamplein an
preenriching nonselective liquid environment, incubation at 37 oC, then seeding in two
enrichingselectiveliquidenvironmentsusingculturesfromthepreenrichingenvironmenton
selective solid media, which after incubation at 37 oC are checked for the presence of
Salmonellasuspectedcoloniesandconfirmedbyidentificationtest.
The main experimental data were statistically processed, calculating: arithmetic average,
variance, average standard deviation, variation coefficient, and the homogeneity of the
experimentalvariancesweretestedbyFishertest(SanduGh.,1995).

RESULTSANDDISCUSSIONS

CharacteristicsofthesortChickenViennasausage
Organolepticcharacteristics

992

UniversitateadetiineAgricoleiMedicinVeterinarIai
ForthefabricationoftheproductChickenViennasausageskinlesschickenbreastand
porklardareused.Therawmaterialisusedchilled,butitcomesbothfromfreshandfrozen
meat.
ChickenViennasausageshavetheappearanceofcylindricalbarswithadiameterof18mm
andalengthof13cm,coatedwithatransparentmembrane,edible.Followingheattreatment
the product color becomes brown. According internal fabrication rules, taste and smell are
pleasant,specifictoproductsboiledandsmoked,enhancedbytheflavorsandspicesusedin
the recipe. Cross cut layout is that of a fine paste of meat, well connected, compact and
uniform, without showing air gaps or agglomerations of fat. In analyzing the product there
werenotnoticeddeviationsfromtheinternalfabricationnormsofthisproduct.
Physicalcharacteristics
pHvalue
pHtestingwascarriedoutona60daysperiod,whichistheshelflifeoftheproduct.Values
obtainedareshownintable1,wherewenoticethedecreaseofthepHvaluefrom6.57,value
obtainedonthefreshproduct,to6.38ontheproductwith30daysconsumedoftheshelflife,
foratitsconclusion,60daysafter,pHvaluetoreach6.22.Calculatedcoefficientsofvariation
havebeenlow,bellow10%.

Table1.pHvaluedynamicsinthestudiedproduct:ChickenViennasausage(n=25)
x sx
V%
Shelflife
Freshproduct
6,570,25 5,34
Productathalfoftheshelflife
6,380,28 4,63
(after30daysstorage)
Productattheendofshelflife
6,220,26 5,42
(after60daysstorage)

Easilyhydrolysablenitrogen
Theresultsacquiredregardingtheeasilyhydrolysablenitrogenarepresentedinthetable
2, these ranging between 19.33 to 24.61 mg NH3/100 g product. The values obtained are
bellowthemaximumallowedbylawlevelof30mgNH3/100gproduct(Ord.560/2007).Low
valuesofthecalculatedvariancecoefficientindicateagoodhomogeneitybetweensamples.

Table2.EasilyhydrolysablenitrogencontentsdynamicsintheproductChickenVienna
sausage(n=25)
x s x (mgNH3/100g) V%
Shelflife
Freshproduct
19,331,05
6,34
Productathalfoftheshelflife
22,310,83
5,21
(after30daysstorage)
Productattheendofshelflife
24,610,72
5,39
(after60daysstorage)

Hydrogensulfide
Thepresenceofthehydrogensulfidewasnotdetectedafterlaboratoryanalysis.
Chemicalcharacteristics
Watercontent
Animportantparameterisanalyzingmeatproductsisthewatercontent.
Intable3arepresentedthevaluesobtainedbylaboratoryanalysisofthewatercontentof
the product Chicken Vienna sausage. One may note that over time the water content

993

Lucrritiinificevol53seriaMedicinVeterinar
decreasesslightly,duetheproductispackagedbyvacuum,theonlylossbeingthosegivenby
juiceexpressionafterpasteurization.The51.76%watercontent,valuerecordedforthefresh
product,endsat51.68%attheendoftheshelflife,themaximum68%watercontent,asitis
legislated, not being exceeded. Coefficients of variance had low values, bellow 10%,
demonstratingthehomogeneityofthestudiedcharacteristics.
Proteincontent
Theboiledandsmokedmeatproductshaveaminimalproteincontentvalueof11%setby
thelegislation.
Inlaboratoryinvestigationsitwasdeterminedthatourproducthasamuchhigherprotein
content,andthatis15.31%forthefreshproduct,15.26%fortheproductathalfoftheshelf
life,after30daysofstorage,and15.19%fortheproductintheexpirationthreshold,asseenin
table 4, exceeding the amount required by law. The variance coefficients calculated for this
parameterwerebellow10%.

Table3.WatercontentofthesortChickenViennasausage(n=25)
x s x (%)
V%
Shelflife
Freshproduct
51,761,25
6,34
Productathalfoftheshelf
life
51,701,16
4,58
(after30daysstorage)
Productattheendofshelf
life
51,681,20
5,34
(after60daysstorage)

Table4.ProteincontentofthesortChickenViennasausage(n=25)
x s x (%)
V%
Shelflife
Freshproduct
15,310,13
6,31
Productathalfoftheshelf
life
15,260,11
5,27
(after30daysstorage)
Productattheendofshelf
life
15,190,12
6,19
(after60daysstorage)

Fatcontent
Maximum allowed fat content for the boiled and smoked meat products, without
structure,asisthecaseinthissituation,is26%(Ord.560/2007).
FortheChickenViennasausages,subjectofourresearch,wehaveobtainedvaluesslightly
bellow the value required, results ranging from 24.53% to 24.6% fat content (table 5). The
variancecoefficientsdidnotexceed10%,whichcorrelatedwithaverygoodhomogeneityof
thestudiedcharacteristic.

994

UniversitateadetiineAgricoleiMedicinVeterinarIai

Table5.FatcontentofthesortChickenViennasausage(n=25)
x s x (%)
Shelflife
V%
Freshproduct
24,530,26
6,21
Productathalfoftheshelflife
24,560,30
5,38
(after30daysstorage)
Productattheendofshelflife
24,600,24
5,49
(after60daysstorage)

Saltcontent
Salt content values obtained were bellow the maximum 3% allowed by legislation, these
oscillating between 1.031.045% (table 6). Calculated variance coefficients were low, which
reflectsthehomogeneityofthestudiedcharacteristic.

Table6.SaltcontentofthesortChickenViennasausage(n=25)
x s x (%)
V%
Shelflife
Freshproduct
1,030,09
5,34
Productathalfoftheshelflife
1,040,08
6,27
(after30daysstorage)
Productattheendofshelflife
1,0450,10
5,43
(after60daysstorage)

Nitritescontent
Nitrites content tests indicated values between 2.732.74 mg NO2/100 g product, data
which may be found in table 7. Compared with outstanding norms, these results fall within
normalvaluesfortheanalyzedparameter.Viewedintermsofvariancecoefficientsvalues,the
resultsarehomogenous.

Table7.NitritescontentofthesortChickenViennasausage(n=25)
x s x (%)
Shelflife
V%
Freshproduct
2,740,11
5,29
Productathalfoftheshelflife(after30daysstorage)
2,730,09
5,81
Productattheendofshelflife(after60daysstorage)
2,730,10
6,02

MicrobiologicalcharacteristicsofthesortChickenViennasausage
Laboratory analysis revealed that the product is fit for consumption, no contamination
withListeriaorSalmonellabeingindicated(table8).

Table8.MicrobiologicalcharacteristicsofthesortChickenViennasausage(n=25)
Salm./25g
List.m./25g
Shelflife
Met.SRENISO
Met.SRISO11290
6579:2003
1:2000
Freshproduct
abs.
abs.
Productathalfoftheshelflife
(after30daysstorage)
abs.
abs.
Productattheendofshelflife
(after60daysstorage)
abs.
abs.

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Lucrritiinificevol53seriaMedicinVeterinar

CONCLUSIONS
From our findings clearly emerges that in our research was used a high quality raw
material, which led to obtain finished goods valued by consumers, especially since all
fabrication technological steps have been followed. All finished products analyzed had high
proteincontent,whilethefatcontentwaslow.Microbiologically,thestudiedproductsdidnot
endangerthehealthofconsumers.Inthecontextreported,theproductsmanufacturedbyus
enjoyedaverygoodappreciationfromallcategoriesofconsumers.
Forthefuture,thecompanywheretheresearchwasperformedaimstofurtherdiversify
itsrangeofchickenmeatproducts,soastocovertheeverincreasingrequirementsof
consumers.Atthesametime,willexpandtherangeofnaturalflavorsfortheproducts
manufacturedsoastomeetthediversifiedrequirementsofconsumersinthisregard.

BIBLIOGRAPHY

***Regulamentul(CE)nr.852/2004alParlamentuluiEuropeanialConsiliuluicuprivirelaigiena
produseloralimentare.
2. ***Regulamentul(CE)nr.853/2004alParlamentuluiEuropeanialConsiliuluidin29aprilie2004.
3. ***Regulamentul(CE)nr.854/2004alParlamentuluiEuropeanialConsiliuluidin29aprilie2004.
4. ***Regulamentul(CE)nr.882/2004alParlamentuluiEuropeanialConsiliuluidin29aprilie2004.
5. ***Publisher:OPOCE;Regulamentul(CE)nr.2073/2005alComisieidin15noiembrie2005.
6. ***Regulamentul(CE)nr.2074/2005alComisieidin5decembrie2005.
7. ***560/1271/339/210/2006Ordinalministruluiagriculturii,pduriloridezvoltriirurale,al
ministruluisntiipublice,alpreedinteluiAutoritiiNaionalepentruProteciaConsumatorilor
ialpreedinteluiAutoritiiSanitareVeterinareipentruSiguranaAlimentelorpentruaprobarea
Normelorcuprivirelacomercializareaproduselordincarne.
8. VacaruOpriI.(coordonator),2000TratatdeAvicultur,vol.I.EdituraCeres,Bucureti.
9. VacaruOpriI.(coordonator),2002TratatdeAvicultur,vol.II.EdituraCeres,Bucureti.
10. VacaruOpriI.(coordonator),2004TratatdeAvicultur,vol.III.EdituraCeres,Bucureti.
11. SanduGh.,1995Metodeexperimentalenzootehnie.EdituraCoralSonivet,Bucureti.
1.

996

INVITROACTIVITYOFSOMENATURALESSENTIALOILSAGAINST
THEYEASTLIKEALGAPROTOTHECA

Bouari(Cuc)Cosmina,Gh.Rpuntean,C.Ctoi,N.Fi,G.Nad,P.Bolf,M.Taulescu,
A.Gal,R.Moussa,A.Nagy,F.Tbran

UniversityofAgriculturalSciencesandVeterinaryMedicineClujNapoca
FacultyofVeterinaryMedicine
Mnturstreet,no35
cosminacuc@yahoo.com
Abstract
ThegoalofthisstudywastodeterminetheinvitrosusceptibilityofProtothecaisolates
to conventional antifungal agents and to essential oils, provide an alternative to
classicaltherapywithnaturalproducts,astrongtendencyinthelastyears.
Twelve Prototheca zopfii isolates from mastitic milk samples and one P. wickerhamii
referent strain (RE4608014 ATCC 16529), from American Type Collectin, were
submittedtoantifungalsusceptibilitytestingbyclassicaldiffusimetricmethod.
ForbothProtothecastrainstested,thehighestefficiencywereregisteredfor:fir,savory
and coconut essential oils. The tested isolates were shown to be resistant to rattle,
marigoldandeucalyptusessentialoils.
Difficulties in treating protothecosis in humans and animals with conventional drugs
andthepotentinvitroactivityofessentialsoilsdemonstratedhereraisetheinterestin
further investigations on the therapeutic use of these nonconventional natural
products.

Keywords:Prototheca,therapy,essentialoils

INTRODUCTION

The genus Prototheca (Trebouxiophyceae, Chlorophyta) includes unicellular


achlorophylous yeastlike microalgae that reproduce by formation of a variable number of
sporangiosporeswithinasporangium(5).ThegenusProtothecawasestablishedbyKrugerin
1894, and five species: Prototheca wickerhamii, Prototheca zopfii, Prototheca stagnora,
ProtothecaulmeaandProtothecablaschkeaehavebeenrecognized(1,7).Inaddition,three
distinctbiotypesofP.zopfiihavebeendefinedonthebasisofphenotypiccharacteristics,and
recentlybymolecularcharacterization(6,7).
Prototheca species are ubiquitous and can be isolated from various environmental
sources,suchasslimefluxoftrees,grass,freshandsaltwater,andwastewater.Apathogenic
potential has been indicated for P. wickerhamii and P. zopfii. Although the former is the
predominantcauseofhumaninfections,thelattercausesinfectionsinanimals,particularlyin
cowsanddogs(2,3).
P. zopfii has been identified as inducing a therapyresistant inflammation of the
mammaryglandindairycowsleadingtoseverelossesinaninfectedherd(1).Thesealgaedo
not respond to routine mastitis therapy and the only control measure to date has been the
eliminationoftheinfectedanimals(3).

997

Lucrritiinificevol53seriaMedicinVeterinar
Recently, the potential antimicrobial effects of essential plant oils have attracted serious
attentionwithinthescientificcommunity.Severalreportshavedocumentedtheefficiencyof
essential oils extracted from various plant species, such as Melaleuca alternifolia (tea tree)
and,morerecently,fromCitrusbergamia(bergamot)(4,8).
The aim of this study was to investigate the in vitro susceptibility of Prototheca isolates to
conventionalantifungalagentsandtoessentialoils.

MATERIALSANDMETHODS

Twelve P. zopfii strains isolated fom mastitic milk samples and one P. wickerhamii
referent strain were used for the study. The isolates were identified on the basis of
microscopic, cultural (growth in glucose broth and on gelose mediums) and biochemical
features (assimilation of glucose, galactose, glycerol, sucrose and trehalose). In vitro
susceptibilitytestingwasperformedbyclassicaldiffusimetricmethod.Eightnaturalextracts:
coconut , polioel 3, eucalyptus (Eucalyptus globules), rattle (Calendula officinalis), marigold
(Hypericum perforatum), fir (Abies alba), aloe (Aloe barbadensis) and savory (Satureja
hortensis) comparative with positive and negative controls (Lincocin forte and distillated
water)weretesteduponbothProtothecaspecies.Twoconcentrationofeachextract(0,1and
0,2%)indistillatewaterwereused.
The vegetal extracts used in the study were plants extracts found in Romania flora
andothercountrieswithsubtropicalclimate.Werepurchasedcommerciallyasrecommended
standardized products to combat various diseases becaused of the bactericidal and
antimycoticpotential.Theseproductshavetheadvantagethatthebactericidaleffecthavea
low environmental impact, the only approved by European Union for use in humans and
animals.
For in vitro testing of these products were used: Petri dishs with glucose agar
medium, wells cut dies, automatic pippets, sterile tips, Prototheca culture in glucose broth
mediumorsuspensioninsalinesolutionperformedfromcoloniesdevelopedonagarsurfaces.
Warkingtechniquewereclassicaldiffusimetricmethodonsolideglucosemedium.

RESULTSANDDISCUSSION

The researches performed in the last period, regarding protothecosis combating


developed function of knowledge level and of technological possibilities. Pharmacological
protocol for therapy of this infection has not been developed yet either in human or in
veterinary medicine, treatement remains a controversial problem. Therefore the establish
therapy,isalongterm,andtheresultseveneffectiveinnitialy,whileprovingthecontraryby
relapses occurring. Basic treatement in this disease remained some drugs which still fairly
wideapplicability.
Almost exclusively drugs therapy utilization (antibiotics and chemiotherapics) has led to the
antibioresistance phenomenon. Very important as finding alternatives to the use of these
products have proven to be harmful to animals and humans by residues accumulation in
animalsproductsandintheenvironment.
Testscarriedoutinthedirectionofestablishingthealgaecideeffectofessentialoils
showed different aspects according to the plant from which it was obtained, extract
concentrationandalsotothespeciesofProtothecatested.
For both Prototheca tested species the best results were registered at extract
concentrationof0.2,the mostefficientprovedto be:fir,savoryandcoconutessentialsoils.

998

UniversitateadetiineAgricoleiMedicinVeterinarIai
Both Prototheca species have shown a reduce sensitivity to aloe vera extract and a total
resistancetorattle,marigold,andeucalyptusessentialoils(fig1and2).

Co
Av

Av

P3
C

P3
C

B
E

Co

E
G

Fig.2.P. wickerhamii vegetal extracts


efficacity: Co coconut, P3 Polioel 3, E eucalyptus, S rattle, G marigold, B fir,
Av aloe, C savory.

Fig.1.P. zopfii (10 strain) vegetal extracts


Co coconut, P3 Polioel 3, E efficacity:

eucalyptus,
S rattle, G marigold, B fir,
Av aloe, C savory.

Table1summarizestheinvitrosusceptibilityprofileoftheProtothecaisolatestothe
plantsextracts intwodifferentconcentrationtested,compareingwithpositiveandnegative
controls.

999

1000

1
2
3
4
P.z
5
6
7
8
9
10
11
12
Averageof
inhibition
area
diameter
(mm)
P.w

Testated
strains

8,33

10

13,25

15

0,1%
8
0
11
9
10
9
10
11
10
0
11
11

0,2%
15
0
18
18
10
15
18
14
18
0
15
18

Aloe

30

0,2%
27
26
24
27
25
25
27
30
27
30
27
30

27,08

18

0,1%
10
15
18
12
10
10
13
15
18
12
18
18

14,08

Fir

0,2%
0
0
0
0
0
0
0
0
0
0
0
0

0,1%
0
0
0
0
0
0
0
0
0
0
0
0

Rattle

0,2%
0
0
0
0
0
0
0
0
0
0
0
0

0,1%
0
0
0
0
0
0
0
0
0
0
0
0

20

21,75

0,2%
20
22
22
23
20
21
24
22
20
22
20
25

10

10,8

0,1%
14
10
18
12
0
14
0
18
12
0
14
18

21

0,2%
20
25
20
20
22
23
21
19
24
25
25
24

22,33

17

19,83

0,1%
17
22
18
20
22
19
20
22
17
18
22
21

Vegetalextractsandconcentrationstestated
Marigold
Coconut
Savory

0,2%
0
0
0
0
0
0
0
0
0
0
0
0

0,1%
0
0
0
0
0
0
0
0
0
0
0
0

Eucalyptus

Vegetalextractsefficacirycomparativewithpositivandnegativecontrols

17

14,25

15

11

Polioel3
0,2%
0,1%
24
22
18
16
20
18
22
20
0
0
24
22
10
0
18
16
15
0
0
0
0
0
20
18

10

12,8

0,2%
10
8
18
0
0
12
20
16
16
20
10
24

10,3

0,1%
8
0
16
0
0
10
17
14
14
17
8
20

+ctrl

Table1

ctrl

0
0
0
0
0
0
0
0
0
0
0
0

Lucrritiinificevol53seriaMedicinVeterinar

UniversitateadetiineAgricoleiMedicinVeterinarIai

In the figure bellow can be observed a graphic representation of the average of inhibition
areas, depending of the concentration used, obtained for each plant extract tested compared with
positivecontrolforbothProtothecaspecies(fig.3,4).

Fig.3,4.Representationofaverageefficacityintermsofessentialoilconcentration,
comparativewithpositivecontrol,onbothProtothecatestedspecies.

FiressentialoilsinbothconcentrationtestedprovedahighestefficiencyforP.wickerhamii,
with a value of average of inhibition area of 30 mm, versus 27 mm for P. zopfii. For both
concentration tested, Prototheca strains showed a higher efficacy compared to conventional drug
usedaspositivecontrol(Lincocin).
Savoryessentialoilas0,2and0,1%concentrationwasmoreefficientforP.zopfiithanforP.
wickerhamii.Forbothspeciesthisextractshowedahigherefficiencycomparedtopositivecontrol.
Thetestedisolateswereshowntoberesistanttorattle,marigoldandeucalyptusessential
oils.
1001

Lucrritiinificevol53seriaMedicinVeterinar
Atherstudywererealisedinordertoselectanewantifungalcompound,ofvegetalorigin.
Was evaluated in vitro the efficiency of the following extracts: Camelia sinensis, Cupressus
sempervirens, Pistacia lentiscus and Glicine soja seeds extracts, upon some strains of Candida
albicans,CriptococcusneoformansandProtothecawickerhamii.AmongthoseCameliasinensis(green
tea) extracts, at MIC of 3004800 g/ml extract solution, which corresponds to a concentration of
1302010 g/ml total poliphenols prouved efficacy upon P. wickerhamii tested strains. Tests
performed in which were used only pure polyphenols present in the extract of C. sinensis showed
that only epicatechin 3O galate (ECG) and epigalocatechin 3O galate (EGCG), prouved anti
Protothecaactivity(10).Asanalternativetousageconventionalantifungals,anotherstudyreported
the efficacy of essentials oils of Maleleuca alternifolia (tea tree) and that of Citrus bergamota
(bergamout) (4,8,9). Plant action is based on these chemical composition, on principals active
components and on synergic activity of all components. Important is that plants, throught there
contentinbioactivesubstancesactsdirectlyonpathogenmicroorganismandonsomephysiological
functions,orindirectlyinthebiologicalprocessesthatmodifiedfavorable,havingpositiveinfluence
on whole body health. Plants act as the antiseptic, antioxidante, antiinflammatory, hepatocytes
stimulating,antimitotics,immunomodulatory,endocrinoregulationandanthelmintic.
Inconclusion,despitetheinvitroactivityofseveralantifungaldrugs,difficultiesintreating
animalswiththeseconventionaldrugsandthepotentinvitroactivityofessentialoilsdemonstrated
here raise the interest in further investigations on the therapeutic use of these nonconventional
naturalproducts.

CONCLUSIONS

The most efficient nonconventional products, for both Prototheca species ware: fir,
savory and coconut essential oil in 0,2% concentration.Those natural products in 0,1 as weel as in
0,2%concentrationshownahigherefficiencythanconventionalantifungaldrugs.
Ontheotherhendrattle,marigoldandeucalypusessentialoilsshowednoeffectonany
Protothecastraintested.

REFERENCES

1.

Buzzini P, Turchetti B, Facelli R et al. 2004, First largescale isolation of Prototheca zopfii from milk
producedbydairyherdsinItaly.Mycopathologia;158:42730.
2. LassFlorlC,MayrA.2007,Humanprotothecosis.ClinMicrobiolRev,20:23042.
3. Marques S, Silva E, Carvalheira J et al. 2006, In vitro antimicrobial susceptibility of Prototheca
wickerhamiiandProtothecazopfiiisolatedfrombovinemastitis.JDairySci;89:42024.
4. MondelloF,DeBernardisF,GirolamoAetal.2003,Invitroandinvivoactivityofteatreeoilagainst
azolesusceptibleandresistanthumanpathogenicyeasts.JAntimicrobChemother;51:12239.
5. PoreRS,1985,Protothecataxonomy.Mycopathologia,90:12939;
6. RoeslerU,MollerA,HenselAetal.2006,DiversitywithinthecurrentalgalspeciesProtothecazopfii:a
proposalfortwoProtothecazopfiigenotypesanddescriptionofanovelspecies,Protothecablaschkeae
sp.nov.IntJSystEvolMicrobiol;56:141925;
7. Roesler U, Scholz H, Hensel A. 2003, Emended phenotypic characterization of Prototheca zopfii: a
proposalforthreebiotypesandstandardsfortheiridentification.IntJSystEvolMicrobiol;53:11959;
8. Romano L, Battaglia F, Masucci L et al. 2005, In vitro activity of bergamot natural essence and
furocoumarinfree and distilled extracts, and their associations with boric acid, against clinical yeast
isolates.JAntimicrobChemother;55:1104.
9. TortoranoA.M.,etal,2008,JournalofAntimicrobialChemotherapy,two:10.1093/jac/dkn107;
10. TurchettiBetal.,2005,PhytotherapyResearch19(1):4449.

1002

ORGANOLEPTICRESEARCHOFSALAMIDURINGTHEVALIDITY
PERIOD(FOODDETERIORATION)

F.M.FOICA ,M.CARPCRARE ,DanaSimonaDRUGOCIU


1
A.I.A.College,araBrseiPrejmerBraov,
2
FacultyofVeterinaryMedicine,Iasi
3
FacultyofVeterinaryMedicine,Bucharest
dr_mikeflavius@yahoo.com

ABSTRACT.Beforeassessingthenutritionalpotential,thatis,checkingiftheproductisnourishing
ornot,theconsumermakesdecisionsbasedonitspsychosensorialeffect,whichbroughtusto
thepresentresearch.Thus,weisolatedseveralmeatproductscoveredinmembrane,whichwere
studiedduringthewholevalidityperiod,theresultsbeingdifferentfromoneproducttoanother
and from one sort to another, with changes that would either shorten the validity period or
extenditforcertainproducts.
Keywords:sensoryanalysis,salami,validity.

The sensory analysis of food products is, basically, as old as mankind, but progress
was made no sooner than the latest three decades, by the intensification of the scientific
researchaimingatobjectivizingit.Thisspecialinterestforthesensoryanalysisoffoodraises
from the progress made in physics, chemistry, microbiology, biochemistry, histology,
technology,merceology,aswellasinotherdomainsofthetechniqueandscience.
The systematic research made during the last decades in the field of sensorics
resultedinarichresourceregardingthewayofapplyingthesensorialanalysisinthecontrol
andqualityassessmentofthefoodproducts.Theinterestintheissuesraisedbythesensorics
is currently very high, which is not at all by chance, as the sensorial properties of food
productsalwaysdrawtheattentionoftheconsumers,whoreactsensitivelyandalsopromptly
toallthebasicchangeswhichaffectcertainproducts,andespeciallythosechangesrelatedto
sensorycharacteristics.
If,formostoftheotherindustrialproducts,qualityischaracterisedbyawelldefined
property or group of physical or chemical properties, in the case of food products quality is
determined by three different essential criteria: innocuity, nutritional value and sensory
quality. Sometimes, food quality is assessed by means of elements such as packaging and
labeling,whichareimportantfortheproductsprotectionandaspect.

MATERIALSANDMETHODS

Weisolatedthreebatchesofproductsfromacompanywhichproducessausagesin
the county of Brasov, a company which occupies an average market segment; the isolation
wasmadeasfollows:
BATCH I fresh salami: pork parizer (thick rosy sausage made of boiled minced
meat),cremwurst,Polishsausagesmadeofchickenbreast
BATCHIIboiledandsmoked:rusticsalami,summersalami,bakedsalami
BATCHIIIairdried:Scelesalami,Chorizosalami,Baciusalami
These products were subject to examinations and sensory assessment based on
scores,andwealsomadeastatisticstudyregardingtheconsumersopinionaboutsausagesin
differentstagesofthevalidityperiod,thatis,atthebeginning,middle,andattheendofthe
validityperiod.

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Lucrritiinificevol53seriaMedicinVeterinar
Thesensorypropertieswillbeexaminedinthefollowingorder:theconditionofthe
transportpackage,outeraspectandshapeoftheproduct,theaspectincrosssection,smell,
tasteandconsistency.
Dimensions are checked immediately after checking the outer aspect and product
shape. In order to check the crosssection aspect, the pieces of meat products are cut
perpendiculartothelongitudinalaxis,eitherinslicesor inpieces,usingeitherathinbladed
andverywellsharpenedknifeorameatcuttingmachine(themachineisnotusedforaspic,
blackpudding,smokedbaconandsausages).
Theorderoftheexaminationisthefollowing:wewillstartwiththelessspicedsorts
(cremwurst, Italian salami, etc.) and we will continue gradually with the more spiced ones
(Agnitasausages,sausageswithcumin,etc.).
Members of the commission are not allowed to exchange opinions, as this might
influencetheresultofthesensoryevaluation.
Atthesensoryevaluationwecouldnotice:
Outeraspect:wholepiecesshowingacleansurface,withoutanyimpuritiesorspots
of mould except for the raw salami or sausages with special noble mould on the surface).
Themembranehastobeneatandwithoutanywrinklesforfreshsemismokedmeatproducts,
it must have wrinkles in the case of semismoked airdried salami (classic summer salami
coveredinnaturalmembrane),itmustadheretothecomposition,andunderthemembrane
thereshouldnotbeanyairholes,meltedfat,juice,larvaeorinsectgalleries.
Crosssection aspect: compact, wellbound composition, with pieces of bacon of
equal size and equally spread throughout the whole composition (for meat products with a
mosaicaspect).Thecompositionmustnothaveairholes,meltedfatblobs,pocketswithjuice
oralbuminprecipitate.
For fresh salami, the composition is juicy, but without letting out liquids when
pressedmoderately.
Forsemismokedsalami,thecompositioniscompact,wellbound,firmandelastic.
For products which last longer, the composition is relatively hard, firm and
homogeneous.
Colour: on the exterior, the colour is specific according to the product and to the
technological process used, as well as to the type of membrane (natural, synthetic or semi
synthetic the last two being able tobecoloured and imprinted). On thecrosssection, the
colourmustbehomogeneous,specifictothesort,withoutareasshowingamodifiedcolour.
Thepiecesofbaconareeitherwhiteorpink,withouthavingagrey,greenoryellowhue.The
semismokedproductsandproductswhichlastlongerhaveareddish,homogeneouscolouron
thecrosssection,withoutanydarkerhueonthemarginsoranyotherchangesinthecolour
towardsthemiddle.
Smell and taste: characteristic to the sort, pleasant, moderately salted or spiced,
withoutnochangedorimpropersmellortaste.
Thesensoryqualityofmeatproductsisgenerallyassessedona110scale,thefirst
fivestepsofthescalerepresentingproductsshowingpositivequalities.(Banuetal.,2007).
Theproductslabelledexceptionalandverygood(grades9and8)areincludedin
the superior category; the products labelled good, better than average or average
(grades7,6and5)areincludedinthe1stcategory.(Banuetal.,2007).
Theareareferingtotheunsatisfactorygraderepresentslessthan20%ofthewhole
(Tableno.2).

1004

UniversitateadetiineAgricoleiMedicinVeterinarIai
Thesensoryanalysisiscompletedwiththephysicochemicalanalysisfortheintegrity
assessment:water,fat,proteins,sodiumchloride,nitrates/nitrites,polyphosphates,including
starch,vegetalproteinsorcollagenproteins.
These evaluations are necessary in order to check if the product observes the
standardsimposedbythecompanyorbranchregardingtherecipes,technologicalprocessor
finalproductparameters.

RESULTS

SensorycharacteristicsofthePORKPARIZER
Validity period: 20 days (stored at 04C and relative humidity of air between 75
80%)givenbytheproducer;itobtainedthefollowingscore:Tableno.1

Tableno.1Valabilityperiodscore
Organolepticproperties
12.08.2009 22.08.2009 01.09.2009 11.09.2009
Outeraspect
8
7
7
7
Colourthroughthesection
6
6
6
5
Flavour
7
6
6
5
Taste
6
6
5
4
Consistency
5
7
6
6
Juiciness
7
7
7
7
Overallassessmentofquality
5
5
5
4
Total
44
44
42
38
Score
6.28
6.28
6
5.42

9
8
7
6

12.08.2009

22.08.2009

01.09.2009
11.09.2009

3
2
1

us
t
G

As
pe

ct
ul
e

xt
er
io

Fig.2.Porkparizercrosssection
duringdifferentstagesofthevalidityperiod

Fig. 1 - Graphic representation of the


organoleptic changes undergone during
different stages of the validity period.

SensorycharacteristicsofthePOLISHSAUSAGESMADEOFCHICKENBREAST
Validity period: 10 days (stored at 04C and relative humidity of air between 75
80%) given by the producer. As a result of the score it obtained, we noticed the changes
presentedinfig.3.

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Lucrritiinificevol53seriaMedicinVeterinar
9
8
7
6

12.08.2009

17.08.2009

22.08.2009
27.08.2009

3
2
1

Fig. 3 - Graphic representation of


the organoleptic changes
undergone during different stages
of the validity period.

G
us
t

As
pe
ct
ul
ex
te
r io

Fig.4.Polishsausagesouteraspect.

SensorycharacteristicsofCREMWURST
Validity period: 10 days (stored at 04C and relative humidity of air between 75
80%)givenbytheproducer;itobtainedthefollowingscore:fig.5
9
8
7
6

10.08.2009

15.08.2009

22.08.2009

Fig. 5 - Graphic representation of


the organoleptic changes
undergone during different stages
of the validity period.

27.08.2009

3
2
1

G
us
t

As
pe
ct
ul
ex
te
r io

1006

UniversitateadetiineAgricoleiMedicinVeterinarIai

Fig.6.Cremwurstouteraspect.

SensorycharacteristicsoftheSUMMERSALAMI
Validityperiod:15days(storedat1012Candrelativehumidityofairbetween75
80%)givenbytheproducer;itobtainedthefollowingscore:fig.7.
10
9
8
7
10.08.2009

17.08.2009

25.08.2009

31.08.2009

Fig. 7 - Graphic representation of the


organoleptic changes undergone during
different stages of the validity period.

2
1

G
us
t

As
pe
ct
ul
ex
te
r io

Fig.8.Summersalamiouteraspect.

SensorycharacteristicsoftheRUSTICSALAMI
Validityperiod:15days(storedat1012Candrelativehumidityofairbetween75
80%)givenbytheproducer;itobtainedthefollowingscore:fig.9.

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Lucrritiinificevol53seriaMedicinVeterinar
10
9
8
7
12.08.2009

17.08.2009

27.08.2009

03.09.2009

3
2
1

Fig. 9 - Graphic representation of the


organoleptic changes undergone during
different stages of the validity period.

G
us
t

As
pe
ct
ul
ex
te
r io

Fig.10Rusticsalamiouteraspect.

SensorycharacteristicsoftheBAKEDSALAMI
Validityperiod:15days(storedat1012Candrelativehumidityofairbetween75
80%)givenbytheproducer;itobtainedthefollowingscore:fig.11.
8

Fig. 11 - Graphic representation of the


organoleptic changes undergone during
different stages of the validity period.

7
6
12.08.2009

17.08.2009

27.08.2009

03.09.2009

2
1

us
t
G

As
pe
ct
ul
ex
te
r io

SensorycharacteristicsoftheBACIUSALAMI
Validityperiod:45days(storedat1012Candrelativehumidityofairbetween75
80%)givenbytheproducer;itobtainedthefollowingscore:fig.12.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
10
9
8
7
10.07.2009

10.08.2009

24.08.2009

03.09.2009

Fig. 12 - Graphic representation of the


organoleptic changes undergone during
different stages of the validity period.

2
1

us
t
G

As
pe
ct
ul
ex
te
r io

SensorycharacteristicsoftheCHORIZOSALAMI
Validityperiod:45days(storedat1012Candrelativehumidityofairbetween75
80%)givenbytheproducer;itobtainedthefollowingscore:fig.13.
9
8
7
6

12.08.2009

17.08.2009

22.08.2009
27.08.2009

3
2
1

Fig. 13 - Graphic representation of


the organoleptic changes
undergone during different stages
of the validity period.

G
us
t

A
sp
ec
tu
le
xt
er

io
r

SensorycharacteristicsoftheSCELESALAMI
Validityperiod:45days(storedat1012Candrelativehumidityofairbetween75
80%)givenbytheproducer;itobtainedthefollowingscore:fig.14.
10
9
8
7
12.08.2009

17.08.2009

22.08.2009

27.08.2009

3
2
1

Fig. 14 - Graphic representation of the


organoleptic changes undergone during
different stages of the validity period.

G
us
t

As
pe
ct
ul
ex
te
r io

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Lucrritiinificevol53seriaMedicinVeterinar
CONCLUSIONS

1.Thebestscoreatthecategoryoffreshmeatwasobtainedbytheparizer,inwhosecase
thevalidityperiodcanbeextendedatleast5days.
2. The cremwurst and Polish sausages showed visible modifications and, therefore, had a
lower score before the end of the validity period, which results in a 4day decrease of the
validityperiod(40%).
3. The baked salami was the one which best kept its organoleptic properties within the
smokedproducts,whereasthelowestscorewasobtainedbyrusticandsummersalami.
4. Raw salamibest preserved thei sensory characteristics, with a score thatwould allowthe
extensionofthevalidityperiodwithapproximately2025%.
5. The results of the sensory analysis of the products quality characteristics must be
correlated to the results of the physicochemical and microbiological analysis, in order to
objectivizethequalitycontrolasawhole.

BIBLIOGRAPHY

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.

ApostuS.,2006Microbiologiaproduseloralimentare,vol.I,volII,volIIIEdituraRisoprint,Cluj
Napoca.
ApostuS.,2004ManagementulCalitiiAlimentelor,EdituraRisoprint,ClujNapoca.
ApostuS.,NaghiuA.,2008Analizasenzorialaaalimentelor,EdituraRisoprint,ClujNapoca.
Advisory Comituitte on the Microbiological Saftey of Food, 2004 Second raport on
Campylobacter,London.
BanuC.,2006Biochimia,microbiologiaiparazitologiacrnii,EdituraAgir,Bucureti.
BanuC.,2007Suveranitate,securitateisiguranalimentar,EdituraASAB,Bucureti.
BrzoiD.,MeicaS.,NeguM.,1999Toxiinfeciilealimentare,EdituraDiaconCoresi,Bucurei.
BrdaanG.,2007Siguranaalimentelor,EdituraIonIonescudelaBrad,Iai.
Buiuc D., Negu M., 2008 Tratat de microbiologie clinic, Ediia a II a, Editura Medical,
Bucureti.
CarpCrareM.,2001Microbiologieveterinar,EdituraVenus,Iai.
Chira A., 2005 Sistemul de management al siguranei alimentului conform principiilor HACCP,
EdituraConteca`94,Bucureti.
DanV.,2001MicrobiologiaAlimentelor,EdituraAlma,Galai.
DanV.,BahrimG.,NicolauA.,2002Microbiologiaproduseloralimentare,ManualulInginerului
nindustiealimentar,volII,p13184,EdituraTehnicBucureti.
FoodSafetyInformation,2006CampylobacterQuestionsandAnswers.
GuerreroI.,ChabelaL.P.,1999Spoilageofcookedmeatsandmeatproducts,Encyclopediaof
FoodMicrobiology,London.
Mencinicopschi G., Cironeanu I., 2006 Produse Romneti din carne, conform cerinelor UE,
procesare,control,retee,EdituraAltPressTour,Bucureti.
MitreaS.I.,2003SiguranaalimentelorprinaplicareasistemuluiHACCP,EdituraBogdana.
SavuC.,2008Igienaicontrolulproduselordeorigineanimal,EdituraSemne,Bucureti.
SavuC.,GeorgescuN.,2004Siguranaalimentelor,EdituraSemne,Bucureti.
JayJ.M.,1998ModernFoodmicrobiology,Arnold,London.
Jay J.M., 2000 Modern Food Microbiology An Aspen Publication, Aspen Publishers, Int
Gaitherburg,Maryland.

1010


RESEARCHCONCERNINGDIARYCOWSWELFARE
INAFARMNEARBUCHAREST

F.Furnaris1,ElenaMitranescu1,L.Tudor1,VioletaSimion2,
C.Togan3,B.Tasbac1,S.Bustani1
1)UniversityofAgronomicalSciencesandVeterinaryMedicine,FacultyofVeterinary
Medicine,105Spl.IndependenteiStreet,Bucharest,Romania;ffurnaris@gmail.com
2)SpiruHaretUniversityBucharest,FacultyofVeterinaryMedicine
3)NationalSanitaryVeterinaryandFoodSafetyAgency

Abstract
Agoodanimalwelfarecouldnotbeconceivedwithoutcontinuemonitoring,basedonpractical,
numericaltools.ThepresentstudyaimstoassessthewelfareofdiarycowsfromHolsteinFrisian
breedinafarmnearBucharestduringMayJune2008period.
As in Romania there an official numeric system for the assessment of animal welfare does not
exist yet, permitting comparisons between different situations and farms, we used the ANI 35L
2000version,theAustrianofficialsystem.Thissystemhadinviewbothanimalbasedparameters
(behavioral) and engineering based parameters (housing conditions, paddock quality,
microclimate).Thereareassessedfiveareasofinfluence:locomotion,socialinteraction,flooring,
light and air, and stockmanship. Points are awarded within each area of influence for several
parameters using five assessment sheets. The researches were done based on the direct
measurements,healthrecordsorusingspecificequipmentastheDragerMiniwarnelectronicgas
meter (CO2 and NH3 analysis) or the Hill catathermometer (draught intensity). The sum of all
pointsforthefiveareasofinfluencegivestheoverallANIscore.Theobtainedfinalscorewas19
points, which indicates a poor welfare. In order to improve cows welfare in the farm up to an
acceptablelevelmeasuresmustbetakenreferringtothecriticalpointsofhousingcondition.

Keywords:diarycows,welfare,ANI35L,gaitscore,hygiene

Ensuringahighlevelofanimalwelfareisnotjustawaytoincreasetheproductionbut
alsoamoraldutyofmanregardingtheanimals.
Defined by Broom as the state of individual as regards its attempts to cope with its
environment, the animal welfare is an issue more and more approached by general public
and world associations and organizations, mainly veterinary, but also in other field of
activities.
AfterjoiningtheEuropeanUnion,inRomaniatheconsumersofanimaloriginproducts
became more interested in raising welfare level of farm animals, resulting in an increased
number of laws concerning this matter. However, in our country there is not used yet a
nationalpracticalsysteminordertoassessthelevelofanimalwelfareatfarmlevel,asystem
permitting comparisons between different situations and farms or to rate farms or shelters
based on livestock welfare. Therefore, in our researches, we used an Austrian integrative
numericsystemofanimalwelfareassessment,respectivelytheAnimalNeedsIndex(ANI)35L.
This system could be applied in our country, because it covers all infield rearing systems
possible: with or without tether system, and all the shelter types: open or closed, with or
withoutpaddockandwithdifferenttypesoffeeding,watering,lightingfacilities.

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Lucrritiinificevol53seriaMedicinVeterinar
MATERIALANDMETHODS

TheresearchesaimedtoassessdiarycowswelfarebasedonANI35Lsystemsinafarm
nearBucharestduringMayJune2008.Thesystemisimportantbyitsintegrativecharacter,
gathering animal based parameters (behavioral) and engineering based factors (housing
conditions,paddockquality,microclimateetc).
According ANI system, the welfare level was assessed on the basis of five areas of
influence: locomotion, socialinteraction, flooring, light and air, stockmanship each area of
influencecontainingseveralfactorswhichareawardedbycomparisonwithreferencevalues,
usingthe5systemsheets.Thesumofallpointsawardedinthefiveareasofinfluencegives
theoverallANIscore,whichindicatesthewelfarelevelofthediarycows.
Theassessmentwasdoneinoneofthefourshedsofthefarm,inwhichwashouseda
numberof168animalsfromHolsteinFrisianbreed(235AnimalWeightUnits=168animalsx
1.4 multiplier), with an average weight of 540 kg, an average body length of 1.95 m and an
average height at withers of 1,55 m. The shed have 72.65 m length and 15.5 m width.
Concerning space internal structure, it is organized in 4 rows of stalls with vertical tether
system. It is used the mixed lighting system, through the 96 windows of 1.2 m/0.85 m,
respectively55m2shedsurfaceand38lightbulbsof100W.Theventilationisnatural,madeby
20airoutletopeningsof30/25cmand4airinletopeningsof50/60cm.
Theresearcheswereconductedusingthefollowingmethods:metricmeasurementsfor
the calculation of available floor area, light index or other parameters; processing animal
healthrecords;specificequipment(DragerMiniwarngasmeterforassessingtheCO2andNH3
levelintheair,Hillcatathermometerformeasuringdraughtvelocity).Inaddition,inorderto
improve the system's methodology (to make more it objective), we used for assessing the
cleanliness ofanimalsamethodproposedbyN.B.Cook(2002) andforconditionsof hooves
thegaitscoringmodifiedbyN.B.Cook(2005).

RESULTSANDDISCUSSIONS

TheresultsarepresentedinTableno.1.
Table1
TheANIoverallscorecalculation
Areaof
influence

Factors
a

I.Loco
motion

Space
allowance
2
(m /AWU)

Lying
down,
lyingand
rising

Stallsizeand
boundaries

Movement
ofthe
tether

Outdoor
yards
access
(days/year)

Pasture
access
(days/year)

Points
awarded

2,5

II.Social
interaction

Space
allowance
2
(m /AWU)

Herd
structure

The
management
oftheyouth

Accessto
outdoor
yards
(days/year)

Accessto
pastures
(days/year)

Points
awarded

2,5

1012

Score
/areaof
influence

2,5

2,5

UniversitateadetiineAgricoleiMedicinVeterinarIai

III.
Flooring

Softnessin
thelying
area

Cleanliness
inthelying
area

Degreeof
slipperiness
inlyingarea

Thequality
ofthe
activity
areaand
passage
ways

Points
awarded

2,5

IV.Light
andair

Daylight

Airquality

Draughtsin
lyingarea

Noise

Points
awarded

0,5

0,5

0,5

V.Stock
manship

cleanliness
of
pens/feeding
/drinking
areas

technical
condition
of
equipment

Points
awarded

0,5

0,5

Outdoor
yardstype
andquality

Pasturetype
andquality

1,5

Accesstooutdoorareas

Days/year

Average
hours/day

conditionof
integument

cleanliness
ofanimals

conditions
ofhooves

technopathies

animal
health

0,5

0,5

0,5

OVERALLANISCORE

5,5

3,5

1
19

Withinthefirstareaofinfluencelocomotionthereareapproached3factors:stall
size and boundaries, movement of the tether and outdoor yards access (days/year). There
could not be approached neither the factors referring strictly to loose housing system: the
space allowance (m2/AWU) and lying down and rising, nor the pasture access (days/year)
becausethecowshavenotaccesstopastures.Stallsizeandboundariesfactorisscoredwith0
pointsstallsbeingratedasrestrictive,becauseareshort(1.8x1.2m)andhavehighandrigid
walls.Movementofthetetherfactorisscoredwith0pointsthebackandforthmovement
allowance being smaller than 0.4 m and the lateral movement smaller than 0.3 m. Outdoor
yardsaccess(days/year)factorisscoredwith2.5pointstheanimalsaccessingthepaddocks
230240days/yearandthepaddockproviding12.37m2/cow.Thus,thescoreforthefirstarea
ofinfluenceisthesumforthe3factorsabove,respectively2.5points.
Within the second area of influence social interaction there are approached 3
factors: space allowance (m2/AWU), herd structure and access to outdoor yards (days/year).
Spaceallowancefactorisscoredwith0points,beingunderthevalueof6m2,respectively3
m2 (1.8 m x 1.2 m stall surface added with manger surface of 1.2 m x 0.7 m). For the herd
structure the score is 0 points, cows being reared in tether system and the changes of stall
allocationarenotfrequent.Outdooryardsaccess(days/year)factorisscoredwith2.5points,
similarwiththescoringwithinthefirstareaofinfluence:locomotion.Thescoreforthesecond
areaofinfluenceis2.5points.
Withinthethirdareaofinfluenceflooringthereareapproached5factors:softness
inthelyingarea,cleanlinessinthelyingarea,degreeofslipperinessinlyingarea,thequality
oftheactivityareaandpassageways andoutdooryardstypeandquality.The firstfactor is
scored with 2.5 points, in the assessed house being used as litter a layer of straw thicker
than 60mm.Forthecleanlinessinthelyingareathescoreis0point(dirtyfloors)andforthe
degree of slipperiness 1 point. For establishing the quality of the activity area and passage
waystherewasobservedthefloorofpassagewaystotheoutsideareas.Becausethefloorof
theseareaarefromconcrete,arigidmaterial,withalowthermalinsulationfactorandwhich
could generates sliding (the rate of lameness was high in the assessed farm: 2022%), the

1013

Lucrritiinificevol53seriaMedicinVeterinar
scoreis0points.Outdooryardstypeandqualityfactorisscoredwith1.5points.Thescorefor
thethirdareaofinfluenceis5points.
Withintheforthareaofinfluencelightandairthereareapproached:daylight,air
quality, draughts in lying area, noise, access to outdoor area in days/year and access to
outdoor areas in hours/day. For the first factor the score is 0.5 points because the shed is
closedandthepercentageofwindowarearelativetofloorareais10%(lightindex1/10).Air
quality is sufficient and it is scored with 0.5 points: the CO2 concentration being lower than
0.2% and the average concentration of NH3 is 14 ppm. The draughts in lying area factor is
scored0.5points,beingregisteredseldomexceedingoftheadmittedlimits(averagespeed
measuredwithHillcatathermometeris1.05m/s).Forthefactornoisethescoreis1point,the
shedbeingnaturalventilated.Theaccesstooutdoorareasindays/yearisscoredwith2points
(230240days)andtheaccesstooutdoorsareainhours/dayswith2points(ameanvalueof
8.5hoursdaily).Thescorefortheforthareaofinfluenceis5.5points.
Within the fifth area of influence stockmanship there are approached 7 factors:
cleanliness of pens/feeding /drinking areas, technical condition of equipment, condition of
integument,cleanlinessofanimals,conditionsofhooves,technopatiesandanimalhealth.The
first factor gets 0.5 points, the hygiene being medium. For the technical condition of
equipment, the score is 0.5 points due to droppings collecting system malfunction. The
condition of integument is good, being observed minor skin lesions in less than 10% of the
stock,sothescoreis0.5points.Thecleanlinessoftheanimalswasassessedusingthemethod
proposedbyN.B.Cookin2002(figure1),thescorebeing0.5points(good).
Conditionsofhooveswasassessedbasedongaitscore(figure2)receiving0.5points
(mediumcondition).Thetehnopatiesincidenceislowerthan15%andthelesionsareminor
sotheANI35Lfactorisscoredwith0.5points.

Figure1Bodyhygienescoringincows(N.B.Cook,2002)

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Figure2Gaitscoring,adaptedbyN.B.Cook(2005)

Theanimalhealthisqualifiedasgoodsoitreceives1point,basedonthelowmortality
(0.8%respectively5cowsfromatotalof623cows)andgoodreproductiveindicators(table
2).
Table2
ReproductiveindicatorsJanuaryJune2008

Co
Hei
T
ws
fers
otal
Totalnumberofcows
480
14
6
3
23
Gestantcowsafterfirst
198
85
2
insemination
74
Obtainedcalves
120
81
2
01
Totalofgestationloss
32
19
5
1

The overall ANI score is of 19 points, showing that the diary cows welfare in the
studiedshedispoor(overallscorebetween16and20).

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Lucrritiinificevol53seriaMedicinVeterinar
CONCLUSIONS

1. The diary cows welfare in the assessed farm is poor, the final ANI 35L score being 19
points.
2. Thecritical pointsofthehousing conditionsarethefollowing: thespatialrestriction, the
useoftethersystem,thelackofpastureaccess,theincreasedvalueofdraughtsintheresting
area, the poor floor hygiene, the equipment malfunction and the slipperiness of the activity
areaandpassageways.
3. In order to get the cows welfare to an acceptable level need to be taken immediate
actionsregardingthecriticalpointsabove.

BIBLIOGRAFY

1.
2.
3.

4.

Bartussek,H.DerTiergerechteitsindex.Berichtberdie8.IGNTagungvom22.24.2.1990an
derLFSSchlierbach,BALGumpenstein,Irdning,p.3436,1990
Broom, D. M. Animal wellfare defined in term of attemptsto cope withthe environment, Acta
Agric.Scand,Sect.A,AnimalSci.Supplementum,no.27/1996:2228
NigelB.Cook,DouglasJ.ReinemannAtoolboxforassessingcow,udderandteathygiene.Pages
3143inProc.Natl.MastitisCounc.Annu.Mtg.Natl.MastitisCounc.Inc.,Verona,WI.AToolBox
forAssessingCow,UdderandTeatHygiene,2007
TeusdeaV.Bunastareasiprotectiaanimalelor,EdituraOmegaPrint,Bucuresti,2005

1016

RESEARCHESREGARDINGAIRQUALITYINNEAMTCOUNTY

F.Furnaris1,ElenaMitranescu1,CatalinaLala2,L.Tudor1,
MagdalenaGoncearov1,Marianaerbea3,L.I.Ilie1
1
UniversityofAgronomicalSciencesandVeterinaryMedicine,FacultyofVeterinary
Medicine,105Spl.IndependenteiStreet,Bucharest,Romania;ffurnaris@gmail.com
2)
RocheRomania.SRL
3
NationalSanitaryVeterinaryandFoodSafetyAgency

Abstract
InanareaoftheoldprovinceofMoldavia, specialbyitsparks andreservations,astudyonair
quality was made. There were assessed gaseous pollutants, heavy metals by spectroscopy,
sedimenting air particulates and noise intensity. The assessments were made for gaseous
pollutantsbyusingthecolorimetermethod,forheavymetalsbyatomicabsorptionspectroscopy,
for sedimenting air particulates by the sedimentation method, and for noise intensity by
sonometer.
Following the study, air quality in Neamt area ranged within the admitted limits in terms of
concentration of gaseous pollutants, sedimenting particulates quantity and concentration of
heavy metals, but noise level was exceeded by 17% in schools, nurseries, kindergartens and by
100%inparksandrailwayareas.

Keywords:air,gaseouspollutants,particulates,noise,heavymetals

INTRODUCTION

In our country environmental protection is legislated, being a matter of national


interest. Accumulation of pollutants in the environment will break the ecological balance,
degradingtheprimordialfactorsoflife:air,water,soil,throughwhichplantsandanimalsare
affected,and,directlyorindirectly,mantoo.
Nowadays, theconceptofenvironmentaldamageisno longerconfinedtogermsand
potentiallyharmfulclimaticfactors,butdamageoccursalsobythermalandsoundpollution,
vibrations,photonicradiationsetc,generatedbyelementswhoseabsenceisinconceivablein
somestageofindustrialandeconomicdevelopment.
The area had in view in the above study (Neamt) is special by its rich vegetation,
national parks (Ceahlau and Hasmas Cheile Bicazului, Vanatori national park), 20 nature
reserves,6naturalmonumentsand2avifaunalspecialprotectionareas,beingverytransited
bybothforeignandRomaniantourists.

MATERIALSANDMETHODS

In order to establish air quality in Neamt there were determined main gaseous
pollutants(NO2,SO2,andNH3),heavymetals(Pb,Cd,Zn,Mn,Cr,Cu,Ni,Co),sedimentingair
particulatesandnoiseintensity.
Gaseous pollutants were established in five control points on a variable number of
samples,from183to204,dependingonthesamplingpoint.
Forthe assessmentofheavymetalsintheairinNeamt area, samples werecollected
fromeightpointsandsedimentingairparticulatesweremeasuredinfivecontrolpoints.

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Lucrritiinificevol53seriaMedicinVeterinar
Fornoiseintensitydeterminationsweremadeinmarkets,retails,outdoorrestaurants,
schools, nurseries, kindergartens, playgrounds for children, parks, recreation areas and
railways.
Gaseous air pollutants were assessed by classical colorimetric methods and heavy
metalsbyatomicabsorptionspectroscopymethod.
Sedimending air particulates were assessed by sedimentation method, and for the
noiseintensityleveltherewasusedBK2250sonometer.
Resultsinterpretationwasdoneaccordingtoregulationsinforce.

RESULTSANDDISCUSSIONS
AverageconcentrationsofgaseousairpollutantsintheareaareshowninTable1.

Table1
AverageannualvaluesofgaseousairpollutantsPiatraNeamtcity
Average
Maximumadmittedlimit
Sampling
Thepollutant No.of
annual
accordingtoOrder
Stationtype
point
type
samples concentration
592/2002andSTAS
(g/m3)
12574/87(g/m3)
Wheather
station

NO2
Background
SO2
station
NH3
NO2
Traffic
station
SO2

202
202
202
203

17,1521
6,8338
27,7798
20,9723

100
250
100
100

203

5,1647

250

NO2
SO2
NH3
NO2
SO2
NH3
NO2
SO2

204
204
204
183
183
183
201
201

10,9611
5,8833
42,1671
7,6502
4,7192
40,9707
16,4097
4,9660

100
250
100
100
250
100
100
250

Dumbrava
Rosie
Fuelstation
Savinesti
Industrial
Center
station
crossroad
Savinesti
Socin

Industrial
station

Turturesti

Industrial
station

Analyzing the data from the table it is noticed that there were no exceedings of the
admittedlimitsrecordedforanygaseouspollutantinanypointofthefivecheckpoints.
InTable2areshowntheannualaveragevaluesofheavymetalsinairfromNeamtarea.
Therewereassessedtheconcentrationsoflead,cadmium,zinc,manganese,chromium,
copper,nickelandcobaltin8controlpointsfromNeamtcounty,respectively:PiatraNeamt,
Savinesti,Roznov,Turturesti,Roman,Bicaz,TascaandViisoara.
Analyzing the values in the table, it can be noticed that there was not registered
averagevaluesovertheadmittedlimitinanysamplingpointforanyassessedelement.
Doing a comparative analysis between sampling points and the assessed elements
concentrationsfindsthat,intermsofconcentrationofPb,thehighestannualaveragevalues
wererecordedinSavinesti,cadmiuminRoman,zincinTasca,manganeseinTasca,chromein
Tasca,copperinSavinesti,nickelandcobaltinTasca.
Sedimenting air particulates average values determined in five points (Piatra Neamt,
Bicaz,Tasca,TarguNeamt,Roman)arepresentedinTable3.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Table2

Area
PIATRA
NEAM

AverageannualvaluesofairheavymetalsPiatraNeamtcity
Averageannualconcentrations(ppm)
Cadmiu
Mangane Chrom Coppe Nicke
Lead
m
Zinc
se
e
r
l

Cobal
t

19.94
16.580 77.315 5
193.06 29.64
21.310 7
5
104.03 28.42
38.660 5
0
109.24 48.50
46.770 0
0
141.81 45.48
25.146 3
4
111.21 33.87
39.250 4
3
122.90 67.48
78.847 5
1
39.03
32.125 85.223 0

15.16
1
20.85
5
17.31
0
15.45
0
24.85
5
12.78
2
41.91
6
16.51
0

980.558 441.915

SVINETI

76.636 6.991
138.30
0
2.817

ROZNOV

84.245 1.575

701.750 416.200

TURTURESTI

99.133 0.356

ROMAN

93.511 4.100

BICAZ

80.028 0.754
104.12
2
0.798

769.633
1250.90
3
1159.43
4
1673.72
9

74.730 1.835

847.900 576.600

TACA
VIIOARA

887.067 622.900

326.500
536.147
500.788
759.790

Table3
AverageannualvaluesofsedimantingairparticulatesPiatraNeamtcity
No.of
Averagevalues
City
Samplingpoint
sample
g/m2/30days
PiatraNeam

Bicaz

Taca

TrguNeam

Roman

19Noiembrie Street
Solidaritatii Street
Ciocarliei Street
Mrceni
Dodeni
House
Housefamily1
Housefamily2
Housefamily3
Mreti Street
MihaiEminescu
AleeaCastanilor
Hospital
Healthcenter 6
Republicii Boulevard
Schoolno.8
Maximumadmittedlimit

1019

34

36

36

27

48

6
6
7
8
7
11
7
12
13
8
10
13
16
15
7
7
17

Lucrritiinificevol53seriaMedicinVeterinar
Analyzing theobtainedresultsfortheconcentrationofsedimentingairparticulatesit
canbenoticedthatinallsamplingpoints,valueswerebelowthemaximumadmittedlimitof
17g/m2/30days.
Valuesclosetothemaximumadmittedlimitwererecordedinthehospitalsandhealth
centersfromRomansamplingpoint.
Noise intensity was determined in open spaces, schools, nurseries, kindergartens and
railwaysareas.TheaveragesvaluesareshowninTable4.
Table4
AverageannualvaluesofnoiseintensityNeamtarea
No.ofassessments
Measuredvalues(db) %Exceeding
Atthe
Atthe
Atthe
Samplingpoint
Interio
Interio
functional
functional
Interior functional
r
r
areaslimit
areaslimit
areaslimit
Markets,retails,
5
3
63,6
67,5
0
0
outdoor
restaurants
Schools,
6
4
75,3
68,2
17
25
nurseries,
kindergartens,
playgroundsfor
children
Parks,recreation 20
8
78,8
64,4
100
25
areas
Railways
48
79,1
100

Analyzingdatafromthetabletherearefoundtobecriticalpointswheretheintensity
noiseexceededadmittedlimitsby17%inschools,kindergartens,playgroundsforchildrenand
by100%inparks,recreationandleisurearea,aswellasinrailwayarea.

CONCLUSIONS

1. Air quality in terms of the gaseous pollutants, the amounts of heavy metals and
sedimentingairparticulatesrangewithintheadmittedlimits.
2. Noise intensity is exceeded in parks and railway areas by 100%, and in schools,
nurseries,kindergartensby17%.

REFERENCES

1. Marcal,W.S.,Nascimento,L.Do.andothersCattleasbioindicatorofenvironmentalpollution,
AHoraVeterinaria22,(129),5356,PortoAlegreBrasil,2002
2. MitranescuElena,TapaloagaDana,Tapaloaga,R.P., Simion VioletaThe impactof industrial
pollution on the air quality in the SouthEastern part of Romania, Haikou ichuk HABM
imehiC.3.UKOSOtom8no,2(29),acmua4,Ucraina,2006,pp.231234
3. Teusdea,V.,ElenaMitranescuProtectiamediului,IVtheditions,OmegaPrintPublishing,2007
4. ***LawoftheEnvironmentLaw137/1995

1020


RESEARCHONAVIANCOLIBACILLOSIS
CONDITIONSININTENSIVEREARING

DianaGalaanu1,T.Perianu2,OanaTnase2
1
MorandiCom.LipovVaslui,2FMVIai
diana.galatanu@gmail.com

Colibacilozele birds are contagious infectious disease that causes economic losses in affected
flocks. They are produced by E. coli, are frequently encountered and superimposed infections
withotherbacteriaand/orotherviruses.
Diseasewasstudiedinaflockof22000chickensbreedIssaBrown.Thepercentageofmorbidity
was CREC and the mortality reached 6.3%. Following isolation of 51 strains of E. coli and
performingantibiogramhasbeenestablishedforfivedaystreatmentwithfloron,resultinginlow
mortalityrate.
Losseswerenotsignificantchicksreachedmaturityattheageofproductiveperformance.

Keywords:colibacillosis,E.coli,intensiverearing,sistem,poultry.

Aviancolibacillosisisaseriousproblemforthepoultryindustryisoneofthemaincauses
of morbidity, and mortality and birds. Pathogenic strains of Escherichia coli in poultry may
cause aerosaculit, omfalitis, peritonitis, salpingitis, synovitis, sepsis, cellulitis, necrotic
dermatitis and granulomatous syndrome in poultry. Considering the frequent presence of
diseaseinflocksofbirdskeptinintensiveandconsequencesofthisdisease(unevennessinfact,
decrease the percentage of laying low body weight, seizure at slaughter) were conducted
epidemiological surveys, clinical and morphological of colibacillosis birds under intensive
growth.
MATERIALANDMETHODS

Epidemiological investigations were conducted on a sample of 22,000 chickens breed


Issa Brown in a poultry farm in Vaslui during MayAugust 2005. Poultry litter are kept
permanentlyonthegroundsunflowerhusksandshavings,andthedensityof15birds/sq.
The first signs of illness occurred at the age of 24 days following vaccination against
pseudopestei.Morbidityrateincrease(upto50%)andmortalityratereached6.3%at48days
afterthat,returntotheclinicalnormal.(Table1)
Diagnosis of avian colibacillosis was confirmed by laboratory tests and by clinical
examinationandnecropsy.Therewereatotalof450samplestakentoestablishadiagnosisand
treatmentoptim.Shaveisolatedanumberof51bacterialstrainsidentifiedasE.colifromthe
liver,heart,airsacs,lung.Wereperformedsowingtheusualmediabrothagarmediumbutalso
specificforthediagnosisofE.coli(Levin).AllAPECstrainsstudiedfermentedlactoseonculture
media, the biochemical character is considered constant at APEC strains. All lactozopositive
strainsweresubjectedtofurthertestingbytheminimumsetTSImuCongored.Colibacillosis
diagnosisisconfirmedbymorphologicalandbiochemicalcharacters.
Necropsy was performed on a total of 650 birds being observant air bags lesions
fibrinous76.9%89.2%fibrinouslesionsofpericarditis,hepatitishemoragiconecrotic15.3%,
fibrinoussalpingitis23%53.8%fibrinpurulentperitonitis.

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Lucrritiinificevol53seriaMedicinVeterinar

Birdsage
25days
26days
27days
28days
29days
34days
35days
38days
40days
41days
42days
43days
44days
45days
46days
47days
48days

Nodeadbirds/day
10
15
30
40
50
70
75
80
100
110
200
280
150
100
50
25
10

Table1.Mortalityintheflockofchickens
Nototalbirds/day
Percentagemortality
10
0,04%
25
0,11%
55
0,2%
95
0,43%
145
0,65%
215
0,97%
290
1,31%
370
1,68%
470
2,1%
580
2,63%
780
3,54%
1060
4,8%
1210
5,5%
1310
5,9%
1360
6,1%
1385
6,2%
1395
6,3%

650 birds from the necropsy lesions were found air sack fibrinous 76.9% 89.2%
fibrinouslesionspericarditis,hepatitis,necrotichemoragico15.3%23%salpingitisfibrinous,
purulentperitonitisfibrin53.8%.(Table2)

Table2.Typeandfrequencyofpathologicalchanges
Birdsage
Nodead
Airsack Pericarditis Hepatitis Salpingitis Peritonitis
birds/day
fibrinous
2534days
290
68,9%
86,2%
15,5%

8,6%
3540days
845
93,4%
94,6%
23,6%
35,5%
76,9%
4148days
335
28,8%
59,7%

14,9%

Movement of poultry material, globally, to disseminate APEC strains and hence the
release of multiple resistance to antibiotics, which requires the monitoring of this view of
APECstrainsisolatedfrombirdsrearedinintensivesystem,thereferencelaboratories,which
inmanycountriesisalreadyinplace.
Followingtheperformancetulpinideantibiogramforthe51E.coliisolates,therewas
increasing resistance to most strains of antibiotic susceptibility and restriction, present in
particular to newer antibiotics. (Table 3). The best results were obtained in florfenicol (90%
sensitivity).
Other 36 strains of E. coli tested werefound tobe sensitive in more than 50%, the
followingantibiotics:flumequin(61.11%),enrofloxacin(58.3%)andnorfloxacin(52.77%).
Regarding antibiotic resistance, the 51 strains tested in the first set proved to be
resistant to more than 50% the following antibiotics: doxicilin (92.57%), tetracycline
(85.13%), neomycin (77.69%), ciprofloxacin and erythromycin (60.34%), spectinomycin
(57.85%).

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Table3.BehaviorresultsfromantibioticstrainsofE.coli
Antibiotics
Sensibile%
Moderatelysusceptible%
Rezistent%
Neomicina
10
12,31
77,69
Enrofloxacin
58,3
23,7
8
Apramicina
68,2
27,2
4,6
Streptomicina
7,5
23,8
68,7
Colistin
75,4
23,4
1,2
Furazolidon
62,4
26,8
10,8
Tetraciclina
6,7
8,17
85,13
Amoxicilina
51,4
13,7
34,9
Spectinomicina
20
22,15
57,85
Lincomicina

100
Gentamicina
40,2
28,3
31,5
Sulfametoxazol+
11,5
34,6
53,9
Trimetroprim
Ampicilina
2,7
21,6
75,7
Fluorfenicol
90
6,4
3,6
Lincospectin
89,5

10,5

Episodetriggeredbyaviancolibacillosisinpoultryfarmbecauseofthemicroclimate
supraalomerare deficiencies, excessive dust, stress, vaccination, mortality losses varying by
them.(Fig.1).

Fig.1Mortalityinaflockofchickens
Diseaseoccursinbirdsaffectedbysneezing,serousocularandnasalcatarrh,cough,
shortness breath and sinus infraorbitale deformation. Birds at necropsy air bags appear fine
depositsoffibrinthatlaterturnsintofibrindepositscaseoustheappearanceofou.Pulmonii
greavesarehiperemiaiandincreaseinvolume,tonguelesionsofpneumonia.Subsequently,
fibrin color of air bags closed envelope. Serofibrinoase serous pericarditis occur and

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Lucrritiinificevol53seriaMedicinVeterinar
subsequently followed by liver and serous inflammation serous abdominal tututror then, all
bodiesarecoveredbyfibrin.(Fig.2,Fig.3).

Fig.2FoamyexudateintheairbagsFig.3Exudatecheeseintheairbags
birdsinfectedwithE.colibirdsinfectedwithE.coli

As the evolution of the disease, liver or dark green, increased consistency and
pectoral muscles are congested increase in volume, brittle and outbreaks of hepatitis
hemoragiconecrotic. It finds and fibrin purulent peritonitis, salpingitis fibrinous. (Figure 4,
Figure 5) have normal bowel inflammation blue, often with the serous intestinal bleeding
points.Bleedingisfoundalsointhepericardium,endocardiumandinotherorgans.

Fig.4LiverhypertrophyinchickensFig.5Aerosaculitandsalpingitisin
chickensaged40days40daysold

A high frequency of aerosaculit lesions, pericarditis, hepatitis, salpingitis and


peritonitis were found at the age of 3540 days. During e evolution of the disease were
thinning birds Hall transfer to other halls and drug treatment was applied following
completionofantibiogramsforfivedayswithfloron200ml/100litriwaterindrinkingwater
sincetheageof40days.After23daysofadministrationofthemortalityratebegantofall.
(Table4).

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Table4.Mortalityratesaftertreatmentwithantibiotics
Birdsage
No. adbirds
Percentagemortality
41days
110
0,5%
42days
200
0,9%
43days
280
1,27%
44days
150
0,53%
45days
100
0,45%
46days
50
0,22%
47days
25
0,11%
48days
10
0,04%

CONCLUSIONS
Research avian colibacillosis in a group of chickens reared in intensive system led to the
followingconclusions:
1. Illness were found in a consignment of 2,200 chickens reared in intensive vaccination
becauseofstressandinappropriateconditionsmicrolimat.
2. Colibacillosis occurred after vaccination because of stress and high density of birds in the
hall.
3. The disease started at the age of 25 days, the morbidity rate is high (50%), but mortality
reached6procentil3%.
4. Colibacillosis diagnosis was established by laboratory tests (by cultivation on agar, broth)
Morphologicalandbiochemicalexaminations,clinicalandnecropsy.
5. There were 51 strains of E. coli isolation and antibiogram was observed after performing
strainsincreasedsensitivitytoflurofenicol.
6. Losses were so significant and have affected poultry flocks, chicks reaching productive
performanceintermsofeggproductionwhenhereachedadulthood.

REFERENCES
1.

SF Altekruse, F. Elvinger, C. DebRoz, FWPierson, JD Eifert, N. Sirangananthan, 2002 Avian


Diseases,46(3),pp562569.
2. KapurV,WhiteDG,WilsonRA,WhittleTS1992Outermembraneproteinpatternsmarkclones
ofEscherichiacoliO2andO78strainsthatcausesaviansepticemia.Infectimmune.April,60(4),
pp16871691.
3. Victoria Martin, 2001 Infectious diseases in domestic animals, Ceres Publishing House,
Bucharest.
4. JJMaurer,CLHofacre,WoolRE,P.Gibbs,R.Froyman,2002,AvianDiseases,46(3),pp704706.
5. Victoria Martin, 2001 Infectious diseases in domestic animals, Ceres Publishing House,
Bucharest.
6. MitrnescuE.IonitaI.,2001Increasedbirdhygiene,nutrition,diseasesandtreatments,masks,
Bucharest.
7. Nettle A., Stanica Andreea, 2004 Sensitivity to antibiotics of strains of E. coli, Salmonella ssp,
PseudomonasaeruginosaandStaphylococcusaureusisolatedinthediagnosticcenterofSN"Pasteur
Institute"intheyear2003,Newsletter15(1),pp1418.
8. VasiuConstantine,2007Infectiousdiseasesinanimals,Mega,ClujNapoca.
9.VidottoMC,MllerEE,deFreitasJC,AlfieriAA,GuimaresIG,SantosDS.,1990,VirulenceFactorsof
avianEscherichiacoli.AvianDiseases,34(3):531538.
10.
NettleA.,StanicaAndreea,2004,sensitivitytoantibioticsofstrainsofE.coli,Salmonellassp,
PseudomonasaeruginosaandStaphylococcusaureusisolatedinthediagnosticcenterofSN"Pasteur
Institute"intheyear2003,Newsletter15(1),pp1418.

1025

RESEARCHCONCERNINGTHEINFLUENCEOFTHERMAL
TREATMENTONTHEQUALITYOFFLORAL
HONEYPRODUCEDINROMANIA

ANCAMARIAGALI,I.OGOE,
L.TUDOR,I.L.ILIE1,MITRNESCUElena
FacultyofVeterinaryMedicineBucharest

Abstract
Romanian honey is processed in different ways in order to extend its shelf life and prevent
crystallization,thustheproducersheatingitattemperatureswithvaluesbelow100oC.
It is believed and recent studies have partially demonstrated that the thermal treatment of
honeydew and floral honey determines a transformation of the hydroxymethylfurfural content
(abbreviatedHMFintheresearchfield).Theformationofthiscompoundishighlyinfluencedby
thewatercontent,totalacidity,pHvalueandmineralsubstancescontent.
The honey samples were subjected to heating at different temperatures, of 50, 75, 100oC for a
period of time between 15 and 60 minutes, afterwards being analyzed for the HMF content by
usingHPLC.
Asitwasalreadyprovedinotherpreviousstudies,asignificantincreaseconcerningHMFcontent
inhoneysampleswasnotobservedevenwhenheatingthesamplesat100oC.ThereasonforHMF
content severe increase is probably due to another factor, probably related to production or
storagestep.
Keywords:floralhoney,thermaltreatment,quality

INTRODUCTION

Sincetheearliesttimes,honeyhasbeenusedasfoodandmedicalproduct,asitisa
naturalsubstanceproducedbyhoneybeesfromthenectarofflowersorexudatesoftreesor
plants (2, 9). Honey is considered a very valuable product and it has frequently been
considered a subject for adulteration by different manufacture practices worldwide (1). The
chemical composition of honeydew honey is different from the floral type, concerning pH,
mineral substances content and sugar profiles (3). In spite of the fact that most scientists
consider that raw honey is the most suitable variant of honey for the consumers to eat, in
time, the heating of honey has become a practical method to prevent the postbottling
crystallization, thus nowadays during the processing, honey is usually submitted to thermal
treatment (4, 10). The main degradative compound of heated honey is considered to be
hydroxymethylfurfural (HMF), the processof HMF formation comprising hexose dehydration
especially at a pH value of 5 or even lower, or through Maillard reaction (5, 6). HMF is an
indicatorofheatstresstofoodproductsduringprocessing,itstoxicologicalnaturebeingstill
uncertain. In high concentrations, HMF is cytotoxic, causing irritation to eyes, upper
respiratorytract,skinandmucousmembranes(7,8,11).Honeycompositionisveryimportant
in significance considering HMF formation. In this investigation, we determined the most
commonpropertiesoffloralhoneysuchas:watercontent,totalacidity,mineralelementsand
HMF,examiningmeanwhiletheinfluencethatthethermaltreatmenthasontheHMFcontent
ofhoney,revealingitsfinalquality.

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Lucrritiinificevol53seriaMedicinVeterinar

Sample

Water

MATERIALSANDMETHODS

Thesampleswerecollectedinnumberof10,fromdifferentproducersalloverthe
southernareaofourcountry,andthevegetationoftheselectedareasismainlycomposedof
mixtureofvariousplants,likelinden,acacia,sunflowerandseveralforesttrees.Thesamples
wereintroducedinglassjarsandstoredat+4oCuntilanalysis.
Thetotalacidity,mineralsubstancescontentandwatercontentwereinvestigated
accordingtoInternationalStandards.
HMFcontentwasanalyzedusingHPLCmethod.Fromeachsample,1gwasselected
andintroducedin9mlofdistilledwater.Thismixturewascentrifugedat10,000rpmfor15
minutes,inordertoremovethefinedebristhatmightbepresentinthesample.Theobtained
supernatant was filtered using a membrane of 0,5 mm and 25 ml of filtrate substance was
introduced in HPLC column, where the mobile phase comprised a mixture of methanol and
water. The HMF quantity was determined by using an external calibration curve, and the
detectorforHPLCwassetat280nm.
Thethermaltreatmentcompriseddifferenttemperaturevalues,from50to100oC,
(50, 75 and 100oC) for a 15, 30, 45 and 60 minutes, cooled and analyzed afterwards. The
samples were introduced in thermal treatment tubes and hermetically sealed, being in
duplicate. There have been included a total number of 240 tubes, due to the number of 10
typesofhoneysamples,andforeachtemperature,wehaveincludedaduplicateofsamples
foreachperiodoftime.Afterheating,thesampleswerecooledat4oC.

RESULTSANDDISCUSSION

The chemical characteristics of the honey samples included in the study are
comprisedinTable1andFig.1.

Table1
Thechemicalcharacteristicsofhoneysamples

Mineralcontent
pH
Acidity
K
Ca
Mg
Na
Fe
Zn
Cu
No.

(%)

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

4,2
3,7
3,6
4,1
4,0
4,4
3,5
3,8
3,4
3,5

19,01
19,16
16,87
16,52
17,98
16,45
16,13
17,81
17,35
19,41

(mmol/ kg
)
8,23
16,44
17,36
21,49
10,84
8,50
13,38
19,05
11,79
15,84

(mg/kg1)

2012,45
1986,78
1980,23
1997,65
1959,45
2029,60
1997,92
2004,59
2007,69
2006,74

1027

157,59
155,48
165,12
160,23
154,15
153,64
150,23
151,87
152,84
156,65

65,61
66,01
67,23
67,51
66,32
65,98
64,85
68,56
66,54
65,40

40,32
41,55
40,98
39,47
38,65
38,12
37,25
39,91
39,76
38,67

15,54
15,01
16,17
15,89
15,81
15,67
15,64
15,73
16,04
15,59

39,80
38,62
38,94
37,62
39,05
39,12
39,56
38,67
38,42
39,09

2,02
1,98
1,64
2,05
2,03
2,01
2,10
2,09
2,05
2,06

UniversitateadetiineAgricoleiMedicinVeterinarIai
Takingintoaccountthefirstparameter,thepH,sampleno.6presentedthehighest
value,of4.4,meanwhilethelowestonewasregisteredtosampleno.9.Consideringthewater
content, in percentage values, sample no.10 presented the highest value, of 19.41, and the
lowestwascharacteristictosampleno.7,withavalueof16.13.Sampleno.4hadthehighest
value of total acidity, 21.49 mmol/kg1, and sample no.1, the lowest, 8.23 mmol/kg1. The
potassium content, was correlated with a 2029.6 mg/kg1 value for sample no.6, and with
1980.23mg/kg1forsampleno.3.Meanwhile,thecalciumquantitywasof165.12mg/kg1for
sampleno.3andof150.23mg/kg1forsampleno.7.Themagnesiumcontentwas65.4mg/kg
1
(the lowest) for sample no. 10 and of 68.56 mg/kg1 for sample no.8. The highest value
registered for sodium content was for sample no.2, with 41.55 mg/kg1 and the lowest for
sample no.7, of 37.25 mg/kg1. The minimum value for iron quantity was 15.01 mg/kg1 for
sampleno.2andthemaximumwasregisteredforsampleno.3,of16.17mg/kg1.Thezincwas
quantifiedat37.62mg/kg1insampleno.4and39.8mg/kg1forsampleno.1,whileforsample
no.3thecoppercontentwas1.64mg/kg1and2.1mg/kg1forsampleno.7.Inotherwords,
sample no. 1 presented the highest value for zinc content, and the lowest for total acidity;
sampleno.2hadthehighestcontentofsodiumandthelowestconsideringiron;sampleno.3
hadthehighestvaluesforcalciumandironcontentandthelowestforpotassiumandcopper;
sampleno.4presentedthehighestvaluesoffortotalacidityandthelowestforzinccontent;
sampleno.6hadthehighestvalueforpHandforthepotassiumcontent;sampleno.7hadthe
highest content of copper and zinc and the lowest value for calcium, sodium and water
content;sampleno.8registeredthehighestvalueofmagnesiumcontent,whilesampleno.9
the lowest pH and sample no.10 presented the highest content of water and the lowest in
magnesium.

Fig.1Thechemicalcharacteristicsofhoneysamplesgraphicandvalues

2100
2000
1900
1800
1700
1600
1500
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
0

pH
Water
Acidity
Mg
Na
Fe
Zn
Cu
Ca
1

K
2

10

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Lucrritiinificevol53seriaMedicinVeterinar
TheHMFcontentinsamplesisincludedintable2andfig.2.

Table2
TheformationofHMFindifferenttypesofhoneysamples
atdifferenttemperatures(mg/kg1)(n=10)

Time
Heatingtemperatures
(min)
50(oC)
75(oC)
100(oC)
Initial
0,54
0,54
0,54
15
1,01
1,67
4,52
30
1,57
2,11
13,41
45
1,82
4,83
21,67
60
2,04
7,64
39,61

Fig.2TheformationofHMFindifferenttypesofhoneysamples
atdifferenttemperatures(mg/kg1)(n=10)graphicandvalues

42
40
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
Initial

At 15 min
50 C

At 30 min
75 C

At 45 min
100 C

At 60 min

Inwhatconcerns theHMFcontent,thehighest valuecanbeobservedatsamples


heated for 60 minutes, these increasing at the same time with temperature value. After
investigating and obtaining the data for HMF content, the main results are that at a
temperatureof50oC,nomatterthetimeusedforheating,theHMFcontentisstable,within
limitsof0.54initiallyto2.04afterthermalprocessingfor60minutes.Atatemperaturevalue
of 100oC HMF content is already 4.52 mg/kg1 after 15 minutes, 13.41 mg/kg1 after 30
minutes,21.67mg/kg1after45minutesand39.41mg/kg1afteronehour.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
CONCLUSIONS

Duetothefactthathoneyissubjectedtoathermaltreatmentinordertoprevent
postbottlingcrystallization,thehydroxymethylfurfuralcontentincreases,duetotheheating
processattemperatureswithintherangeof50to100oC.ThedatashowthatHMFcontentis
directlyrelatedtothetemperaturevalueusedforthermalprocessingofhoneyandlesswith
theprocessingtime,becausehoneyheatedat50oCfor60minuteshasanHMFcontentof2.04
mg/kg1, in comparison to 39,61 mg/kg1 for honey heated at 100oC for the same period of
time.

REFERENCES

1.Bath,P.K.,Singh,N.AcomparisonbetweenHelianthusannuusandEucalyptuslanceolatushoney.
FoodChemistry,67,1999,389397.
2.DurlingLouise,BuskLeif,BjrnE.EvaluationoftheDNAdamagingeffectoftheheatinducedfood
toxicant 5hydroxymethylfurfural (HMF) in various cell lines with different activities of
sulfotransferases,FoodandChemicalToxicology,Volume47,Issue4,2009,880884
3.FreitasM.C.,PachecoA.M.G.,Ferreira1E.,NutrientsandotherelementsinhoneyfromAzores
and mainland Portugal Journal of Radioanalytical and Nuclear Chemistry, Vol. 270, No.1,2006,
123130
4. Husy T., Haugen M., Murkovic M., Jbstl D., Stlen L.H., Bjellaas T., Rnningborg C., Glatt H.,
Alexander J. Dietary exposure to 5hydroxymethylfurfural from Norwegian food and
correlations with urine metabolites of shortterm exposure, Food and Chemical Toxicology,
Volume46,Issue12,December2008,Pages36973702
5. Janzowski, C., Glaab, V., Samimi, E., Schlatter, J., and Eisenbrand, G., 5Hydroxymethylfurfural:
assessmentofmutagenicity,DNAdamagingpotentialandreactivitytowardscellularglutathione,
FoodChem.Toxicol.38,2001,801.
6. Jose Miguel AlvarezSuarez, Sara Tulipani, Stefania Romandini, Enrico Bertoli, Maurizio Battino,
Contributionofhoneyinnutritionandhumanhealth:areviewMediterr.J.Nutr.Metab.,2009,
3:1523;
7.Karkacier,M.,Gurel,F.,Efendi,Y.,Yaygin,H.,&Mutaf,S.,Effectsofdifferentstorageconditionson
thequalityofflowershoneyandhoneydew,JournalofFacultyofAgriculture,AkdenizUniversity,
1995,3543.
8. Kukurov K., Karoviov J., Grief G., Kohajdov Z., Lehkoivov J., Determination of 5
HydroxymethylfurfuralafterWinklerandbytheHPLCMethodforAuthenticationofHoney,2006,
InstituteofChemistry,SlovakAcademyofSciences,1169600600348
9. Qi X., Watanabe M., Aida Taku, Smith R. Catalytical conversion of fructose and glucose into 5
hydroxymethylfurfural in hot compressed water by microwave heating , Catalysis
Communications,Volume9,Issue13,2008,22442249.

10.Spano Nadia,CasulaLucia,PanzanelliA.,PiloMaria,PiuPaola,ScanuRoberta,TapparoA.,Sanna
G.AnRPHPLCdeterminationof5hydroxymethylfurfuralinhoney:Thecaseofstrawberrytree
honey,Talanta,Volume68,Issue4,2006,1390139
11.TeixidE.,SantosF.J.,PuignouL.,GalceranM.T.,Analysisof5hydroxymethylfurfuralinfoodsby
gaschromatographymassspectrometry,JournalofChromatographyA,Volume1135,Issue1,24
November2006,Pages8590

1030


RESEARCHCONCERNINGTHEMOISTURECONTENTAND
VISCOSITYOFROMANIANHONEYSTOREDATDIFFERENT
TEMPERATURES

ANCAMARIAGALI,I.OGOE,
L.TUDOR,I.L.ILIE,MITRNESCUElena
*FacultyofVeterinaryMedicineBucharest

Abstract
The moisture content is the quantity of water contained in a material. In order to see how the
moisture content of honey and the storage temperature affect the final quality of honey, we
measuredtheviscosityof5typesof floral honey, specific to the southern part of Romania. We
startedbymeasuringtheinitialwatercontent,andafterwardsweincreasedthisparameterto20
%,25%,30%and35%.Eachtypeofprocessedhoneywasstoredatdifferenttemperatures:20
o
o
o
o
o
C, 25 C, 30 C, 35 C and 40 C. The results showed variable values concerning this parameter,
withrangesfrom0,581to20,165Pas,andtheconclusionisthatviscosityisdefinitelyinfluenced
bymoisturecontent,temperatureandevenitsbotanicalorigin.

Keywords:floralhoney,moisturecontent,temperature,viscosity.

Honeywithaveryhighqualitypresentsalowwatercontent.Ifthewatercontentis
greater than 19%, honey will ferment and lose its freshness, because raw honey
(unpasteurized) contains yeast strains, that produce fermentation, this producing a high
acidity, deteriorating the honey.(1, 3, 7) Besides that, honey has the property of being very
hygroscopic,beingabletoeasilyabsorbmoisturefromtheair.
Viscosity is considered one of the most important characteristics of honey, being
abletoaffectthefinalqualityofthisproduct(2,6).Theimportanceofthisparameterishigh
especially in the sector of honey production, starting from the extraction stage and ending
withthepackingone(8,9).
Theoptimumtemperatureforhoneystorageis1016oC.Someresearchersconsider
that the ideal temperature for crystallization is 14oC, so their recommendation is to store
honey at 2023oC (4, 5). Meanwhile, at low temperatures crystallization is slowed down.
Nevertheless, honey quality decreases with increasing the temperature, the HMF content
increasingaswell,whiletheenzymeactivitydecreases(10).
The mainobjectiveofthisstudywastoinvestigatethe effectofmoisturecontent
andstoragetemperature,relatedstrictlytoviscosity,onthefinalqualityofhoneydestinedfor
humanconsumption.

MATERIALSANDMETHODS

Fiverawtypesofhoneywerestudied,namelylinden,forest,acacia,sunflowerand
polifloral honey. The samples were obtained directly from the producers, and not from the
market.Theweightofeachoriginalhoneysamplewasapproximately1000g.
The water content of the samples was measured with a digital 2522 HHR2N
refractometerandafterwards,therefractivevalueswereconvertedtomoisturecontents.

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Lucrritiinificevol53seriaMedicinVeterinar
Eachsamplesviscositywasmeasuredinitiallyattheoriginalmoisturecontent,and
afterthisstep,anamountofdistillerwaterwasaddedtoeachsample,inordertoincreasethe
watercontentvalueto20%,25%,30%and35%.TheviscositywasmeasuredwithaSchott
rotationalviscometer,whichisequippedwithanincorporatedLCDthatshowstheviscosityin
mPas. In order to analyze the honey at the maximum precision, the samples of 50 ml each
were heated at 45oC for 2 h, in an incubator, to remove entirely the air bubbles and the
crystalsthatwerealreadyformed.After24hours,viscositywasmeasuredat20oC,25oC,30oC,
35 oC and 40oC, for each of these storage temperature values, a number of 10 samples was
used,induplicate.

RESULTSANDDISCUSSION

The initial moisture content of honey samples is included in table 1. From the
obtaineddata,acaciahoneyseemstohavethehighestvalueconcerningmoisturecontent
18,7%whilepolifloralhoneyhadtheminimumfromallfivetypesofsamples,only16,2%.

Table1
Theinitialmoisturecontent(averagevalue)ofthesamples
(attheinitialamountofsampletakendirectlyfromtheproducer)

Sample
Moisture(%)
Lindenhoney
16,7
Foresthoney
15,6
Acaciahoney
18,7
Sunflowerhoney
18,1
Polifloralhoney
16,2

Concerning the viscosity data (showed in table 2 and revealed in figure 1), we
analyzeditbyapplyingdifferentpredefinedmoisturecontentvaluesof20%,25%,30%and
35%,muchabovethenormalmoisturecontentvaluesthatthehoneyshows.Correlatedwith
the moisture content values, we analyzed the viscosity as a function of the storage
temperature,byapplying5differentvaluesofthisparameter:20oC,25oC,30oC,35oC,and40
o
C.
Theresultsshowedthatthevaluesofviscosity(measuredinPascalsecondsPas)
were comprised in ranges from 25,654 Pa s measured for linden honey, with a moisture
contentof20%andstoredatatemperaturevalueof20oC,to0,431Pas,forpolifloralhoney,
with a moisture content of 35 % and stored at 40oC. These results show that the viscosity
decreasesduringtheincreaseoftemperatureandmoisturecontent,soitsfluidityisraising.

1032

UniversitateadetiineAgricoleiMedicinVeterinarIai
Table2
Thevaluesforviscosity(Pas)forthedifferenttypesofanalyzedhoney,
asafunctionofmoisturecontentatdifferenttemperatures

Linden

Forest

Acacia

Sunflower

Polifloral

Moisture
(%)
20
25
30
35
20
25
30
35
20
25
30
35
20
25
30
35
20
25
30
35

20oC

25 oC

30 oC

35oC

40 oC

25,654
16,549
10,625
5,458
24,857
18,521
10,627
5,321
21,628
17,684
12,458
8,667
18,510
15,519
11,258
7,027
18,006
14,611
8,887
5,127

20,628
11,498
8,624
3,726
19,362
12,327
8,629
4,218
18,569
14,623
8,746
5,941
15,812
12,157
8,449
3,227
14,176
12,113
6,941
3,934

14,627
8,961
6,375
1,834
13,384
6,519
3,337
2,994
15,627
10,289
6,210
3,319
10,917
7,142
4,617
1,394
10,914
7,517
4,283
1,307

8,147
6,321
2,746
1,003
7,612
3,631
2,006
1,089
7,615
5,912
4,252
2,113
6,819
4,618
2,176
0,687
6,701
3,515
1,275
0,560

4,632
2,617
1,587
0,627
2,034
1,981
1,527
0,942
4,216
3,378
2,080
1,001
3,397
2,178
1,127
0,887
3,803
1,567
1,492
0,431

35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0

Lin

n
de
re
Fo

st
a
Ac

cia
nf lo
Su

40 C

35 C

30 C

r
we

25 C

li
Po

20 C

ral
flo

Moisture (%)

Fig.1Viscosityvaluesofdifferenttypesofhoneywhenmoisturecontentwasartificially
modifiedanddifferenttemperaturevalueswereapplied

1033

Lucrritiinificevol53seriaMedicinVeterinar

CONCLUSIONS

1. Themoisturecontentofhoneyisinfluenceddirectlybyitsbotanicalorigin.Thehighest
moisture content of nonprocessed honeywas observedfor Acaciahoney (18,7 %) and
thelowestonewasobtainedforpolifloralhoney.
2. Theviscosityofhoneyvarieswithtemperatureandmoisturecontent,andindirectlyby
itsbotanicalorigin.Theviscosityoflindenhoneywasdefinitelyhigherthantheviscosity
shownfortherestofthehoneytypes.Thelowestvaluesforviscositywereobtainedfor
polifloralhoney,nomatterthestoragetemperaturesapplied.
3. Eventhoughpolifloralhoneyhadaspecificmoisturecontentof16,2%,itsvaluesforthis
parameterwereincreasedartificiallyanditsviscositydatawerealsothelowestfromall5
species investigated in this study. On the other side, even though its original moisture
contentwasonly16,7%,lindenhoneypresentedthehighestviscosityvaluesofall.

REFERENCES

1.

2.
3.

4.
5.

6.

7.

8.

9.
10.

CaviaMara,FernndezMuioM.A.,AlonsoTorreSara,HuidobroJ.F.,SanchoMaraEvolutionof
acidity of honeys from continental climates: Influence of induced granulation, Food Chemistry,
Volume100,Issue4,2007,17281733.
Cereser Camara V., Laux D. Moisture content in honey determination with a shear ultrasonic
reflectometer,JournalofFoodEngineering,Volume96,Issue1,2010,9396.
ChudzinskaM.,BaralkiewiczD.Estimationofhoneyauthenticitybymultielementscharacteristics
usinginductivelycoupledplasmamassspectrometry(ICPMS)combinedwithchemometrics,Food
andChemicalToxicology,Volume48,Issue1,2010,284290.
CuiZ.W.,SunL.J.,ChenW.,SunD.W.Preparationofdryhoneybymicrowavevacuumdrying,
JournalofFoodEngineering,Volume84,Issue4,2008,582590.
Gomes, Susana, Dias, G.L., Moreira L.L., Rodrigues, Paula, Estevinho, Leticia Physicochemical,
microbiological and antimicrobial properties of commercial honeys from Portugal, Food and
ChemicalToxicology,Volume48,Issue2,2010,544548.
Lachman J., Kolihov D., Miholov D., Koata J., Titra D., Kult K. Analysis of minority honey
components:Possibleusefortheevaluationofhoneyquality,FoodChemistry,Volume101,Issue
3,2007,Pages973979.
Migda W., Owczarczyk H. B., Kedzia B., HoldernaKedzia E., Madajczyk D. Microbiological
decontamination of natural honey by irradiation, Radiation Physics and Chemistry, Volume 57,
Issues36,2000,285288.
RuizMatute A.I., Brokl M., Soria A.C., Sanz M.L., MartnezCastro I. Gas chromatographicmass
spectrometriccharacterisationoftriandtetrasaccharidesinhoney,FoodChemistry,Volume120,
Issue2,2010,637642.
Turkmen N., Sari F., Poyrazoglu E. S., Velioglu Y. S. Effects of prolonged heating on antioxidant
activityandcolourofhoney,FoodChemistry,Volume95,Issue4,2006,653657.
WonS.R.,LeeD.C.,KoS.H.,KimJ.W.,RheeH.I.Honeymajorproteincharacterizationandits
applicationtoadulterationdetection,FoodResearchInternational,Volume41,Issue10,2008,952
956.

1034

AUDITSYSTEMATICANDINDEPENDENTEXAMINATIONOF
EFFECTIVEIMPLEMENTATIONOFSANITATIONPROGRAMS

MAGDAGONCIAROV1,R.POPA2,L.TUDOR1,DOINALUPU2,
ELENAMITRNESCU1
1)U.S.A.M.V.,FacultyofVeterinaryMedicineBucharest,105SplaiulIndependentei,District5,
Bucharest,050097,magdagonciarov@yahoo.com
2)SanitaryVeterinaryDirectionandfortheFoodSafety,

Summary

The audit is the systematic and independent examination to determine whether the activities
and results comply with planned programs are implemented effectively and are suitable for
meetingtargets.
Theauditisacomprehensiveassessmentofafoodprocessor.Theauditisconductedbyoneor
moreauditors,whoarecompetenttoperformaudits.Oneoftheauditorsshallbeappointedas
chief auditor of the audit team leader .If necessary , make the team with expert technical
knowledgeoftheareaofactivityaudited.

Keywords:foodsafetyaudit,inspection,HACCP,risk

Theauditplanincludesareas/activitiesaudited,thedatescheduledauditfunctionsof
thesystemtobeaudited,theapplicablereferencedocuments(auditcriteria).

Duringtheauditfindingswillbecommunicatedtotheauditduringtheauditinorder
toavoidfurthermisunderstandings.

Thefindingsshouldbewritten,mustcoincidewiththerequirements.Inspectionsare
unannounced,auditisannounced.Theauditisdirectedtowardscontinuousimprovement.(2)

Auditworkofgoodhygienepracticesshallverifythatfoodbusinessoperatorsapply
procedurescontinuouslyandproperlyconcerningatleast:
checkstheinformationfoodchain;
designandmaintenanceoffacilitiesandequipment;
personalhygiene;
trainingonproceduresandhygiene;
pestcontrol:
waterquality
temperaturecontrol;
checksonentryandexitoffood/andtheundertakingandanyaccompanyingdocuments.

Thenatureandintensityofauditingtasksforeachunitarebasedonestimatedrisk.In
thisrespect,thecompetentauthorityshallevaluateperiodically:
theriskstopublichealthand,whereappropriate,animalhealth;
ifslaughterhouses,animalwelfareissues;
typeofprocessingperformedandresults;
previousrecordofthefoodbusinessoperatorinrespectoflegislationonfood.(1)

European Commission experts may carry HACCP system in order to verify food
business operators apply these procedures permanently and properly. They determine in

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Lucrritiinificevol53seriaMedicinVeterinar
particularwhetherproceduresprovideguaranteestothepossibleextent,thatproductsareof
animalorigin:
complywithmicrobiologicalcriterialaiddowninCommunitylegislation;
are in accordance with Community legislation on residues, contaminants and prohibited
substances;
nophysicalrisksuchasforeignbodies.(4)

Theimplementationofaudittaskscompetentauthorityshallinparticular:
toensurethatallstagesofproduction,onpersonnelanditsactivitiesintheunitcomplies
withrelevantrequirementsofCommunitylaw.Tocompletecontrol,thecompetentauthority
mayconductproficiencycheckstoensurethat,intermsofskills,staffmeettheparameters
specified;
toverifyallrelevantrecordsoffoodbusinessoperators;
totakesamplesforlaboratoryanalysis,ifnecessary;
tojustifytheitemsconsideredandtheauditfindings.

European Commission, general and specific audits in all Member States , mainly
aimedatcheckingifofficialcontrolsarecarriedoutaccordingtocontrolplans(TheNational
Framework for Inspection and Control) and whether the food legislation regarding feed is
respected.

IfthechecksarecarriedoutinRomania,TheNationalSanitaryVeterinaryandFood
Safety experts to ensure all necessary assistance to fulfill their duties. The European
CommissionwillinformtheNationalSanitaryVeterinaryandFoodSafetyresultsofthechecks.

Ifaseriouslyrisktoanimalhealthisidentifiedduringanauditoraninspectionofthe
European Commission, The National Sanitary Veterinary and Food Safety Authority must
immediatelytakeallnecessarymeasurestoprotectanimalhealth.Ifthesemeasuresarenot
taken or if they are deemed insufficient, the European Commission, in accordance with
comunitary procedure, adopt measures necessary to protect animal health and inform the
NationalSanitaryVeterinaryandFoodAuthorityonthis.(3)

Documentation required for audit farm classification requires consideration of


existingdocumentsintheunit:
veterinaryauthorizationissued(original);
controlnotes,evaluationsheetsfollowingpreviousinspections;
recommendations,sanctionsorothermeasuresofficiallyarranged;
officialresultsofsampling;
commercial/economicdocumentation
(contracts/invoices/listssuppliers/distributionlists,etc).

Documents shall specify the amount and results of activities ,including data useful
new risk assessment and new planning. For all controls, including controls ad hoc or
emergency,theremustbepaperson:thetypeofcontrol,controlresultsandthemeasures/
improvement.

In addition to general requirements concerning audits for good practice in terms of


hygiene, the official veterinarian must ensure that the food businessoperator times its own
procedures for each collection, transportation, storage, handling, processing and use or
disposal of animal byproducts animal origin, including specific risk materials are the
responsibility of food business operator. Besides the general requirements of the HACCP
based audits the official veterinarian must ensure that operators provide assurances that
procedureswherepossible,themeat:
doesntpresentpathologicallyabnormalitiesordeterioration;

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UniversitateadetiineAgricoleiMedicinVeterinarIai

doesnotcontainspecifiedriskmaterial,unlessthisisprovidedbylaws,andwereproduced
inaccordancewithCommunitylegislationintheTSE.(5)

If it meets the duties of the inspection, the official veterinarian must take into
account the results of audits performed .It is appropriate, if necessary ,he shall identify the
inspectiondutiesaccordingly.

The official veterinarian must monitor and analyze relevant information from the
registersholdingoforiginofanimalsforslaughterandtotakeintoaccounttheresultsofthis
checkandjustifiedgroundsofthisanalysiswhenperformingantemortemandpostmortem.
Ifitmeetsthedutiesoftheinspection,theofficialveterinarianmusttakeintoaccountofficial
certificates accompanying the animals and any statements of veterinarians engaged in the
primacyproductioncontrols,includingtheofficialveterinarian.Ifthefoodbusinessoperators
involved in the food chain take additional measures to reassure on food safety , by
independentthirdpartiesorbyothermeansand,wheresuchmeasuresaredetailedenough
and the animals included in these systems can be identified with certainty, the official
veterinarianmayconsiderallthisinthedutiesofinspectionandverificationproceduresbased
onHACCPsystem.

Stepsforconductingtheauditare:
openingsession;
auditofthedepartments;
documentedfindingsanddiscussofthemwithaudited;
meetingofauditors;
closingmeeting;
draftingtheauditreport.

Audit team leader has the final decision on how to conduct the audit and the
formulationofthefindings,prepareauditreport,coordinatetheworkofauditors;

Auditors:
make/create audit objectively, in accordance with established audit plan and moral
principles;
reviewwiththeaudittoobtainconcreteevidence,sothattheobjectivesandsufficientto
assessthefunctionalityoftheHACCPsystem.

Itisnecessarytoestablishaprogramofaudits,andfrequencyofauditsshouldtake
intoaccount:
sizeoftheorganizationauditedandtechnologicalfeatures;
thenumberofitemsorgroupsofproductsobtained;
riskgroupofproducts;
numberofnoncomplianceofthepreviousaudit.

Theauditorsfoundthattheprocedures,workinstructionsandotherdocumentsare
known,available,understoodandappliedbythestaffoftheorganization.

Requirementsforauditors:
tohaveaspiritofobservation;
toadapttodifferentsituationsandpersonalbeingaudited;
tobestrong,balanced,tenacious,diplomatic,withmoderation;
tohavecriticalthinking;
tobefamiliarwiththeauditcriteria;
knowassessmentauditprocess;
befamiliarwithHACCPprinciplesandactivities;
toassesswhetherthestepsareproperlyimplementedHACCPsystem;
howtoassesswhethermonitoringoftheCCPisappropriate;

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Lucrritiinificevol53seriaMedicinVeterinar
toidentifyandmakenonconformity;
tomakesomerecommendations;
tomakecommentonthefindings.(6)

CONCLUSIONS

1. The audit is a comprehensive assessment of a food processor. The audit is


conducted by one or more auditors, who are competent to perform audits. One of the
auditorsshallbeappointedaschiefauditoroftheauditteamleader.Ifnecessary,theteamis
completedwithanexpertintechnicalknowledgeoftheareaofactivityaudited.
2. The audit represents systematic and independent examination to determine
whether the activities and results comply with planned programs and if these programs are
implementedeffectivelyandaresuitableformeetingtargets.
3. European Commission, general and specific audits in all Member States, mainly
aimed for checking if official controls are carried out according to control plans (egg The
NationalFrameworkforInspectionandControl)andwhetherthelegislationonproductsfood
isrespected.

REFERENCES

1.
2.
3.
4.
5.

6.

Gonciarov Magda, veterinary and health legislation for food safety Ed. Printech, Bucharest,
2008
Savu,C.,andcol.,Healthcheckfood,CeresPublishingHouse,Bucharest,2005,
Tudor,L.,veterinarycontrolandanimalproducttechnology,EdPrintech,Bucharest,2005
Lawno.150ofMay14,2004onfoodsafety,republish,publishedinOfficialGazettenumber959
datedNovember29,2006.
Regulation (EC) no.178/2002 of the European Parliament and Council of January 28, 2002,
establishing the principles and requirements of food law, establishing the European Food
SafetyAuthorityandlayingdownproceduresinmattersoffoodsafety.
Regulation(EC)no.2073/2005of15November,2005onmicrobiologicalcriteriaforfoodstuffs.

1038

OFFICIALCONTROLCONTROLMETHODFORCHECKING
COMPLIANCEWITHFOODLAW

MAGDAGONCIAROV1,R.POPA2,L.TUDOR1,DOINALUPU2,
ELENAMITRNESCU1
1)U.S.A.M.V.,FacultyofVeterinaryMedicineBucharest,105SplaiulIndependentei,District5,
Bucharest,050097,magdagonciarov@yahoo.com
2)SanitaryVeterinaryDirectionandfortheFoodSafety,

The official controls should be carried out without prior warning, except audit work where is
requiredpriornoticetotheoperatorforanimalfooddomainorthefoodsector.Also,officialad
hoccontrolsmaybemade.
Theofficialcontrolsarecarriedoutinanyofthestagesofproduction,processinganddistribution
ofanimalfoodorfeedandanimalproducts.
Officialcontrolshavetobeperformedusingappropriatecontrolmethodsandtechniquessuchas:
monitoring,supervision,checking,auditing,inspection,samplingandanalysis.
Keywords:food,control,audit,risk,monitoring

Theofficialcontrolrepresentsanykindofcontrolmadebythecompetentauthority
ortheEuropeanCommissionandEUmemberstates,toverifycompliancewithfoodlaw.
Must ensure that official controls are carried out regularly, based on a risk analysis
andanappropriatefrequency,givenby:
1) Identified risks associated with animals, animal food, feed, business activity in animal feed, food
businessenterprises,useoffeedor animalfoodorwithanyprocess,material,substance,activityor
operationthatcaninfluenceanimalfoodorfeedsecurity,healthoranimalwelfare;
2)Previousrecordsofanimalfoodorfeedoperators,oncompliancewithlegislationinthefeedorfood
lawortherulesofhealthandanimalwelfare;
Thereliabilityofthealreadydoneselfchecks;
Anyinformationthatmayindicatenoncompliance.(5)
Officialcontrolsshouldbecarriedoutwithoutapriorwarning,exceptaudit
activities,wherethepriornoticeofthefoodorfeedoperatorisnecessary.Also,officialchecksmaybe
madeadhoc.

Officialcontrolsarecarriedoutinanyofthestagesofproduction,processinganddistribution
ofanimalfoodorfeedandanimalproducts.

Theseshouldincludechecksonbusinessactivityinthefeedandfoodbusiness,regardingthe
use of feed and animal food, food and feed storage or any process, substance, activity or operation
includingtransportfeedorfoodandliveanimals.Competentauthoritiesforperformingofficialcontrols
shouldmeetanumberofoperationalcriteriainordertoensuretheirimpartialityandeffectiveness.They
shouldhaveenoughstaff,withadequatequalifyingandexperienceandtopossessadequatefacilities
andequipmenttoproperlyperformtheirduties.

Competentauthoritiesshouldensurethat,incasesinwhichtheofficialcontrolsrequiresthe
participationofdifferentcontrolunits,theyapplyandeffectivelyimplementappropriatecoordination
procedures. Competent authorities should also ensure that, in cases in which the competence of
performing official controls has been delegated from a central level to a regional level, between the
centralandtheregionalorlocallevelexistsanefficientandeffectivecoordination.(1)

Laboratoriesparticipatingintheanalysisofofficialsamplesshouldoperateinaccordanceto
certaininternationallyapprovedproceduresorrulesbasedonperformancecriteriaandtouseanalysis

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Lucrritiinificevol53seriaMedicinVeterinar
methods that were considered valid, as far as this was possible. These laboratories should provided
especiallywithequipmentsthatallowthecorrectdeterminationofthestandards,likethemaximum
levels of residue set by legislation. Also, the national and community reference laboratories should
contributetoachievingahighlevelofqualityandconsistencyofanalyticalresults.Thisobjectivecanbe
achieved through activities such as application of validated analytical methods, the availability of
referencematerials,organizingcomparativetestingandforminglaboratorystaff.Thereshouldbeproper
proceduresforcooperationofthecompetentauthoritiesofamemberstateandofdifferentmember
states,especiallywhereofficialcontrolsrevealthatfoodissuesconcernmorethanonememberstate.In
ordertofacilitatethiscooperation,memberstatesshouldassignoneormoreliaisonbodieswiththe
purposeofcoordinatingsendingandreceivingrequestsforassistance.

Officialcontrolsonproductsofanimaloriginshouldincludeallmattersthatareimportantin
theprotectionofpublichealthand,whereappropriate,animalhealth.Thesecontrolsshouldbebased
onthemostrecentrelevantinformationandthereforeshouldbepossibletoadapttheminlightofnew
relevantinformationavailable.Officialcontrolsonmeatproductionarenecessaryinordertoverifythat
foodbusinessoperatorsandinspections,includingchecksofcontrolsevenoperators.

Memberstatesshallensurethatfoodbusinessoperatorsprovideallnecessaryassistanceto
ensure effective implementation of official controls by the competent authority. The competent
authorityshallcarryoutcontrolstoensurethatfoodbusinessoperatorscomplywiththerequirements
ofRegulation(EC)no.852/2004,Regulation(EC)no.853/2004andRegulation(EC)no.1774/2002.(4)

ThecompetentauthorityinRomaniaasadestination,cancontroltheauthoritiesresponsible
toitsfoodcompliancewithfoodlawthroughdiscriminatorycontrols.Forreasonsstrictlynecessaryfor
theorganizationofofficialchecks,thecompetentauthoritymayrequireoperatorswhohavetheirgoods
thatweresuppliedformtheEuropeanUnionmemberstatestoreportthearrivalofsuchgoods.

If during a check destination or during storage or transport in Romania, the competent


authorityfindsnoncompliance,itmaytakestepswhichmayincludereturnofEuropeanUnionmember
stateoforigin.

Thecompetentauthoritiesmustensure:

theeffectivenessandappropriatenessofofficialcontrolsonfoodinallstagesofproduction,
processinganddistribution;

thatthosecarryingoutofficialcontrolsareoutofanyconflictofinterest;

thattheyownorhaveaccesstoadequatelaboratorycapacityfortestingandstaffsuitability
qualifiedandexperiencedenoughsothatofficialcontrolsandcontrolresponsibilitiestobecarriedout
efficientlyandeffectively;

thattheyhavefacilitiesandequipmentmaintainedinproperconditiontoensurethatstaffcan
performofficialcontrolsefficientlyandeffectively;
thattheyhavelegalpowerstocarryoutofficialcontrolsandtotakeappropriateaction;

thattheyhavecontingencycomplainedplaceandreadytoimplementsuchcomplaintsthe
situationofneed;

thatfoodbusinessoperatorsareobligedtoundergoanyinspectioncarriedoutandassiststaff
incarryingoutitsauthority.
The competent authorities must Ensure that all their staff performing official
controls:
a)receive,forcompetence,appropriatetrainingallowinghimtoperformcompetentlyandto
carryoutofficialdutiesconsistently.
b)informthepurposeanddaystofollow,ifnecessary,additionaltrainingregularly;
c)provideskillsformultidisciplinarycooperation.(3)
The official checks shall be performed using appropriate control methods and
techniques, such as: monitoring, supervision, checking, auditing, inspection, sampling and

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UniversitateadetiineAgricoleiMedicinVeterinarIai
analysis. Monitoring is achieving planned sequence of observations or measures in order to
obtainageneralassessmentofthelevelofcompliancewithfoodlaw;

Supervision is a careful examination of one or more businesses with food business


operators to work food or their activities. Monitoring means observing the detail of various
aspectsofproductsandunitswhichfallwithinthecompetenceofANSVSADSVSA,following
theincidenceofpotentiallyhavingimpactonfoodsafety.

Verification is control, by examination and consideration of evidence, in terms of


meetingtherequirementsspecified.Verificationisamethodoftestingbyexaminingobjective
evidenceandanalysisonlegislativerequirements.

Theauditisthesystematicandindependentexaminationtodeterminewhetherthe
activities and results comply with planned programs and these programs are implemented
effectivelyandaresuitableformeetingtargets.

Inspectionistheexaminationofanyaspectoffeed,food,healthandwelfare,toverify
thatsuchmattersareinaccordancewiththerequirementsoflegislationonfeedandfoodlaw
andhealthandwelfarerulesanimals.
The inspection is based on the element of surprise. If it is considered that, by explaining
whythecompletionoftheinspectioncandistorttheresult,willnotcommunicateaparticular
view.Samplingforanalysisissamplingoffeedorfoodorothersubstancesrepresentativeof
the production, processing and distribution of feed or food or animal health (including
environmental) in order to verified by analysis of compliance with legislation in the feed or
foodlaworanimalhealthrules;
Sampling methods and analysis used in official controls must comply with EU rules
intonationallaw,or:
a)ifsuchrulesdonotexist,withinternationallyagreedrulesandprotocols,asthosethatthe
European Committee for Standardization (CEN) has accepted or those agreed by national
legislationor
b) where those referred to above, other methods for their purpose or developed in
accordancewithscientificprotocols.
Officialcontrolsonfoodshouldinclude,thefollowingactivities:
1.examiningcontrolsystemsthatfeedbusinessoperatorsandthosewithpetfoodbusiness
haveimplementedandresultsachieved;
2.inspection:
primaryproduction facilities, business activity in feed and food business enterprises,
including their surroundings, facilities, offices, equipment, facilities and equipment, vehicles,
andfeedandfood;
raw materials, ingredients, processing auxiliary agents and other products used in the
preparationandproductionoffeedandfood;
semiproducts;
materialsandarticlesintendedtocomeintocontactwithfood;
productsandprocessesforcleaningandmaintenanceandpesticides;
labeling,presentationandadvertising;
3.controlsonhygieneinbusinessactivityinfeedandfoodbusinessenterprises;
4. evaluating Procedures for Good Manufacturing Practice (GMP), good hygiene practices
(GHP), good agricultural practice and HACCP (Hazard Analysis and Critical Control Point),
taking into account the use of guides established in accordance with national legislation
transposingEUlegislationspecifies;
5. examining written materials or other records that may be relevant to assess compliance
withlegislationinthefeedandfoodlaw;

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Lucrritiinificevol53seriaMedicinVeterinar
6.interviewswithanimalfeedbusinessoperators,withtheactivityinfoodandtheirstaff;
7. reading values recorded by measuring instruments business activity in the feed or food
businessenterprises;
8.controlsowninstrumentsmadebythecompetentauthoritytoverifythemeasurements
madebyoperatorsofanimalfeedbusinessandthosewithfoodbusiness.(4)

Multiannualnationalcontrolplanshouldcontaingeneralinformationonthestructure
andorganizationoffoodcontrolsystems,inparticularon:
- strategicobjectivesoftheplanandhowtocontrolprioritiesandallocatingresources
reflecttheseobjectives;
riskclassificationofthoseactivities;
designationofcompetentauthoritiesandtheirfunctionsatnational,regionalandlocal
levelsandtheresourcesavailabletotheseauthorities;
managementandgeneralorganizationofofficialcontrolsatnational,regionalandlocallevel,
includingofficialcontrolsinindividualcompanies;
controlsystemsapplyingtodifferentsectorsandcoordinationbetweenvariousservices
ofthecompetentauthoritiesresponsibleforofficialcontrolsinthesesectors;
whereappropriate,delegatedtaskstoothercontrolbodies;
methodstoensurecompliancewithoperationalcriteria;
stafftrainingforperformingofficialcontrols;
documentedprocedures;
organizationandoperationofcontingencyPlansforemergencyanimaldiseaseorincase
offoodpoisoningduetocontaminationoffoodandothersituationsofpublichealthrisk;
organizationcooperationandmutualassistance.

CONCLUSIONS
1.Officialcontrolmeansanyformofcontrolthatmadehimcompetentauthorityor
theEuropeanCommissionandEUMemberStates,toverifycompliancewithfoodlaw.
2.Responsibleauthoritiesforofficialcontrolsshouldmeetanumberofoperational
criteriatoensuretheirimpartialityandeffectiveness.
3.Officialcontrolsmustbecarriedoutwithoutpriorwarning,exceptauditworkthat
is required prior notice to the operator for animal feed business activity or the food. Also,
officialchecksmaybemadeadhoc.Official
4. Controls must be made in any of the stages of production, processing and
distributionoffeedorfoodandanimalproducts.
5.Officialcontrolsmustbemadeusingappropriatecontrolmethodsandtechniques,
suchas:monitoring,supervision,checking,auditing,inspection,samplingandanalysis.

REFERENCES
1.Savu,C.,andcol.,Veterinaryfeedsanitarycontrol,CeresPublishingHouse,Bucharest,2005.
2.Tudor,L.,veterinarycontrolandanimalproducttechnology,PublisherPrintech,Bucharest,2005.
3.Regulation(EC)no.599/2004of30March2004adoptingaharmonizedmodelcertificateand
inspectionreportrelatingtoCommunitytradeinanimalsandanimalproducts;
4.Regulation(EC)no.854/2004oftheEuropeanParliamentandEUCouncilof29April2004layingdown
specificrulesfortheorganizationofofficialcontrolsonproductsofanimaloriginforhuman
consumption;
5.Regulation(EC)no.882/2004oftheEuropeanParliamentandEUCouncilof29April2004onofficial
controlsperformedtoensureverificationofcompliancewithfeedandfoodstandardsandanimalhealth
animalwelfare

1042

ANTIMICROBIALSENSITIVITYOFE.COLISTRAINSISOLATEDFROM
PIGSEPTICEMICCOLIBACILOSIS

V.HERMAN,CorinaPASCU,LuminiaCOSTINAR,B.FAUR,IoanaVDUVA,AncaSURPAT,
SorinaIRIMIE
FacultyofVeterinaryMedicineTimioara
Str.CaleaAraduluiNo.119,300645,Timisoara,Romania
Email:cabesti@yahoo.com

Abstract
Inthepaperarepresentedtheresultsconcerningantimicrobialsensitivityfor23strainsofE.coli
type1isolatedfrompigswithsepticaemiclesions.
In vitro testing by diffusimetric method for sensitivity to 12 antimicrobials (ceftiofur, colistin,
amoxicillin, amoxicillinclavulanic acid, enrofloxacin, gentamycin, penicillin, oxytetracycline,
lincospectin, tiamulin + tetracycline, erythromycin, doxycicline) showed less susceptible strains
andmanystrainsresistanttotestedantimicrobials.
Keywords:E.coli,antimicrobialsensitivity.

Diseases caused by E. coli produce important economic losses in pigs. To institute


appropriate therapy is necessary to isolate bacteria that can cause the disease and to know
thebehaviortotherapeuticsubstances,forchoosingthemosteffective(2,4).
Antibiogram by using diffusimetric method is quick and easy to realize. Not always
have the highest accuracy, but provide a rapid response to the recommended antibiotic
therapy(1,3,4,5).
ThispaperpresentsE.colistrainssensitivitytodifferentantibiotics.

MATERIALSANDMETHODS

Between 2008 and 2009 were examined piglets that provided from fattening farms
fromthewestareaofthecountry,whichatthenecropsywerereportedsepticaemiclesions.

Culturingwasmadefromparenchymalorgans,usingtheusualmedia.Sampleswere
incubated24hoursat37C.IdentificationofE.coliwasbasedonculturalcharactersonLevin
media,afterpassageofisolatesfromprimarycultureontheusualmedia.
For testing susceptibility of E. coli strains were used discs with antimicrobial
substances produced by different companies. The discs are placed in tubes of 50
microcomprimates,eachtubeisspecifiednameandquantityoftheantimicrobialsubstance:
micrograms(g)orIU(2,4).
Following the known principles, using diffusimetric method, was carried out
antibiogram. From the liquid medium that contained pure culture, after 24 hours, using
Pasteur pipette was taken a quantity of 1 to 1.2 ml, which was deposited on the Mueller
Hintonagarsurface.Theplatewasintroducedinthermostatfor15minutes,thenwasputthe
microcomprimates on plate, and the result was read and interpreted after 24 hours of
incubationat37C.
Studied E. coli strains were tested against the following antimicrobials: ceftiofur,
colistin, amoxicillin, amoxycillin/clavulanic acid, enrofloxacin, gentamycin, penicillin,
oxytetracycline,lyncospectin,tiamuline+tetracycline,erythromycin,doxycicline.
Results reading and interpretation consisted in inhibition zone size induced by
antimicrobials, area where microbial colonies are lacking. Diameter of inhibition zone is

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Lucrritiinificevol53seriaMedicinVeterinar
directly proportional to the sensitivity of that germ. Interpretation of the results was done
usingtablesfortheinterpretationofthebacteriasensitivity,asspecifiedbythesupplierfirms.

RESULTSANDDISCUSSIONS

Antimicrobial susceptibility testing results of the 23 strains of E. coli isolated are


presentedintable1.
As seen from table 1 were less strains of E. coli sensitive to tested antibiotics.
Susceptibility of E. coli strains to some antimicrobials is due to their recent introduction in
medicinepractice.

Table1
Antibiogramresults

Results
No.oftested
Antimicrobials
strains
S
I
R
ceftiofur
9
5
3
17
colistin
3
10
3
16
amoxicillin
1

18
19
amoxicillinclavulanic
7
5
5
17
acid
enrofloxacin
6
6
11
23
gentamycin
5
9
9
23
penicillin

14
14
oxytetracycline

5
5
lincospectin

1
17
18
tiamuline+tetracycline

8
8
erythromicyn

13
13
doxycicline

2
10
12
Legend:Ssensitive;Iintermediate;Rresistant

For some antimicrobial that are commonly used in pig farms (oxytetracycline,
lincospectin, tiamuline + tetracycline, erythromycin, doxycicline) was observed that strains
showedconsistentlyresistance.
Itwasnotedphenomenonofmultipleresistancetotestedantibioticsfor15strainsofE.
colifromthe23isolatedstrain.Intheliterature,multipleantibioresistanceisdefinedasthe
resistance of the same strain of bacterial to four or more antimicrobials. Antimicrobial
resistance is due to genetic mutation mechanisms and recombination, after frequent use of
antimicrobials(2,4,5).
Multipleresistanceofthe15strainsofE.coli,areasfollows:
2strainsto8antimicrobials;
5strainsto7antimicrobials;
4strainsto6antimicrobials;
1strainto5antimicrobials;
3strainsto4antimicrobials.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
CONCLUSIONS

Were isolated 23 strains of E. coli type 1 from pigs with septicaemic lesions. In vitro
testing by diffusimetric method for susceptibility to 12 antimicrobials (ceftiofur, colistin,
amoxicillin, amoxicillinclavulanic acid, enrofloxacin, gentamycin, penicillin, oxytetracycline,
lincospectin,tiamulin+tetracycline,erythromycin,doxycicline)showedlesssusceptiblestrains
and many strains resistant to tested antimicrobial substances. 15 strains of E. coli, were
presentedmultipleresistanceofantimicrobialsubstances.
Toestablishaneffectivetherapyisrecommendedtobeperformedpriorantibiogram,
consideringthedifferentbehaviortothestudiedantimicrobials.

REFERENCES

1.

2.

3.

4.

5.

Amezcua, R., Frienship, R.M., Dewey, C.E., Gyles, C., Fairbrother, J.M., Presentation of
postweaning Escherichia coli diarrhea in southern Ontario, prevalence of haemolytic E. coli
serogroupsinvolved,andtheirantimicrobialresistancepatterns,Can.J.Vet.Res.2002,66,73
78.
Choi, C., Ham, H.J., Kwon, D.,Kim, J., Cheon, D.S., Min, K., Cho W.S., Chung, H.K., Jung, T.,
Jung,K.,Chae,C.,AntimicrobialsusceptibilityofpathogenicEscherichiacoliisolatedfrompigs
inKorea,J.Vet.Med.Sci.,2002,64,7173.
Hendriksen, R.S., Mevius, D.J., Schroeter, A., Teale, C., Jouy, E., Butaye, P., Franco, Alessia,
Utinane,Andra,Amado,Alice,Moreno,M.,Greco,Christina,Stark,Katharina,Berghold,C.,
Myllyniemi, AnnaLiisa, Hoszowski, A., Sunde, Marianne, Aarestrup, F., Occurrence of
antimicrobialresistanceamongbacterialpathogensandindicatorbacteriainpigsindifferent
European countries from year 20022004: the ARBAOII study, Acta Veterinaria Scandinavica,
2008,50,19.
Herman, V., Corina Pascu, Luminia Costinar, Faur B., Ioana Vduva, Anca Surpat, Sorina
Irimie, Maria erbescu, E. coli strains` characterization isolated from pig septicemic
colibacilosis,Lucr.t.Med.Vet.Timioara,2010,XLIII(inpress).
Teale,C.J.,Cobb,S.,Martin,P.K.,Watkins,G.,VLAAntimicrobialSensitivityReport,Norwich,
2002,1921.

1045

STUDYCONCERNINGTHESEASONALVARIATIONOF
CATTLEMILKPHVALUES
ANDTHEOVERALLINFLUENCEONMILKQUALITY

L.I.ILIE,L.TUDOR,AncaMariaGALI,ElenaMITRNESCU,R.F.POPA
FacultyofVeterinaryMedicineBucharest

Abstract
This research had the main objective of evaluating the seasonal variations in cattle milk pH
values,onalotof10dairycows,fromafarmnearBucharest,animportantproviderofmilkand
milkproductsforthecity.Fromthetotalnumberofcows,weselectedagroupof10cows,from
whichwecollectedonesampleperweek.Thesesampleswereanalyzedusingamultiparameter
device, designed to perform physicochemical analysis of milk. The data that we obtained were
used in statistical formulas, in order to calculate the average values per week and month, the
frequency of certainintervalvalues,andthestandarddeviation.Theresultsrevealedanoverall
pHvalueof6,82.Thefrequencyanalysisshowedthat41%ofthesampleshadvaluesofpHof6.8
6.9,25%withpHvaluesof6.86.9and23%with6.97.0.Wealsoobtainedanoverallstandard
deviation value of 2,765. This last value shows the way that the values differ from the overall
average value. The main conclusion is that pH values vary with weather conditions, physical
statusoftheanimals,thequalityoffeedandtheshelterandmaintenancehygiene.ThepHvalues
variedeachmonth,withnotendencyofincreasingordecreasingforalargerperiodoftime.The
monthofAugustpresentedthehighestvalue,6,9whileMaythelowest6,77.Consideringthefact
thatfreshmilk(immediatelyaftermilking)hasapHvaluesituatedverycloseto7,andthatthis
decreasesintimeduetoprocessingto6,7(beforepasteurization)and6,6afterprocessingandat
refrigerationtemperature,thefinalconclusionisthatthemilkproducedbytheselectedfarmis
suitedforhumanconsumptioninanycommercialform.
Keywords:milkpH,seasonalvariation,standarddeviation,quality.

INTRODUCTION

Milkcontainsa largenumberofbuffer substancesandconsequentlybehavesasa


buffer solution. Fresh cows milk has a pH value close to 7, this decreasing during raw milk
refrigeration to 6.76.5, while for cold processed milk the values are situated between 6,59
and6.33(1,6,8).Becausemilkisabuffersolution,considerableaciddevelopmentmayoccur
beforethepHchanges.ApHlowerthan6.5indicatesthatconsiderableaciddevelopmenthas
takenplace.Thisisnormallyduetobacterialactivity.ThepHmeasurementsareoftenusedas
acceptancetestsformilk(4,5).
Measuringmilkacidityisanimportanttestusedtodeterminemilkquality.Acidity
measurementsarealsousedtomonitorprocessessuchascheesemakingandyoghurtmaking
(2,7). The titratable acidity of fresh milk is expressed in terms of percentage lactic acid,
becauselacticacidistheprincipalacidproducedbyfermentationaftermilkisdrawnfromthe
udderandfreshmilkcontainsonlytracesoflacticacid.However,duetothebufferingcapacity
of the proteins and milk salts, fresh milk normally exhibits an initial acidity of 0.14 to 0.16%
whentitratedusingsodiumhydroxidetoaphenolphthaleinendpoint(3,9).
In this study, we investigated the variation of milk pH values over a ten months
periodoftime,onagroupof10cowsinordertoobservethetendencyofthisparameter,and
toevaluatetheinfluenceonoverallmilkquality.

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MATERIALSANDMETHODS

A number of 10 dairy cows were selected and the milk samples were prelevated
onceaweek,foranperiodof10months,selectingeachmonththesamedates:the5th,the
12th,the19thandthe26th.Theexperimentalgroupcomprised10cowsofalmostthesameage,
with the same diet and bodyweight, and entering together at almost the same time in the
lactation period. The milk samples were collected from each cow, one per cow each week,
withatotalof4samples/month,and40samplesperentireexperimentalperiod/cow,witha
totalof400samplesanalyzedperentireperiod/lot.
Theselectionoftheexperimentaldairycowsfollowedseveralcriteria,like:
the absence of mastitis or any kind of disease that may interfere with the milk
analysis;
abodyweightofalmost500550kg;
amilkproductionof800010000kg.
The samples were introduced in 50 ml plastic bottles, introduced at refrigeration
andanalyzedinamaximumperiodof2hoursfromthecollectionmoment.
The analysis was carried out using EcoMilk Ultrasonic Analyzer, a multiparameter
calibrateddevicedesignedforrapidanalysisofrawandprocessedmilk.Thesampleswereleft
for 510 minutes at room temperature before proceeding with the analysis, and afterwards,
using an automatic pipette with a 5 ml tip, a quantity of 15 ml from each sample was
introducedintheanalysistubes.

RESULTSANDDISCUSSIONS

Theresultsshowedslightvariation,butincreasinganddecreasingfromamonthto
another.ThehighestvaluewasregisteredforthemonthofAugust6,90whilethelowest
wasobservedinMay.InFebruary,thepHhadanaveragevalueof6,83,thisdecreasingto6,80
in March, increasing again in April, reaching 6,77 in May. June came with an increase of pH
value to 6,81, in July observing again a decrease to 6,78 and in August reaching the highest
value,of6,90.Forthenextthreemonth,thepHvaluewasmaintainedinthesamerange,with
6,83 in September, 6,84 in October and 6,82 in November. All the data are shown in table
no.1,fig.1andfig.2.

Table1
TheaveragevaluesofmilkpHpermonthandforeachweek

Month/Week
1
2
3
4
Monthlyaverage
February
6,78
6,83
6,85
6,87
6,83
March
6,76
6,82
6,81
6,81
6,80
April
6,81
6,83
6,86
6,83
6,83
May
6,78
6,78
6,73
6,80
6,77
June
6,81
6,77
6,85
6,81
6,81
July
6,81
6,8
6,72
6,81
6,78
August
6,84
6,89
6,92
6,95
6,90
September
6,91
6,86
6,73
6,82
6,83
October
6,86
6,81
6,81
6,89
6,84
November
6,89
6,82
6,81
6,78
6,82

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Fig.1TheaveragevaluesofmilkpHpermonth
7
6,95
6,9
6,85
6,8
6,75
6,7
6,65
6,6
6,55

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6,5

Fig.2Theaveragevaluesofmilkdensitypereachmonthandweek

February
7

6,95

6,95

6,9

6,9

6,85

6,85

6,8

6,8

6,75

6,75

6,7

6,7

6,65

6,65

6,6

6,6

6,55

6,55

March

6,5

6,5
W1

W2

W3

W4

W1

W2

April

W3

W4

May
7

6,95

6,95

6,9

6,9

6,85

6,85

6,8

6,8

6,75

6,75

6,7

6,7

6,65

6,65

6,6

6,6

6,55

6,55

6,5

6,5

W1

W2

W3

W4

W1

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June

July

6,95

6,95

6,9

6,9

6,85

6,85

6,8

6,8

6,75

6,75

6,7

6,7

6,65

6,65

6,6

6,6

6,55

6,55

6,5

6,5

W1

W2

W3

W4

W1

W2

W3

W4

W3

W4

W3

W4

Consideringinmonthvariation,theaveragevaluespresentedanincreasetendency
fromweek1toweek4inFebruaryandAugustandadecreasetendencyinNovember,with
mixed tendency during the rest of the months. Except for the month of September, when
betweenweek2andweek3thedifferenceishigherthanintherestofthecases,thevariation
presentedslightslopes.

August

Septem ber

6,95

6,95

6,9

6,9

6,85

6,85

6,8

6,8

6,75

6,75

6,7

6,7

6,65

6,65

6,6

6,6

6,55

6,55
6,5

6,5
W1

W2

W3

W1

W4

October

6,95

6,9

6,9

6,85

6,85

6,8

6,8

6,75

6,75

6,7

6,7

6,65

6,65

6,6

6,6

6,55

6,55

6,5

November

6,95

W2

6,5

W1

W2

W3

W4

W1

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From the total number of samples, 164 (41 %) presented values between 6,8 and
6,9,99samples(25%)hadvaluesbetween6,7and6,8,and90samples(23%)hadvaluesof
6,97,0. From the rest of the milk samples, 40 (10 %) presented values of 6,66,7 and only
seven(2%)hadvaluesof6,56,6.Thesedataareincludedintable2,fig.3andfig.4.
Table2
ThefrequencyofthemilkpHvalues
(numberofsamplesandpercentageforeachcategory)

Month/Valuecategory
6,56,6
6,66,7
6,76,8
6,86,9
6,97,0
February
0
4
8
17
11
March
3
6
10
15
6
April
0
4
10
14
12
May
3
6
13
17
1
June
1
3
12
21
3
July
0
4
18
10
8
August
0
1
2
15
22
September
0
3
8
23
6
October
0
3
8
17
12
November
0
6
10
15
9
Total
7
40
99
164
90
Percentage
2%
10%
25%
41%
23%

Fig.3Thenumberofsamplesforeachcategoryoffrequency,permonthofanalysis

25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
February

March

April

May
6,5-6,6

June
6,6-6,7

July
6,7-6,8

August
6,8-6,9

September

October

November

6,9-7,0

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Fig.4TheoverallpHvaluespercentageforthenumberofsamplesincluded
ineachcategoryoffrequency

2%

23%

10%

25%

41%

6,5-6,6

6,6-6,7

6,7-6,8

6,8-6,9

6,9-7,0

Consideringthestandarddeviationofthevaluesweobtained,thisspecificstatistic
indicatorpresentedrangevaluesfrom0,0202608to0,04552472,asshownintable3.The
lowestvalueofthisindicatorwasobservedinAugust,correlatedwiththehighestmonthlypH
averagevalue,of6,90,whilethehighestvalueofstandarddeviationwasobservedinMarch.
TheslightestdifferencewasobservedduringthemonthsofJuneandJuly.FromFebruaryto
June,thetendencywasdifferent,fromanincreaseinFebruarytoadecreaseinMay,while
betweenJulyandAugustthedecreasethatweregisteredwasveryabrupt.Thetendencyof
incrementwasobservedonlyinthelatestfourmonths,fromAugusttoNovember.

Table3
ThestandarddeviationvaluesformilkpHvalues

Standarddeviationofmilk
Month/Statisticindicator
pHvalues
February
2,932138725
March
3,56432848
April
2,8384745
May
3,23874011
June
2,77415528
July
2,779826883
August
1,873966382
September
2,252429244
October
2,61092656
November
2,78796521
Averageperperiod
2,765295137

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Fig.5Thestandarddeviationgraphicfordairycowsmilkdensityvalues
foraperiodof10months

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4
3,9
3,8
3,7
3,6
3,5
3,4
3,3
3,2
3,1
3
2,9
2,8
2,7
2,6
2,5
2,4
2,3
2,2
2,1
2
1,9
1,8
1,7
1,6
1,5

CONCLUSIONS

Milk pH is considered a very important physicochemical trait, among others like


addedwater,superficial tension, viscosity, freezing point, conductivity anddensity, being an
instrument of milk quality evaluation. Fresh milk presents a pH of 7 (immediately after
milking),andintimethisvaluetendstodecreaseto6,7(afterrefrigeration)andeven6,5after
processing and refrigeration storage. For fresh milk, like the one included in the samples of
this study, a pH of 6,5 or lower indicates a high bacterial load, with acid development. In
comparisontothisvalue,onlyafewofthesamplespresentedvaluesbetween6,5and6,6(7
samples)and10%ofthesamplesvaluesbetween6,6and6,7,thisindicatingapossiblenon
detectedsubclinicalmastitis.Thelowestvalueregisteredoverallwas6,51.Ofcourse,through
processing and refrigeration storage, the bacterial load reduced to minimum, this milk not
beingconsideredintheendathreattohumanhealth.
Overall,thefrequencyofthesampleswithoptimalpHvalues(over6,7)wasof88%
(41%between6,86,9,25%between6,76,8and23%ofthemwithvaluesof6,97,0).From
thequalitycontrolpointofview,theseresultsmaybeconsideredappropriate,andthemilk
maybethereforeusedafterprocessinginmanywaysforhumanconsumption.
Also,thestandarddeviationshowedvalueswhichcantakeustotheconclusionthat
milkpHvaluesdonotvaryabruptlyovertime,thusmilkcompositionbeinginfluencedbythe
weatherconditions,feedquality,maintenanceconditionsandshelterhygiene.Tosumup,the

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Lucrritiinificevol53seriaMedicinVeterinar
pH values cannot be considered as dispersed in a high degree, with normal variations and
optimaldifferencesfromaperiodtoanother.

REFERENCES

1. MaY.,BarbanoD.M.Serumproteinandcaseinconcentration:effectonpHandfreezingpoint
ofmilkwithaddedCO2,JournalofDairyScience,vol.86,2003,p.15901600.
2. CanabadyRochelle L.S., Sanchez C., Mellema M., Bot A., Desobry S., Banon S. Influence of
calcium salt supplementation on calcium equilibrium in skim milk during pH cycle, Journal of
DairyScience,vol.90,2007,p.21552162.
3. MaY.,BarbanoD.M.EffectoftemperatureofCO2injectiononthepHandfreezingpointof
milkandcreams,JournalofDairyScience,vol.86,2003,p.15781589.
4. DeMarchiM.,FaganC.C.,ODonnellC.P.,CecchinatoA.,DalZottoR.,CassandroM.,PenasaM.
Predictionofcoagulationproperties,titrableacidityandpHofbovinemilkusingmidinfrared
spectroscopy,JournalofDairyScience,vol.92,2009,p.423432.
5. Ikonen T., Morri S., Tyriseva A.M., Ruottinen O., Ojala M. Genetic and phenotypic
correlations between milk coagulation properties, milk production traits, somatic cell count,
caseincontentandpHofmilk,JournalofDairyScience,vol.87,2004,p.458467.
6. MaY.,BarbanoD.M.MilkpHasafunctionofCO2concentration,temperatureandpressurein
aheatexchanger,JournalofDairyScience,vol.86,2003,p.38223830.
7. KowalchykA.W., Olson N.F.EffectsofpH and temperatureonthe secondaryphaseofmilk
clottingbyrennet,JournalofDairyScience,vol.88,2005,p.665672.
8. GouldI.A.,FrantzR.S.SomerelationshipsbetweenpH,titrableacidityandtheformoltitration
inmilkheatedtohightemperatures,JournalofDairyScience,vol.86,2003,p.154161.
9. Ould Eleya M.M., Desobry Banon S., Hardy J. A comparative study of pH and temperature
effectsontheacidiccoagulationofmilkfromcows,goatsandsheep,JournalofDairyScience,
vol.85,2002,p.227235.

1053

RESEARCHCONCERNINGTHESEASONALVARIATIONOF
CATTLEMILKDENSITYVALUES

L.I.ILIE,L.TUDOR,AncaMariaGALI,ElenaMITRNESCU,F.FURNARIS
FacultyofVeterinaryMedicineBucharest

Abstract
Inthisstudy,wecollectedandanalyzedaseriesofmilksamples,inordertoobservethevariation
ofthedensity,duringaperiodoftenmonths.Weselectedagroupoftencows,fromwhichwe
collected one sample/week. The milk samples were analyzed using a multiparameter device,
designed to perform physicochemical analysis of milk. After obtaining the data, the average
formulaswereusedinordertoobservethevariationoftheaveragevaluespereachweekduring
a monthofstudyandper monthfor the entireperiod.The monthly average valueshad ranges
between1,0280and1,0295.Thefrequencyanalysisrevealedthefactthatonly51%(anumberof
201)ofthesampleswereconsideredappropriatebasedonthemilkstandardvaluerangesof
1,029 to 1,031. Also, using the data already obtained for this parameter, we calculated the
standard deviation. The results showed that the difference from the average value increases
duringthemonthoftransitionfromwintertospring,decreaseswhenthedairycoworganismhas
adjustedtothenewweatherconditions,increasedoverthelastmonthsofspringandduringthe
summerandautumnitpresentedareduction.Onlyduringthelastmonth,weobservedagainan
augmentation.
Keywords:milkdensity,seasonalvariation,quality,standarddeviation.

INTRODUCTION

Amongotherphysicochemicalcharacteristicsofmilk,densityhasproventobeone
of the main indicators of milk adulteration during the last century. Milk density is a species
trait,beingsituatedasgeneralvaluesbetween1,026and1,034.Cattlemilkdensitycomprises
valuesof1,029and1,031,throughadulterationbyaddingwater,thisvaluehavingatendency
ofdecreasingto1(1,4,6).
Milkdensityisgenerallyusedfor:convertingthevolumeintomassandviceversa,
estimating the solid content of milk (in some situations) and calculating other physical
properties, like viscosity (9, 10). Density values depend on: the temperature at the time of
measurement, the temperature history of milk (storage temperature), the composition
(especially the fat content) and the inclusion of air (a problem when analyzing viscous milk
products)(2,3,7,8).
Themainobjectiveofthisstudywastoobservethevariationofthedensityvalues
foraperiodof10months,byanalyzingthemilkcomingdirectlyfromtheselecteddairyunit
withacalibratedmultiparameterdevice,onceaweek.

MATERIALSANDMETHODS

A number of 10 dairy cows were selected and the milk samples were prelevated
onceaweek,foranperiodof10months,selectingeachmonththesamedates:the5th,the
12th,the19thandthe26th.Theexperimentalgroupcomprised10cowsofalmostthesameage,
with the same diet and bodyweight, and entering together at almost the same time in the
lactation period. The milk samples were collected from each cow, one per cow each week,

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Lucrritiinificevol53seriaMedicinVeterinar
withatotalof4samples/month,and40samplesperentireexperimentalperiod/cow,witha
totalof400samplesanalyzedperentireperiod/lot.
Theselectionoftheexperimentaldairycowsfollowedseveralcriteria,like:
the absence of mastitis or any kind of disease that may interfere with the milk
analysis;
abodyweightofalmost500550kg;
amilkproductionof800010000kg.
The samples were introduced in 50 ml plastic bottles, introduced at refrigeration
andanalyzedinamaximumperiodof2hoursfromthecollectionmoment.
The analysis was carried out using EcoMilk Ultrasonic Analyzer, a multiparameter
calibrateddevicedesignedforrapidanalysisofrawandprocessedmilk.Thesampleswereleft
for 510 minutes at room temperature before proceeding with the analysis, and afterwards,
using an automatic pipette with a 5 ml tip, a quantity of 15 ml from each sample was
introducedintheanalysistubes.

RESULTSANDDISCUSSIONS

Theresultsshoweddifferentvariationsduringtheexperimentalperiodoftime,with
lowervaluesduringthespringandsummermonths,andhigheronesduringautumn.Forthe
month of February, the average value of density was 1,0280, this increasing to 1,0282 and
remainingatthatpointforthenexttwomonths.InMay,weobservedanincreaseto1,0284,
possiblyduetothechangingofseason,thisconsistingofhighertemperatures,greenpasture
feeding and a different physical status of the animals. In June, the values increased again,
reachingahighvalue,of1,0294,whileinJulywehavealowervalue,of1,0290.InAugust,the
value increases to 1,0295. In the autumn months, starting with September, the value is
maintainedto1,0295.InOctober,sincetheweatherchangesabruptlyandthetemperatures
are lower, the value decreases to 1,0294. Again, in November there can be observed an
increase, to 1,0295, due to the fact that the dairy cows have succeeded in adjusting and
preserving the physiological parameters, and maintaining an optimal milk production, with
physicochemicalpropertiesthatsuitthestandards.Allthedataareshownintableno.1,fig.1
andfig.2.

Table1
Theaveragevaluesofmilkdensitypermonthandforeachweek,forthe
10dairycowsgroupselectedforanalysis

Month/Week
1
2
3
4
Monthlyaverage
February
1,0286
1,0287
1,0275
1,0273
1,028
March
1,0309
1,027
1,0275
1,0277
1,0282
April
1,0279
1,028
1,0284
1,0287
1,0282
May
1,0276
1,0283
1,0294
1,0285
1,0284
June
1,0293
1,0296
1,0297
1,0293
1,0294
July
1,0293
1,029
1,0292
1,0286
1,029
August
1,0293
1,0293
1,0298
1,0298
1,0295
September
1,0293
1,0299
1,0293
1,0295
1,0295
October
1,0291
1,0299
1,0294
1,0295
1,0294
November
1,0298
1,0292
1,0295
1,0297
1,0295

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1,0297
1,0296
1,0295
1,0294
1,0293
1,0292
1,0291
1,029
1,0289
1,0288
1,0287
1,0286
1,0285
1,0284
1,0283
1,0282
1,0281
1,028
1,0279
1,0278
1,0277
1,0276
1,0275
1,0274
1,0273
1,0272

Fig.1Theaveragevaluesvariationofmilkdensitypermonth

February
1,031

1,031

1,0305

1,0305

1,03

1,03

1,0295

1,0295

1,029

1,029

1,0285

1,0285

1,028

1,028

1,0275

1,0275

1,027

1,027

1,0265

1,0265

1,026

1,026

1,0255

1,0255

March

1,025

1,025
W1

W2

W3

April

1,031

W1

W4

1,0305

1,03

1,03

1,0295

1,0295

1,029

1,029

1,0285

1,0285

1,028

1,028

1,0275

1,0275

1,027

1,027

1,0265

1,0265

1,026

1,026

1,0255

1,0255

1,025

W3

W4

W3

W4

May

1,031

1,0305

W2

1,025
W1

W2

W3

W4

W1

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Lucrritiinificevol53seriaMedicinVeterinar
June

1,031

July

1,031

1,0305

1,0305

1,03

1,03

1,0295

1,0295

1,029

1,029

1,0285

1,0285

1,028

1,028

1,0275

1,0275

1,027

1,027

1,0265

1,0265

1,026

1,026

1,0255

1,0255
1,025

1,025
W1

W2

W3

August

1,031

W1

W4

W2

W3

W4

W3

W4

W3

W4

Septem ber

1,031
1,0305

1,0305
1,03

1,03

1,0295

1,0295

1,029

1,029

1,0285

1,0285

1,028

1,028

1,0275

1,0275

1,027

1,027

1,0265

1,0265

1,026

1,026

1,0255

1,0255
1,025

1,025
W1

W2

W3

October

1,031

W1

W4

W2
November

1,031
1,0305

1,0305
1,03

1,03

1,0295

1,0295

1,029

1,029

1,0285

1,0285

1,028

1,028

1,0275

1,0275

1,027

1,027

1,0265

1,0265

1,026

1,026

1,0255

1,0255

1,025

1,025
W1

W2

W3

W4

W1

W2

Fig.2Theaveragevaluesofmilkdensitypereachmonthandweek,forthe
10dairycowsgroupselectedforanalysis

Consideringthestandardvalues,intable2weincludedtheresultsforthefrequency
ofthevaluesthatweobtained.Fromthedata,wecanseethatfromthetotalnumberof400
samples collected and analyzed during the 10 months experimental period, a number of 27
sampleshadvaluesbetween1,026and1,027(7%),57ofthemwerebetween1,027and1,028
(14%),anumberof115hadvaluesbetween1,028and1,029(29%).From400samples,only
201(51%)couldbecategorizedasframedinthestandard,asthevaluesofdensityforcow
milk are situated between 1,029 and 1,031, therefore, a number of 118 samples had values

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between 1,0291,030 (30 %) and 83 of them presented values of 1,0301,031 (21 %), as
comprisedinfig.3and4.
Table2
Thefrequencyofthemilkdensityvaluesobtainedforthe10dairycowsgroup
selectedforanalysis(numberofsamplespereachcategory)

Month/Value
1,026
1,027
1,028
1,029
1,030
category
1,027
1,028
1,029
1,030
1,031
February
11
7
16
2
4
March
9
13
9
2
7
April
1
16
15
6
2
May
5
8
13
11
3
June
1
5
7
11
16
July
0
4
18
10
8
August
0
4
8
14
14
September
0
0
10
20
10
October
0
0
11
20
9
November
0
0
8
22
10
Total
27
57
115
118
83

November
October
September
August
July
June
May
April
March
February
0

1,026-1,027

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

1,027-1,028

1,028-1,029

1,029-1,030

1,030-1,031

Fig.3Thenumberofsamplesforeachcategoryoffrequency,permonthofanalysis

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Lucrritiinificevol53seriaMedicinVeterinar

7%
21%
14%

1,026-1,027
1,027-1,028
1,028-1,029
1,029-1,030
1,030-1,031

29%

30%

Fig.4Thepercentageforthenumberofsamplesincludedineachcategoryoffrequency

Consideringthestandarddeviationofthevaluesweobtained,thisspecificstatistic
indicator presented range values from 0,0202608 to 0,04552472, as shown in table 3. The
lowestvalueofthisindicatorwasregisteredinOctober,whilethehighestonewasobservedin
March. The latter is considered a transition month considering the seasons, from winter to
spring, while October may be regarded as a month when the specific weather conditions of
autumnmaycontributetothebodypreparationforwinter.
Table3
Thestandarddeviationvaluesformilkdensity
overthe10monthsofexperimentalanalysis

Month/Indicator
Standarddeviation
February
0,038577843
March
0,04552472
April
0,02714314
May
0,03405877
June
0,03669809
July
0,030951575
August
0,030430248
September
0,021130547
October
0,0202608
November
0,02304344
Averageperperiod
0,030781917

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Considering the differences from a month to another, we observed a major


decreasefromMarchtoApril,possiblyduetothespringspecificweatherconditionsandthe
transformations that occur in the organism. The changes considering the feed may also
contribute in a higher or lower degree. Over the next months, from April to June, we
registered an augmentation of the standard deviation value, and from June to October, a
continuousreductionofthisindicator.Thisisaverygoodindicatorwhentakingintoaccount
milkstandards,thereforeobservingagrowthofthequalityofmilkbasedonthemilkdensity
values and the standard deviation calculated for them. As for the last month of study, an
increase was registered, showing us that the diary cows are preparing for winter conditions
andalsobyconsuminganothertypeoffeed,withspecificchangesinthecompositionofmilk
andpropertiesofmilk.

Au
gu
st
Se
pt
em
be
r
O
ct
ob
er
N
ov
em
be
r

Ju
ly

Ju
ne

M
ay

Ap
ri l

h
M
ar
c

Fe
br
ua
ry

0,05
0,0475
0,045
0,0425
0,04
0,0375
0,035
0,0325
0,03
0,0275
0,025
0,0225
0,02
0,0175
0,015
0,0125
0,01
0,0075
0,005
0,0025
0

Fig.5Thestandarddeviationgraphicfordairycowsmilkdensityvalues
foraperiodof10months

CONCLUSIONS

Milkdensityrepresentsavaluableparameterusedasanindicatorwhenanalyzing
thequalityofmilk.Inthisstudy,weuseditalsoasanindicatorforthemilkqualityvariations
registered during several seasons of the year. Monthly average values varied in a range of
1,0280to1,0295,showingusaloweroverallvalueofmilkdensitythanthestandardsrequire,
taking into account that the minimum standard value of this parameter is 1,0290. Also, we
calculated the value frequency, observing that only 51 % (or 201) of the samples could be
consideredwithintherangesofthemilkstandardsforquality.Andbycalculatingthestandard
deviation of these values, for a better interpretation of the seasonal variation, we observed
that milk samples vary within ranges of 0,0202608 to 0,04552472, the monthly values
increasing during theperiodwhendairycows organism neededanadjusting to theweather
conditionsandthechangesinfeed,anddecreasing(beingclosertothestandardvalue)during
the periods of time when the parameters of the dairy cows organism maintained the same
pattern.

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Lucrritiinificevol53seriaMedicinVeterinar

REFERENCES

1.Cheeseman, T. The effect of milk composition, density and temperature on quantity


measurement,InternationalJournalofDairyTechnology,Volume31,2007,Pages6568.
2.ElSamragy Y.A., Hansen C.L., McMahon D.J. Production of ultrafiltered skim milk retentate
powdercompositionandphysicalproperties,JournalofDairyScience,volume88,2005,p.852
859.
3.KinsenllaJ.E.Physicalpropertiesoffoodandmilkcomponents:researchneedstoexpanduses,
JournalofDairyScience,volume89,2006,p.29102915.
4.PalmerL.S.,DahleC.D.Astudyofthechemicalandphysicalpropertiesofremademilk,Journal
ofDairyScience,volume88,2005,p.30683074.
5.Rice F.E., Markley A.L. The relaion of natural acidity in milk to composition and physical
properties,JournalofDairyScience,volume91,2008,p.12991306.
6.Short,A.L.Thedensityofprocessedmilk,InternationalJournalofDairyTechnology,Volume9,
2007,Pages8186.
7.StirnF.E.,ElvehjemC.A.,HartE.B.Preliminaryobservationsoncertainseasonalvariationinthe
physicalpropertiesandnutritivevalueofcowsmilkserum,JournalofDairyScience,volume88,
2005,p.42314238.
8.Tsioulpas,A.,GrandisonA.S.,LewisM.J.Changesinphysicalpropertiesofbovinemilkfromthe
colostrumperiodtoearlylactation,JournalofDairyScience,volume90,2007,p.50125016.
9.Watson, B.D., Tittler R.P. The density of milk at low temperatures, Journal of Dairy Science,
Volume90,2007,p.559567.
10. WhitakerR.,HilkerL.D.Theeffectofhomogenizationatdifferenttemperaturesonsomeof
the physical properties of milk and cream, Journal of Dairy Science, volume 89, 2006, p.3878
3884.

1061

ASTUDYONTHEBEHAVIOROFBROWNBEAR(URSUSARCTOS)
FROMZOOLOGICALGARDENTIMISOARA

C.Fl.LZRESCU1,AdinaBAIAS1,M.AFRENIE2
1
BanatsUniversityofAgriculturalSciencesandVeterinaryMedicine,Facultyof
VeterinaryMedicine,CaleaAradului,119,300645Timisoara,Romania
2
ZooGardenTimisoara
emali:cristi_lazarescu@yahoo.com

ABSTRACT
Therealizationoftheethograminordertoestimatethelevelofwellbeingisrepresentedbythe
establishment of the inventory of all the activities that form the behavioral repertoire of the
speciesthatisunderobservationofthebehavioralflux.Thestructureofthebehavioralpatternsis
geneticallydetermined.Thecaptivityaffectsmostthemechanismsthatreleaseorthatguidethe
typicalactionsofthespeciesandtoalesserextenttheirshape[2,3].Inordertofullyunderstand
andcorrecttheinterpretationsoftheobservationsitisnecessaryfortheethologisttounderstand
thenormalbehaviorofthestudiedanimals.

Keywords:brownbear,behavior,ethogram,zoogarden.

INTRODUCTION

Theaimofthisstudyistheelaborationofanethogramofthespecimensofbrownbear
(Ursusarctos)thatliveincaptivityatthezooinTimisoara.
AccordingtotheConstitutionoftheEuropeanAssociationofZoosandAquaria(E.A.Z.
A.), the zoos are permanent institutions. They are open to the public and they are publicly
managed in order to promote nature conservation and in order to ensure the education,
information and relaxation of the public, by presenting and conservation the life of wild
animals. Ensuring the demands concerning the manifestation of the natural behavior of the
animalsfromthezoosrepresentsaverycomplexanddifficulttoattainobjective[6].
Assessmentofthewelfareofanimalsisverydifficultandthereareseveralreasonswhy
weaffirmthis;firstly,becauseanimalwelfarerepresentsthestateoftheindividualconcerning
histrialtocopewithhisitsenvironment.Fromtherein,therearedifferentlevelsofwelfareto
conspecificindividual.
The realization of the ethogram in order to estimate the level of wellbeing is
representedbytheestablishmentoftheinventoryofalltheactivitiesthatformthebehavioral
repertoireofthespeciesthatisunderobservationofthebehavioralflux.Thestructureofthe
behavioralpatternsisgeneticallydetermined.Thecaptivityaffectsmostthemechanismsthat
releaseorthatguidethetypicalactionsofthespeciesandtoalesserextenttheirshape[2,4].
Inordertofullyunderstandandcorrecttheinterpretationsoftheobservationsitisnecessary
fortheethologisttounderstandthenormalbehaviorofthestudiedanimals.

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Lucrritiinificevol53seriaMedicinVeterinar
MATERIALSANDMETHODS

Inordertodrawuptheethogramforthespecimensofbrownbear(Ursusarctos)that
areincaptivityatthezoologicalgardenfromTimisoara,theirbehaviorhasbeenstructuredon
threedifferentcategories:thesolitarybehavior,thebehaviorrelatedtotheirfeedingandtheir
socialbehavior.
Everycategoryimpliesaseriesofbehavioralpatterns.Inordertoquantifythewayin
which the animal spends its time it is compulsory for the studiedanimals to be observed at
leastsixhours[6].Theanimals(amaleandtwofemales)wereunderobservationforaperiod
of60minutes,tenminutesperhours,forsixhours.Thebehavioroftheanimalswasnoted,
timedandframedintheadequatebehavioralpattern.Duringtheobservationwerenotedthe
atmospheric conditions and the possible unusual situations. The animals were under
observationbetween740and2400.Withinthebehavioralrepertoireatthebrownbearthefirst
category is represented by the behavioral activities such as sleeping, rest, performing body
hygiene, swimming, exploring the environment, urination, defecation. Another category
describesthebehaviorrelatedtofood.Thisentailsthefeedingbehavior(foodconsumption),
thefoodsearchingbehaviorandthedrinkingbehavior.Thesocialbehaviorreferstotheaction
of an individual of coming closer to another at a distance of less than three meters. It also
includes the playful behavior and the aggressive behavior. The playful behavior means
engaginginanactivityofinteractionwithoutanaggressivecharacterwithotherindividualsas
wellasotheractivitiesthatexpressarelationofclosenessbetweentwoormoreindividuals.

RESULTSANDDISCUSSIONS

Threeindividualswereunderobservation:amale(M)andtwofemales(F1,F2).Asfor
thesleepingbehavior,17periodsofsleepingwerenoted,atdifferenthours,forsevendays.In
the first day of observation, a single period of sleeping was noted to the F1 female
between12401250.Intheseconddaytwoperiodsofsleepwerenoticedaround8oclockand
from2140.Inthethirddayfiveperiodsofsleepingwerenoticedat:745755,14301440,15251535,
22152225,22502300.Inthefourthdaythreeperiodsofsleepingwerenoticedaround8oclock
and 22 and 23. Between 22 and 23 (at 23 observations were closed) the sleep was
uninterrupted. In the fifth day three periods of sleep were noticed between 10201030, 2300
2310and23452355.Inthelastofobservation,observationsweremadebetween740and1840.
Twosleepintervalswerenoticedbetween740750and9501000.Asfortherestintervals,wecan
saythyalternatewiththesleepingperiods.Mostoftherestintervalswerenoticedbetween
1130and1530.
Observationsshowthatanimalsliketosleepandrestallatthesametime.Ninetimes
outofthe17sleepintervalsthatwerenoticedanimalsweresleepingatthesametimebutnot
inthesameplace.Insevencasestwoanimalswereseensleepinginthesametimeandintwo
casestwoanimalswereactive.Thesamethingcanbenoticedintherestperiodcases.During
the observations 11 rest intervals were noticed and in seven cases the three animals were
restingatthesametime.Asforterrestriallocomotion,duringtheobservation42sequencesof
the same behavioral pattern were noticed. When animals came at a distance shorter than
threemetersthisfactwasregisteredseparatelybecauseitentailsacertainmotivation.From
the observations the male manifested this behavior towards both females. Within the 41
sequences,themaleshowedthisbehaviorin16casesaswellastheF1femalewhileF2female
showedthisbehaviorinninecases.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Asfortheswimminglocomotionandbathing,observationsshowthatanimalsgointhe
pool quite often. The pool is used for swimming, bathing and playing. Animals were seen
playinginthepoolbuttheywerealsoseenusingdifferentobjectssuchaswoodenpiecesor
tires.Theplayfulbehaviorwasalsoseenoutsidethepool.Animalswereseentogetherwhich
proves the existence ofclose relationships among the individuals. Moreover, non aggressive
manifestationswerenoticedduringtheobservation.Asfortheexplorationbehavior,itiswell
represented. It has different forms of expression and different motivations. Animals were
noticedwhensearchingfortheirfood:theydugholesandthisbehaviorisquiteknowntothis
omnivorous species. The searching food behavior is also well represented during the
observations.Itisfollowedbytheactualfoodconsumption.Theeatingintervalswerenoticed
alloverthedaywithagreaterintensitybetween12002000.Themalewasnoticedeatingafter
2200oclock.
Table1
Ethogrammodelforthebrownbear(Ursusarctos)

Typeof
Behavior
Behavior
Descriptionofthebehavior
behavior
code

I.Solitary
behavior

II.Food
related
behavior

Bodyhygiene

Ig

Animalsmakethebodyhygiene(theyarrange
theirfur,theylicktheirfur)

Sleep

Theanimaladoptsspecificpositionsforsleep
anddoesnotreacttoenvironmentalchanges

Rest

Locomote

Swim

Defecation

Urination

Eat

Searchfor
food

C.H.

Drink

Play

Closeseness

Ap

Agressive

Ag

III.Social
behavior

Theanimalstaysstillbutreactseasilyto
environmentalchanges
Theanimalmovesfromoneplacetoanother
(terrestriallocomotion)
Theanimalmakesmovesinthewater
Adoptingthecharacteristicpositionandthe
effectuationofdefecation
Adoptingthecharacteristicpositionandthe
effectuationofurination
Theanimaleatsthefoodfromhishabitat
Theanimalinvestigateshishabitatinorderto
findfood
Theanimaldrinkswaterorotherliquidsfound
inhisenvironment
Theanimalinteractswithotherindividualsin
activitiessuchas:locomotion,searching,
playingwithobjects,interactionswithoutan
agressivecharacterwithotherindividualsfrom
thesamespecies,otheractivitiesthatexpressa
relationbetweentwoormoreindividuals
Anindividualdrawscloserthan3metersto
another
Theanimalengagesinaphysicalconflictwith
anotheranimalfromhisenvironment

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Lucrritiinificevol53seriaMedicinVeterinar
CONCLUSIONS
We can conclude on the basis of our observations that all the three individuals of
brownbearbehavednormallywithinthebehavioralpatternofthespecies;
Nostereotypicalmovementsandnoaggressivemanifestationswerenoticed;
The behavioral patterns related to the exploration of the environment as well those
relatedtoplayfulbehaviorprovethattheindividualsareclosetoeachother;
Inordertoestablishatimeintervalforeachsequencefromthebehavioralrepertoireof
thespecieslongerperiodsofobservationaswellasvideocamerausearenecessary.

REFERENCES

1.
2.
3.
4.
5.
6.

BROOM D. M. (1999): Animal welfare: the concept and the issues, n: Attitudes to animals:
viewsinanimalwelfare,ed.F.L.Dolins,CambridgeUniversityPress.
BALLENGERT,L.(2002):UrsusArctos.AnimalDiversityWeb.
COCIUM.(1999):Etologie.Comportamentulanimal.EdituraALL,Bucureti.
COCIU M. (1979) Viaa n zoo. Introducere n biologia captivitii. Editura tiinific i
Enciclopedic,Bucureti.
DECUNM.(2004):Etologia,bunstareaiproteciaanimalelor.EdituraMirtonTimioara.
MAUCK, R. (2008):The Ethogram: quantifying behavior and testing hypotheses. Courses of
DepartamentofBiologyatKenyonCollege,Ohio.

1065


RESEARCHESREGARDINGIMPLEMENTATIONOFA
COMPUTERIZEDSURVEILLANCEPROGRAMOFMAMMARYGLAND
INADAIRYCOWFARM

SoranaTeodoraMatei,I.Groza,L.Bogdan,SimonaCiupe,
AnamariaPetrean,SandaAndrei

UniversityofAgriculturalSciencesandVeterinaryMedicine,ClujNapoca,Romania
FacultyofVeterinaryMedicine

ABSTRACT: The purpose of this study was to supervise the activity and health of cow
mammary gland, in a unit specialized in milk production, and implementation of a
computerizedsurveillanceprogramofmammarygland.Theresearchewascarriedout
during 20092010 on a total of 489 cows. The activity and health of cow mammary
gland was performed using the most advanced management software worldwide for
dairyfarms,AfiFarmprogramandBoviVetindicators.Cowsdiagnosedwithsubclinical
mastitis were treated with 3 therapeutic protocols: Masti Veyxym, Synulox LC and
Mastiker E. After monitoring the activity of the mammary gland, were identified a
number of 29 cows (5.93%) who noticed an increase in elctrical conductivity, ranged
between10.6and13.0MMHO.Clinicalexaminationshowedthatonlyonequarterwas
affected.Also,weobtainedalowmilkproduction,rangedbetween2.3and9.3litersof
milk.AfterasingleadministrationofMastiVeyxymanumberof19(65,52%)cowswere
cured, 5 (17,24%) cows needed two administrations of Mastiker E and the other 5
(17,24%) cows were recovered after one treatment with Synulox LC. The AfiFarm
program allows detection of mastitis diagnosis only when we correlated the cows
dynamicactivity,milkproduction,electricalconductivityandmilkingtime.

Keywords:subclinicalmastitis,diagnosis,milk,cow.

INTRODUCTION

Worldwide,mastitisisoneofthemostimportantdiseasesinmilk,continuingtobea
costlydiseasefacingdairyproducers,causingsignificanteconomiclosses.Subclinicalmastitis
is difficult to control since it is a disease caused by multiple factors [OviedoBoyso Javier,
2006].Mosttimes,mastitisnegativelyinfluencesthemilkqualityhavingconsequencesforthe
dairyindustry[Groza,2006].Latesttrendsinthemilkproductionismainlybasedonobtaining
ahighermilkqualityandlessongettingamajormilkquantity.Animportantroleinmonitoring
mammaryglandhealthandproductionofhighermilkqualityisattributed totheveterinary
service[Seegers,2003].
Thepurposeofthispaperwastosupervisetheactivityandhealthofmammarygland
in dairy cows in a unit specialized in milk production in Cluj county, and to implement a
computerprogramformammaryglandhealthsurveillance.

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Lucrritiinificevol53seriaMedicinVeterinar
MATERIALANDMETHODS

Theresearchwascarriedoutduring20092010,onatotalof489cows.Supervision
of activity and mammary gland health at the farm level was performed using a specialized
programcalledAfiFarmandBoviVetindicatorpaper.
AfiFarm program (figure 1) is the most advanced management software worldwide
for dairy farms. This program collect and develop necessary information to optimize the
productivityofanmodernfarmer`sfarm.Theapplication,developedbyIsraelispecialists(N1
inworldmilkproduction),itprovesareliablepartnerforrealfarmer,processinganddelivering
in short time complete data: milk production, herd health, milk electrical conductivity,
detectionofmastitis,milkseparation,etc.Thesoftwarerecordsandoutstandingperformance
when food its controlled, health monitoring, automatic sorting of cows for treatment or
artificial insemination, thus allowing a more efficient management. Also, efficiency increase
becauseofstaffdrasticallyreducedtoserveafarm.Keycomponentsofthemoduleare:daily
reportsonreproductionandfertilityanalysis,healthmonitoring,reportsofmilkingefficiency,
monitoring of veterinary checks, including veterinary diagnostic reports, medication and
treatments,monitoringproceduresformedicaltreatment,milkproductionplanninginterms
ofcowsnumber.

Figure1:AfiFarmcomputerprogramschedule

Bovivetpaperisanindicatorofsubclinicalmastitis.Themethodconsistinplacinga
few drops of milk on the special paper then looking for color change which indicates an
infectedquarter.Duetoacidityofmilk,thepaperwillchangecolorfromyellowtogreenor
bluegreen(figure2).
Cowstreatmentwasperformedwith3therapeuticprotocols:MastiVeyxymbased
onproteolyticenzymesandSynuloxLC,MastikerE(antibioticproducts).

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Figure2:BoviVetIndicatorpaper

RESULTSANDDISCUSSIONS
Table1ParametervaluesobtainedusingtheAfiFarmprogram
Registration
Milk
Electricalconductivity
Activity
Crt.no.
no.
(liters)
(MMHO)
(no.steps/h)
1.
145900
5.2
12.0
47
2.
124452
4.3
11.0
56
3.
125889
8.9
10.9
49
4.
125767
2.5
12.0
51
5.
144132
6.8
10.8
50
6.
238127
6.1
11.2
47
7.
145501
6.0
11.9
48
8.
125127
9.3
10.6
60
9.
125134
5.1
10.8
66
10.
125909
4.2
11.7
63
11.
124780
2.9
12.1
49
12.
125477
3.2
11.7
56
13.
145554
2.9
11.1
59
14.
125222
4.0
11.4
62
15.
125434
4.4
11.0
54
16.
125612
5.4
10.8
52
17.
125177
5.6
10.8
61
18.
145111
3.0
11.4
67
19.
145212
4.8
12.4
63
20.
125303
3.1
10.7
59
21.
145554
2.3
13.0
46
22.
145617
5.2
11.7
56
23.
145509
3.7
10.7
53
24.
125120
2.8
10.8
63
25.
145066
3.6
11.7
60
26.
136767
4.6
10.9
53
27.
134454
6.0
11.2
55
28.
145556
4.9
12.1
47
29.
145434
6.4
10.8
58

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Lucrritiinificevol53seriaMedicinVeterinar

After monitoring the activity of the mammary gland with the AfiFarm computer
programwereidentifiedanumberof29cows(5.93%)whonoticedanincreaseinelectrical
conductivity
(table1).Clinicalexaminationshowedthatonlyonequarterwasaffected.

Electrical conductivity in the 29 cows diagnosed with subclinical mastitis ranged


between 10.6 and 13.0 MMHO (graphic 1), correlated with significantly reduced milk
productioninsubclinicalmastitis.
After data provided by AfiFarm program and extensive surveillance of cattle we
obtained and low milk production. In the 29 cows that electrical conductivity was increased
(>10MMHO),milkproductionvaluesframedbetween2.39.3litersofmilk(graphic2).With
this latest software cowswere diagnosed with 23 daysbefore the occurrence of subclinical
mastitis by electrical conductivity changes, being assigned to code 9. Electrical conductivity
mustalwaysbecorrelatewithmilkproduction,becauseincreaseofelectricalconductivity,not
always occur due to subclinical mastitis. This increase may also depend on high levels of
chlorideinsomefeedandadvancedlactation.

Graphic1:Graphicalrepresentationofelectricalconductivity

Graphic2:Individualmilkproductioninthedetectionofmastitist

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Tohighlighttheevolutionofparametersincowsdiagnosedwithsubclinicalmastitis,
withAfifarmprogram,wepresentainspectionsummaryofonecase.
Cow with registration number 145554, in group 5, presented the highest value of
parametersobtained.Duringmonitoring,werecordedhighvaluesofelectricalconductivityon
days3839(11MMHO),day52(11MMHO),day68(12,8MMHO)andthehighestvaluewas
recordedonday118(13.00MMHO),milkproductiondecreasedat2.3l,andthenumberof
steps/hat46(figure3).

Figure3:GraphicalmodelsobtainedbyprocessingparametersusingtheAfiFarmprogram

AfterasingleadministrationwithenzymebasedproductMastiVeyxym,atotalof19
cows(65,52%)werecuredandatthenextmilkingelectricalconductivitydecreasedbelow10
MMHO(graphic3).
The remaining 10 cows that have not healed after the first treatment were
transferred to Synulox LC and Mastiker E (antibiotic products). The healing rate was 17,24%
aftertreatmentwithSynuloxLCproductafterthefirstadministrationand17,24%responded
positivelyafter2dosesofMastikerEproduct(graphic3).
Cowsdiagnosedwithsubclinicalmastitisandtreatedwithantibioticswerepassed
on7code,thustheyarenolongermilkedbymilkingmachine,butdirectlytothedrum.These
codesappearedonthedisplaywhichisplacedinfrontofthemachinemilkingcow,sothestaff
willknowwheretodirectthemilk.

Graphic3:Efficiencyofsubclinicalmastitisproductsusedintreatment

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Lucrritiinificevol53seriaMedicinVeterinar
CONCLUSIONS

1. Lowincidenceofsubclinicalmastitis(5.93%)ispossiblebecausethefarmimplementeda
computerizedsurveillanceprogramofmammaryglandAfiFarmprogram.
2. Diagnosis of subclinical mastitis showed an increase of electrical conductivity, ranged
between10.6to13.0MMHOandan1520%decreaseofmilkproduction.
3. Early diagnosis of subclinical mastitis allowed the recovery of 19 cows (65,52%) after the
firstadministrationofMastiVeyxymproduct.
4. Coststreatmentofmammaryglanddiseaseswereinsignificant.
5. ThroughthemostadvancedmanagementsoftwareworldwidefordairyfarmsAFIFARM
PROGRAM, the diagnosis of subclinical mastitis can be establish more precisely while a
correlation it is done between the cows dynamic activity, milk production electrical
conductivityandmilkingtime.

ThisworkwassupportedbyCNCSISUEFISCSU,projectPNIIIDEI,1482/2009

BIBLIOGRAPHY

1. BANSAL, B.K., J. HAMANN, N.T. GRABOWSKIT, K.B. SINGH, 2005. Variation in the composition of
selected milk fraction samples from healthy and mastitic quarters and its significance for mastitis
diagnosis.JournalofDairyResearch,72:144152;
2. BARKEMA,H.W.,Y.H.SCHUKKEN,R.N.ZADOKS,2006.InvitedReview:Theroleofcow,pathogenand
treatment regimen in the therapeutic successof bovine Staphylococcus aureus mastitis. Journal of
DairyScience,89:18771895;
3. DINGWELL,R.T.,LESLEIK.E.,SCHUKKENY.H.,SARGEANTJ.M.,2004Associationofcowandquarter
level factors at drying off with new intramammary infection during the dry period. Preventiv
veterinarymedicine;
4. DELRIO, D., AMANDA J. STEWART , PELLEGRINI N., 2005 A review of recent studies on
malondialdehydeastoxicmoleculeandbiologicalmarkerofoxidativestress,Nutrition,Metabolism
&CardiovascularDiseases,15:316328;
5. GROZA,I.S.icol.,Ginecologie,andrologieiobstetricveterinarCompendiu,EdituraAcademiei
Romane,2006,Bucureti;
6. OVIEDOBOYSOJAVIER,VALDEZALARCNJUANJ,CAJEROJUREZM.,OCHOAZARZOSAA.,LPEZ
MEZAE.,BRAVOPATIOA.,BAIZABALAGUIRREV.,2006TheJournalofinfection2007;54(4):399
409;
7. PICCININI, R., BRONZO V., MORONI P., LUZZAGO C., ZECCONI A , 2002 Study on the relationship
between milk immune factors and Staphylococcus aureus intrammary infections in dairy cows, J.
DairyRes.,66(4):501510;
8. SEEGERS,H.,C.FOURICHON,F.BEAUDEAU,2003.Productioneffectsreleatedtomastitisandmastitis
economicsindairycattleherds.VeterinaryResearch,34:475491;
9. SEGUYA,A.G., P.D.MANSEL,2000.An avaluation ofahandheld electricalresistancemeterforthe
diagnosis of bovine subclinical mastitis in late lactation under Australian condition. Australian
VeterinaryJournal,78:2127.
10. www.afimilk.com

1071

SPECTROPHOTOMETRICDETERMINATIONOFNITRITEAND
NITRATEINMEATPRODUCTS

MirelaMICLEAN ,CeciliaROMAN1,LauraPARLAPAN2,IoanStefanGROZA2
1
INCDOINOE2000,ResearchInstituteforAnalyticalInstrumentation,
DonathSt.,no67,400293ClujNapoca,Romania,icia@icia.ro
2
UniversityofAgriculturalScienceandVeterinaryMedicine,ClujNapoca,
MnsturSt.no.35,400372ClujNapoca,Romania,contact@usamvcluj.ro
1

Abstract
Nitrateand/ornitritesaltsconstitutethecuringagentsofredmeatand,inparticular,
processedorpreservedmeatproducts.Highdietaryintakesofnitrateandnitritehave
been implicated in the etiology of human gastric, oesophagus, colorectal and bladder
cancer. Also, nitrate and/or nitrite could act as endocrine disruptors, affecting the
androgenproductioninadultvertebratemales.
Thispaperreportsthecontentsofnitriteandnitrateinprocessedmeatsamplesusinga
spectrophotometric method, following aqueous extraction and filtration. Nitrite was
determineddirectlyusingaGriessIlosvaydiazocouplingreaction.Simultaneousnitrite
and nitrate determinations were based on nitrate reduction in a micro column
containingcadmium.Theanalysedsampleswerecommerciallyavailablemincedmeat,
sausages,Frankfurtandsalami.Thenitrateandnitritecontentsrangedfrom0.72to68
mg/kg and from 0.90 to 61 mg/kg, respectively. Also, moisture, lipid, proteinand salt
contentweredeterminedfornutritivequalitiescharacterisation.

Keywords:nitrite,nitrate,curedmeat,spectrophotometry

Meatandmeatproductsareoneofthemaincomponentsofthehumandiet.Sodium
orpotassiumnitrateand/ornitriteareusuallyaddedtomeatasapreservative.Theeffective
substance is nitrite acting as an inhibitor for some microorganisms, also it has strong
antioxidantproperties,inhibitlipidoxidation,producesthecharacteristicpinkcolourofcured
meats,andcontributetotheflavourofmeatproducts(1).Nitratemaybereducedtonitritein
rawmeatproductsbymicroorganisms(2).Nitriteis10timesmoretoxicthannitrate(3).The
redcolourisdevelopinginanumberofcomplicatedreactionstepsuntilNOmyglobin(Fe2+)is
formed(3).

Nitritecouldresultinformationofcarcinogenicnnitrosamines,byaminespresentin
meatathighertemperatures(4).Theunderlyingmechanismisendogenousnitrosation,which
in the case of nitrate must be preceded by reduction to nitrite. Nitrate and nitrite are
interconvertible in vivo. Nitrosating agents that arise from nitrite under acidic gastric
conditions react readily with nitrosatable compounds, especially secondary amines and alkyl
amides,togenerateNnitrosocompounds(3).

Theconsumptionofprocessedorpreservedorcuredmeats(sausages,ham,bacon,
hot dogs, etc.), has been related to the incidence of colorectal, gastric, oesophagus and
bladder cancer in several epidemiological studies (2, 5). In 2007, a report of World Cancer
ResearchFundandAmericanInstituteofCancerResearchrecommendedtoavoidprocessed
meats based on epidemiological studies showing increased risk of colorectal cancer with

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Lucrritiinificevol53seriaMedicinVeterinar
increasedintakeofprocessedmeats(6).Besides,nitrateshavehighertoxicologicalpotential
as they can worsen the physiological low affinity of haemoglobin for oxygen, producing
methaemoglobinaemia(7).Also,ahighintakeofnitritecanposeahumanhealthriskdueto
thepossibleallergenicandvasodilatoreffects(8).Animalstudieshaveshownthatnitrateacts
asanendocrinedisrupteraffectingtheandrogenproductioninadultmales(9).
The regulation regarding the processed food includes a series of tests determining
theirnutritionalcompositionandhowtheyrelatetoahealthydiet.Oneofthemostimportant
nutrientsisproteinandthemonitoringofitsamount,throughthedeterminationofnitrogen,
must be accurate to establish the nutritional quality of these meat products. Also, total fat
content is probably the most topical of all nutritional components in foods, endangered by
obesityandtheassociatedheartdiseaseanddiabetes.Thesaltscontainedinmeatproducts
include common salt (NaCl), nitrite and nitrate, each exerting useful properties to the food
(10).
Theaimofthisstudywastoinvestigatethemostconsumedtypesofprocessedmeat
products:mincedmeat,sausages,Frankfurt,salamiinordertodeterminetheconcentrations
ofnitriteandnitrate,duetothehealthconcern.Also,thecontentofprotein,lipid,moisture
and sodium chloride was analysed for quality control purposes. Because of the health
implication,themethodforquantificationofnitriteandnitrateshouldbefast,sensitiveand
reliable.

MATERIALSANDMETHODS

Samplingandpreparation
Thirtycommerciallyavailablemeatproductsweresampledfromlocalsupermarkets
situatedinClujNapoca,Romania:5mincedmeat,9sausages,7Frankfurtand9salamis.
All meat products were sampled freshly before lipid extraction to prevent any
hydrolysisoroxidationbeforeitsdispersioninthesolvent.Thefoodmeatproductswerecut
using a stainless steel knife, and then minced in a porcelain mortar with pestle for
homogenization.

Chemicalanalyses
All the reagents used were of analytical grade, acquired from Merck (Darmstadt,
Germany) and double distilled water produced by distiller system (GFL, Germany) was used
throughout the experiment. The nitrite stock solution was prepared at 5 g/l level in double
distilled water and stored at 4 C. The stock solution was gradually diluted at the working
concentrationlevelsandusedforexternalmultilevelcalibration.
Salts,moisture,lipidandproteincontentsweredeterminedaccordingtotheofficial
Romanianstandardizedmethodsforanalysisofmeatproducts.
Forthesalts(nitrite,nitrateandsodiumchloride)contentdetermination,the
aqueousextractofmeatproductwasperformedbytrituratingthehomogenizedmeatsample
withdistilledwaterandthenthesamplewasfilteredthroughanS&Sfilter5892(Scheicher&
SchuellGmbH,Germany)toremovethematrixcomponentspresentinthemeatsamples.
For determination, extracted nitrite reacted with GriessIlosvay reagent forming a
diazonium salt with the sulfanilic acid. This salt was coupled then to alfanaphtylamine, an
aromatic aminetoformthecolouredazodye,analyzedspectrophotometrically (CaryUVVis
spectrophotometer,Varian,USA)at520nmwavelength.Nitrateisunreactiveinthissystem.
Nitrate was reduced to nitrite with cadmium metal put in a micro column, activated with
dilutedhydrochloricacidandthendeterminedasdescribedabove.Bothanionswerepresent

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UniversitateadetiineAgricoleiMedicinVeterinarIai
inthesamemeatproduct,soextractednitritewasdeterminedfirst,thennitratewasreduced
to nitrite, which was remeasured, and the original quantity of nitrate was calculated by
difference. A single chromogenic reagent (GriessIlosvay) containing all the necessary
reactantswasusedinbothmethods.

Sodiumchlorideismeasuredbytitrationasthechlorideionthroughitsreactionwith
silverionaddedassilvernitratetoextractsofmeatproducts.
Moisturecontentwasdeterminedbydehydrationat1053Cuntilconstantweight,
in an drying oven (Memmert, Germany) with automated electronic control of temperature
andtime.
Total lipid content was determined by automatic Soxhlet extraction (Behr Labor
Technic extractor, Germany) of dried sample by repeated percolation in petroleum ether,
under reflux conditions, followedby evaporation of solvent and measuring of the remaining
lipidbygravimetricdetermination.
For protein determination, the method implied the measuring of nitrogen quantity,
duetothefactthatnitrogen ispresentin allaminoacids,andthentheuseamultiplication
factor (6.25 for meat and meat products) to calculate the quantity of protein. The used
methodwasKjeldahldigestion(DigestionSystemK431,withascrubber,BCHILabortechnik
AG, Switzerland) at high temperature with concentrated sulfuric acid, sodium sulfate and
coppersulfateascatalyst,toconvertnitrogenoussubstancesinthemeattoammoniumsalts.
Addition of concentrated alkali then converts the ammonium salts to free ammonia that is
distilled with steam and collected in hydrochloric acid containing suitable colored pH
indicators.Theacidexcessisbacktitratedtoneutralwithsodiumhydroxidesolution.

RESULTSANDDISCUSSIONS

Theoxidationofnitritetonitrateinmeatalsoexplainswhyinmeatproductstowhich
only nitrite has been added, nitrate will be found in considerable concentrations. The
concentrationsofnitriteandnitrateininvestigatedmeatproductsareshowninTable1and
Table2,respectively.

Table1
Crt.No.
Meatproduct
Concentrationofnitrite,mg/kg
Minimum,mg/kg
Maximum,mg/kg Mean,mg/kg
1
Mincedmeat
0.90
33.4
16.4
2
Sausage
8.66
51.2
25.8
3
Frankfurt
5.75
46.5
20.2
4
Salami
16.2
61.0
32.3

The concentrations of nitrite were below the maximum admitted level (MAL) in all
studied samples, according to Romanian legislation (SPC40198, SPC40298, SPC70198,
SPC70298, and SPC40698). The value of MAL was set as 70 mg/kg in all cured meat
products. The obtained mean concentrations of nitrite, in the investigated meat products,
decreasedinthefollowingorder:salami>sausage>Frankfurt>mincedmeat.

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Lucrritiinificevol53seriaMedicinVeterinar
Table2
Crt.No.
1
2
3
4

Meatproduct
Mincedmeat
Sausage
Frankfurt
Salami

Concentrationofnitrate,mg/kg
Minimum,mg/kg
Maximum,mg/kg Mean,mg/kg
0.72
26.3
11.6
0.98
68.0
26.7
1.48
22.4
12.9
1.45
54.2
21.9

Themeanconcentrationsofnitrate,intheinvestigatedmeatproducts,decreasedin
thefollowingorder:sausage>salami>Frankfurt>mincedmeat.Theconcentrationsofnitrate
werelowerthantheconcentrationsofnitriteinmincedmeat,Frankfurtandsalamiandhigher
in sausage samples. European legislation (Council Directive 95/2/EC on food additives other
thancolorsandsweeteners,Directive,1995)settheMALasaresidualamountfornitratein
meat products, at 250 mg/kg. The obtained nitrate concentrations were below MAL, for all
investigatedmeatsamples.
The mean concentrations of nitrite obtained in this study were lower than those
obtained by Hsu et al., 2009, for Frankfurt (83.9 mg/kg) and higher for minced meat and
salami and the mean nitrate concentrations recorded in the present study were lower for
Frankfurt(54.9mg/kg),salami(142.5mg/kg)andmincedmeat(18.7 mg/kg)(2).Also,mean
concentrations of nitrite obtained in the present study were higher than those obtained by
Akyuz et al., 2009, for salami (3.81 mg/kg) and sausage (7.66 mg/kg) and mean nitrate
concentrations were lower for salami (123.35 mg/kg) and sausage (296.4 mg/kg) (11). The
meanconcentrationsrecordedinthispaperwerebelowtheconcentrationsachievedbySiuet
al.,1998,insalamisamplesfornitrite(108mg/kg)andnitrate(98.5mg/kg)(12).
Themeancontentsofmoisture,sodiumchloride,fatandproteinforthecuredmeat
productsareshowninTable3.
Table3
Meanprotein
Crt.No. Meatproduct Meanmoisture
MeanNaCl
Meanfat
content,%
content,%
content,%
content,%
1
Mincedmeat

25.6

2
Sausage
46.2
1.45
30.8
15.2
3
Frankfurt
59.2
2.06
19.5
12.7
4
Salami
52.0
2.54
26.2
13.5

For minced meat, the contents of moisture, salt and protein were notregulated by
Romanianlegislation.Meanfatcontentvariedintherange23.031.45,withanaveragevalue
of25.6%andalltheobtainedvalueswerebelowMAL(32%)setbyRomanianlegislation.
Forsausagesamples,thecontentsofmoisture,salt,fatandproteinrangedbetween
36.560.7%,0.942.40%,27.535.2%and11.618.2,respectively.Inonesamplethecontentof
lipidswashigherthanMAL(32%).ThevaluesofMALformoistureandsalt,andtheminimum
admitted level for protein set by the Romanian legislation were: 63%, 3%, and 11%,
respectively.
In Frankfurt samples the values were in the range 50.475.2% for moisture, 1.38
2.73%forNaCl,17.324.1%forlipidsand8.7914.5%forproteincontent.Inonesample,the
moisturecontentwashigherthanMAL(70%)andinanothersampletheproteincontentwas
lowerthantheminimumadmittedlevel(11%).
Insalamisamplestheobtainedvaluesvariedinthismanner:48.271.0%formoisture,
1.682.66% for salt, 23.636.8% for fat and 10.214.8% for protein content, respectively.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Similar to Frankfurt products, in two salami samples the moisture content was higher than
MAL(68%)andinanothersampletheproteincontentwaslowerthantheminimumadmitted
level(12%).

CONCLUSIONS

The contamination with nitrite/nitrate has the potential to alter the endocrine,
nervous and immune systems of the organism, resulting in changes in gene expression and
phenotypes, therefore the addition of nitrite/nitrate in processed meat must respect the
maximumadmittedconcentrationsforasafehumanconsumption.
Inthisstudy,the contentsofnitriteandnitrateinprocessed meatproduct(minced
meat, sausages, Frankfurt and salami) were determinedusing a spectrophotometric method
following aqueous extraction and filtration for removing the interfering matrix components
suchasproteinandfatbasedsubstances.Thestudiedspectrofotometricmethodrepresentsa
fast,reliable,sensitiveandlowcostanalysisofnitriteandnitrateincuredmeatproduct.The
highestmeanconcentrationsofnitrite(32.3mg/kg)andnitrate(26.7mg/kg)wereregistered
insalamiandsausages,respectively,andallthevalueswerebelowMALsetbylegislation.
Most of the analytes concentrations in studied meat products complied with the
Romanianregulations,exceptforfivesampleswheremoisture,proteinorlipidcontentwere
notconformtothelegislation,loweringthenutritionalqualityoftheproducts.

BIBLIOGRAPHY
1. ZhangX.,KongB.,XiongY.L.,(2007).ProductionofcuredmeatcolorinnitritefreeHarbinred
sausagebyLactobacillusfermentumfermentation,MeatScience,77,593598
Hsu J., Arcot J., Lee N. A., (2009). Nitrate and nitrite quantification from cured meat and
vegetablesandtheirestimateddietaryintakeinAustralians,FoodChemistry,115,334339
3. KarlOttoHonikel,(2008).Theuseandcontrolofnitrateandnitritefortheprocessingofmeat
products,MeatScience,78,6876
4. SebranekJ.G.,BacusJ.N.,(2007).Curedmeat productswithoutdirectadditionofnitrate or
nitrite:whataretheissues?,MeatScience,77,136147
5. Ingested nitrates and nitrites, Available at: http://monographs.iarc.fr/ENG/Meetings/94
nitratenitrite.pdf
6. Milkowski A., Garg H. K., Coughlin J. R., Bryan N. S., (2010). Nutritional epidemiology in the
context of nitric oxide biology: A riskbenefit evaluation for dietary nitrite and nitrate, Nitric
Oxide,22,110119
7. MerusiC.,CorradiniC.,CavazzaA.,BorromeiC.,SalvadeoP.,(2010).Determinationofnitrates,
nitrites and oxalates in food products by capillary electrophoresis with pHdependent
electroosmoticflowreversal,FoodChemistry,120,615620
8. Marco A., Navarro J. L., Flores M., (2006). The influence of nitrite and nitrate on microbial,
chemicalandsensoryparametersofslowdryfermentedsausage,MeatScience73,660673
9. HansenP.R.,TaxvigC.,ChristiansenS.,AxelstadM.,BobergJ.,KiersgaardM.K.,NellemannK.,
HassU.,(2009).Evaluationofendocrinedisruptingeffectsofnitrateafterinuteroexposurein
rats and of nitrate and nitrite in the H295R and Tscreen assay, Toxicological Sciences, 108,
437444
10. HuiY.H.,NipWK,RogersR.W.,YoungO. A.,(2001).Meat scienceandapplications,Marcel
DekkerInc.,NewYork,USA,p.108115
11. Akyuz,M.,Ata,S.,(2009).Determinationoflowlevelnitriteandnitrateinbiological,foodand
environmentalsamplesbygaschromatographymassspectrometryandliquidchromatography
withfluorescencedetection,Talanta,79,900904
12. Siu,D.C,Henshall,A.,(1998).Ionchromatographicdeterminationofnitrateandnitriteinmeat
products,JournalofChromatographyA,804,157160
2.

1076

STUDIESCONCERNINGLEPTINSINFLUENCEONTHESCCIN
BUFFALOMILK

M.MIHAIU1,ALEXANDRALAPUSAN1,,CRINATEODORACARSAI2,
CARMENJECAN1,ROMOLICAMIHAIU2,S.D.DAN1
1)
UniversityofAgriculturalSciencesandVeterinaryMedicine,FacultyofVeterinaryMedicine,
MnturstreetNo.3,400372,ClujNapoca,Romania,email:m.mihaiufmv@yahoo.com
3)
BabesBolyaiUniversity,FacultyofEconomicsandBusinessAdministration,ClujNapoca,
Romania,2)UniversityofAgriculturalSciencesandVeterinaryMedicine,FacultyofAnimal
HusbandryandBiotechnology

SUMMARY
The progress in animal breeding may be improved by combining traditional performance data
with molecular genetic information. Leptin is a protein hormone expressed in the mammary
glandofvariousspecies,includinghuman(SmithKirwinetal.,1998),mouse(Aokietal.,1999),
cattle(Silva etal.,2002;Smithand Sheffield, 2002; Chelikani et al., 2003; SayedAhmedet al.,
2003;Barthaetal.,2005),andsheep(Laudetal.,1999;Bonnetetal.,2002).Theinfluenceofthis
hormoneonmilkproductiontraitswasstudiedatcattles(Buchananetal.,2002),goats(Whitley
N. C. et al. 2009),, sheep (Zhou H. et al. 2009). The presence of leptin in milk raises questions
concerningtheabilityofthemammaryepithelialcellstotransferleptinfromthebloodand/orto
synthesizeit,beforeitssecretion.Inordertogainabetterinsightandunderstandingofleptins
influence on milks quality traits this paper focuses on the possible variations of the SCC
accordingtothegeneticpolymorphismofthishormone.Themethodusedinidentifyingleptins
genotype was PCRRFLP, and SomatosM for assessing the SCC. After the PCR we managed to
identifythefavorablealleleCandTofleptingene.Thisstudyconfirmstheresearchesdonein
this field which concluded also that this hormone may be a factor that influences production
traits.TheassociationbetweenleptinpolymorphismwithmilksqualityindicatorssuchasSCC,
suggeststhatthismarkermaybeusefulforselectionbasedonmolecularinformation.

Keywords:leptin,polymorphism,buffalo,milk,SCC.

MATERIALSANDMETHODS

The research has taken into study 43 milk samples, collected and processed from
buffalosraisedinprivatehouseholdsfromBrasov andSalajcounties.Themethodsused for
DNA extraction from milks somatic cells, were the following: DNA extraction from blood
using the solutions from the extraction kit; the DNA extraction from the somatic cells using
the rapid extraction with PBS. After the DNA extraction we identified the electrophoresis
profile of leptins gene using the PCR RFLP technique. This technique consists in repeated
cycles of heating and cooling so as to melt and enzimatically replicate the DNA. The short
fragmentsofDNAthathavecomplementarysequences,alongwithaDNApolymeraseregion
are the key components in allowing the selective and repeated amplification. By the PCR
method we obtain a progress, in generated DNA this being used as a replication model,
establishing in movement a chain reaction in which the DNA pattern is exponentially
amplified.ThePCRcanbemodifiedonalargescalesoastomakealargeamountofgenetic
manipulations.
The DNA extraction was done according to the following steps: 40l raw milk was
centrifuged at 2000 rotations/min for 30 minutes. After centrifugation the pallet was

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Lucrritiinificevol53seriaMedicinVeterinar
immersed in one ml PBS(phosphate buffersaline), pH 7,4, and centrifuged again at 4000
rot/minute10minutes,andthenwesuspendedagainthepalletina49mllysessolution(0,32
M sucrose, 10mMTrisHCl, 7,5mM MgCl1%, TritonX100). Afterwards we centrifuged the
palletsnucleiat2465gxg10minuteandthenwashedthemtwicewith10mlNaCland0,025M
EDTA.Thepalletwassuspendedagainin3ml10mMTrisHClsolution,pH8,and2mMEDTA,
afterwhichweadded100ml10%SDSand40mlproteinaseK910mg/ml).Thenucleislysiswas
incubated for an hour at 65oC. After incubation we added 500ml NaCl, the precipitated
proteinswerecentrifugedat1800gxg10minutes.Weadded6mlisopropanolafterwhichwe
centrifugedfor10minutesat1800gxg..Thepalletwasdriedataroomtemperatureandre
dissolved in 500ml TrisEDTA(10 Mm TrisHCl, 1mM EDTA solution , pH 7,5. After following
thesestepsthetotalquantityoftheDNAextractedfrommilkwasinbetween14.2ng/l156.8
ng/l.Thismethodisnotsoindicatedbecauseitinvolvestheusingofsometoxic,cancerous
solventslikephenolchloroform,isoamilicalcohol,etc.
The rapid extraction of the DNA from milk and colostrum was realised in the
followingway:2mlofmilkfromeachsamplewereputinatubeandafterwardsweadded
0,4mlPBSsolution,thencentrifugedit45timesat3000rpmfor20minutes.Afterthisstep
weleftthesamplesinabathwaterfor15minutestimeinwhichthesomaticcellsarebroken
down and freeing the DNA quantity. The DNA quantity obtained was then read at the
Nanodrop spectrophotometer. The DNA extracted from milk using this method was in
between67ng/l347ng/l.
After the DNA extraction we processed the samples according to the following
protocol:
InanEppendorftubeweput16.2lwater,2.5lbuffer,1lDNTP,primers(amcrse):
sense primer 1l, antisense primer 1 l; TAQpolymerase 0.3l, MgCl21l, extracted
genomicDNA.WeusedtheamplifyingprogramatTermocyclerthattakes1x95oC5minutes
(the alteration) the attaching of the primers in several stages at 40x95oC1 minute, at
40x64oC1minuteweretheattachmenttakesplaceusually,andat40x72oC1minute.After
thisattachmentprotocolthefinalextractiontakesplaceat1x72C10minutes.
The enzyme restriction was done with the Kpn21 (BspEi) enzyme and followed the
nextsteps:weaddedH2O6,5ml,afterwhichatamponsolution2,5mlandafterwardsKpn21
(BspEi)enzyme1l.Weobtainedareactionmixforthesamplesusedandthenweadded20l
PCRproductappliedbythePCR.
The enzyme has an activity at 55o C; the samples were put in the thermostat for 4
hours in a water bath. The restrictions role was to digest the amplified fragment from the
leptinsgeneinordertodifferentiatetheimplicatedalleleinthebuffalosmilkquality.
The DNA samples migration was made by an electrophoresis reaction in agars gel,
forcoloringandvisualizingtheproductsweused2lethidiumbromide.
InordertoanalyzetheSomaticCellCountnumberfromthesesamplesweusedthe
SomatosMandFossomaticmachine.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
RESULTSANDDISCUSSIONS

AftertheDNAextractionfrommilkweobtainedvaluesrangingbetween14.2ng/l
347ng/l.

Fig.1.DNAquantityvaluesobtainedfromthebuffalomilksamples

In the majority of the samples we obtained the electrophoresis profile by gel


migrationandatthereadingofthebandsrevealedinthegelweobservedtheappearanceofa
singleclearbandof94bpcharacteristictoTTgenotype,twoclearbandsof75bpand19bp
characteristictoCCgenotypeandthreeclearbandsat94pb,75pb,19pbcharacteristictoCT
genotype. Allele C from cytosine and allele T from thiamine are obtained by a cytosine
thiamine mutation. Following this mutation the two alleles are differentiated, representing
three genotypes: CC, TT, and CT. According to the protocol described by Buchanan et al.
(2002), after PCR reaction with specific primers from gene leptin we should obtain the
followingfragments:thelengthoftheamplifiednonrestrictedfragmentshouldbeof94bp
andfollowingtherestrictionwiththeKpn2Irestrictionenzymeweshouldobtainthefollowing
fragments: TT with 94 bp the non restricted alleles by the enzyme, CT which will have the
fragments75bp,94bpand19bpandCCgenotypewith75bpand95bp.

LTTTTTTTTTTTTTTTTTTTTLCTCTCTCTCTCTCTCT
19bp

94bp

75bp
94bp

Fig.2:Theelectrophoresisprofileof94Fig.3:Theelectrophoresisprofileof94bp,
basepairfromleptingenebpand19bpofleptingene
LCCCCCCCCCCCCCC

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Lucrritiinificevol53seriaMedicinVeterinar

75bp,19bp

Fig.4:Theelectrophoresisprofileof75bpand19bpofleptingene
At the analyisis of the SCC we noticed that the differences in the values of the
samplesweresignificant.Wementionthefactthatallthesampleswerekeptunderthesame
temperature conditions and the SCC was analysed as soon as possible in order to obtain a
representativevalue.

Fig.5TheSCCvaluesobtainedfromthebuffalomilksamples

Comparing these SCC values to the genotypes of each individual, buffalos


homozygousfortheTalleledemonstratedanincreasednumberinSCClinearscoreincontrast
withthebuffaloeshomozygousfortheCallele,andheterozygousCT.Inthefieldstudies,TT
genotypewasfoundtoberesponsibleforahighermilkyieldandqualitytraits.HigherSCCcan
besymptomaticformastitisinfectionbutalsothismayreflectapossibleincreaseinmastitis
incidence associated with higher milk yield influenced by leptin or the role of leptin in
modulatinganimmuneresponse.
Ourstudyconfirmstheresearchesdoneinthisfieldwhichconcludedalsothatthis
hormonemaybeafactorthatinfluencesproductiontraits.

CONCLUSIONS

InthepopulationstudiedtheindividualshomozygousforTTgenotypearein
majority,theCTandCCgenotypebeingfoundonlyin12samplesfrom43examined
ThesamplesthathadahigherSCCwerecorrespondenttotheoneswithTTprofile.
Theassociationbetweenleptinspolymorphismwithmilksqualityindicatorssuchas
SCC,suggeststhatthismarkermaybeusefulforselectionbasedonmolecularinformation.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
BIBLIOGRAPHY
1.
2.
3.

4.
5.

6.

7.
8.

9.

AokiN.,M.KawamuraandT.Matsuda,Lactationdependentdownregulationofleptinproduction
inmousemammarygland,Biochem.Biophys.Acta(1999),pp.298306.
BarthaT.,A.SayedAhmedandP.Rudas,Expressionofleptinanditsreceptorsinvarioustissuesof
ruminants,Domest.Anim.Endocrinol.(2005),pp.193202.
M.Bonnet,C.Delavaud,J.RouelandY.Chilliard,Pregnancyincreasesplasmaleptininnulliparous
butnotprimiparousgoatswhilelactationdepressesit,Domest.Anim.Endocrinol.(2005),pp.216
223.
ChelikaniP.K.,D.R.GlimmandJ.J.Kennelly,Shortcommunication:tissuedistributionofleptinand
leptinreceptormRNAinthebovine,J.DairySci.(2003),pp.23692372.
LaudK.,I.Gourdou,L.Belair,D.H.KeislerandJ.Djiane,Detectionandregulationofleptinreceptor
mRNAinovinemammaryepithelialcellsduringpregnancyandlactation,FEBSLett.(1999),pp.
194198.
SayedAhmedA.,S.E.Elmorsy,P.RudasandT.Bartha,Partialcloningandlocalizationofleptinand
leptinreceptorinthemammaryglandoftheEgyptianwaterbuffalo,Domest.Anim.Endocrinol.
(2003),pp.303314.
SilvaL.F.P.,M.J.VandeHaar,M.S.WeberNielsenandG.W.Smith,Evidenceforalocaleffectof
leptininbovinemammarygland,J.DairySci.(2002),pp.32773286.
SmithKirwinS.M.,D.M.OConnor,J.DeJohnston,E.D.Lancey,S.G.HassinkandV.L.Funanage,
Leptinexpressioninhumanmammaryepithelialcellsandbreastmilk,J.Clin.Endocrinol.Metab.
(1998),pp.18101813.
SmithJ.L.andL.G.Sheffield,Productionandregulationofleptininbovinemammaryepithelial
cells,Domest.Anim.Encocrinol.(2002),pp.145154.

1081

STUDYCONCERNINGMOLDOVARIVERWATER
QUALITYINNEAMTCOUNTY

ElenaMitranescu1,CatalinaLala2,F.Furnaris1,L.Tudor1,
Marianaerbea3,D.Mitrnescu4,VioletaSimion5

1
UniversityofAgronomicalSciencesandVeterinaryMedicine,FacultyofVeterinaryMedicine,
105Spl.IndependenteiStreet,Bucharest,Romania;mitranescuelena@gmail.com
2
RocheRomania.SRL
3
NationalSanitaryVeterinaryandFoodSafetyAgency
4
PenitentiariesGeneralDepartment,Bucharest
5
SpiruHaretUniversity,FacultyofVeterinaryMedicine

Abstract
Water is a complex environment where aquatic organisms live, whose chemical composition
resultsfromcomplexinteractionsbetweenatmosphere,hydrosphereandbiosphere.
Moldova River water quality, in Neamt County area, was established based on water samples
collectedfromtwosections:TimisestiandRoman.
Fromthecollectedsamplestherewereassessed:physicalindicators(temperature,pH),chemical
indicators(dissolvedoxygen,OBC5,COCMn),nutrients(NNH4+,NNO2,NNO3totalphosphorus,
totalammonianitrogen),generalions,salinity(fixedresidue,Cl,SO42,Ca2+,Mg2+,Na+,Fe,Mn),
metals(Cr,Cu,ZnanddangeroussubstancesCd,Hg,Ni,Pb),micropollutants(cyanides,phenols,
anionicdetergents),andbiologicalparameters.
Waterqualitydependingonthevaluesobtainedforthefollowedparameterswasrangedintofive
qualitycategories.
Physicalchemical parameters were assessed using NOVA 60 photo colorimeter and results
interpretationinaccordancewithOrder161/2006.
Moldova River along Neamt County territory is ranged, according to the physical chemical
parameters, within the class I throughout the analyzed territory, except for nutrients in section
TimisestiandoxygensysteminRomansection,whicharesuitableforClassIIofquality.
ThebiologicalelementsrangeMoldovaRiverwaterwithinClassIIofquality.
Moldova water pollution on Neamt County territory, in Roman section, is based on the alluvial
deposits presence, the pollution of Roman city and the effluents of two wastewaters treatment
plants.

Keywords:qualityparameters,water,maximumadmittedlimits

INTRODUCTION
Water is an essential element of life. Considered as an environmental factor, water
hasafundamentalrolebyitsactiveparticipationinthephysicalandchemicalprocessesfrom
air and soil and by the involvement in the main atmospheric phenomena that determine
differenttypesofclimate.
Water is used as source of raw materials, source of driving power in industry,
transportingallthewaste,beingaworkingtooletc.
Providing quantitative and qualitative intake of water for human and animal
consumption of natural water sources (underground, surface, and meteoric) and the
vulnerability of water from these sources to different types of pollution require monitoring
andassessingwaterquality.

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Lucrritiinificevol53seriaMedicinVeterinar
In this context, monitoring and characterization activity of the aquatic environment
state is an important tool in water integrated management, providing protection and
sustainableuseofthewaterasthemainenvironmentalfactor.

MATERIALSANDMETHODS

In this paper, Moldova Rivers water quality was assessed along Neamt County
territorybytakingwatersamplesintwosections:TimisestiandRoman.
Assessing the ecological status of Moldova river water was done according to the five
ecologicalstateswhichexpressfiveclassesofwaterquality:ClassIverygood,classIIgood,
ClassIIImoderate,ClassIVlow,ClassVpoor.
Surfacewaterqualityassessmentandclassificationinthe5qualityclasseswasdonebasedon
maximumadmittedlimitscorrespondingtoeachclassasfollows:
ClassIindicatesalowcontaminationlevel,classIIamoderatecontaminationandClassIII,IV
andVreflectacontaminationfrommoderatetoveryhigh.
FortheassessmentofMoldovaRiverswaterqualityonchemicalbasis,thefollowinggroupsof
indicatorswereanalyzed:
1.Physicalindicators(temperature,pH)
2.Oxygenregime:(dissolvedoxygen,OBC5,COCMn)
3.Nutrients(NNH4+,NNO2,NNO3,totalphosphorus,totalammonianitrogen)
4.Generalions,salinity(fixedresidue,Cl,SO42,Ca2+,Mg2+,Na+,Fe,Mn)
5.Metals(Cr,Cu,ZnanddangeroussubstancesCd,Hg,Ni,Pb)
6.Micropollutants(cyanides,phenols,anionicdetergents).
In order to establish the ecological status of Moldova River was used the saprob index
calculation.PhysicalchemicalparameterswereassessedusingNOVA60photocolorimeter.
ResultsinterpretationwasinaccordancewithOrder161/2006.

Resultsanddiscussions
Table1
ThedynamicsofbiologicalindicatorsinmonitoringsectionsofMoldovaRiverswaterin2007
Indicators
Control
section

Date

Phytoplankton
Taxons
no.

Timieti

Taxons

No./mx104

no.

Zoobenthos
Taxons

No./m

no.

07.05

27

1.070.000

14

2.850

20

13.10

23

2.650.000

23

160.750

210

25

1.860.000

19

81.800

115

12.03

28

475.000

08.05

30

740.000

18

11.400

45

14.08

17

840.000

18

9.500

30

14.10

20

1.725.000

28

62.000

105

24

945.000

21

27.630

60

Averagevalue

Roman

No./l

Phytobenthos

Averagevalue

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UniversitateadetiineAgricoleiMedicinVeterinarIai
MoldovaRiveralongNeamtcountyterritorycrossestheMoldavianPlatformdeposits
which,geologically,arecomposedofrelativelyhorizontalSarmatiandeposits.
The results of qualitative and quantitative analysis of Moldova River's phytoplankton,
phytobenthosand zoobenthos in the 2 sections, Timisesti and Roman, are shown inTable 1
andTable2.

Investigationshaverevealedthepresenceof39planktonictaxonsinTimisestisectionand53
inRomansection,andzoobenthos:8speciesinsamplesfrombothsections.
The averagevaluesofthesaprobindexshowagoodecological statusfortheanalyzedriver
section,accordingtothebiologicalqualityelements.
Table2
ThedynamicsofsaprobindexvaluesinmonitoringsectionsofMoldovaRiverswaterin2007
Phytoplankton
Phytobenthos
Macrozoobenthos
Control
Date Saprob Class EcologicalSaprob Class EcologicalSaprob Class Ecological
section
of
of
of
index
status index
status index
status
quality
quality
quality
07.05 1,83 II

Good

1,95 II

Good

2,5

13.10 1,70 I

Very
good

1,56 I

Very
good

1,89 II

Good

1,77 I

Very
good

1,76 I

Very
good

2,20 II

Good

12.03 1,95 II

Good

08.05 1,95 II

Good

2,21 II

Good

2,54 III

14.08 1,94 II

Good

2,43 III

Moderate 3

14.10 2,31 III

2,03 II
Moderate

Good

2,12 II

Good

Good

Good

2,55 III

Moderate

Timieti

Averagevalue

III

Moderate

Moderate

Roman

Averagevalue

2,04 II

2,22 II

III

Moderate

MoldovaRiverwatertemperatureandpH(averagevalues)areshowninTable3.

Table3
WatertemperatureandpHaverageannualvaluesforMoldovaRiverNeamtCounty
Assessedparameters
Globalqualityclass
Controlsection No.ofsamples
Temperature (C) pH
Timieti

220

13,2

8,2

Roman

232

10,5

8,3

Maximumadmittedlimitaccording Notdefined
toOrder161/2006

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Lucrritiinificevol53seriaMedicinVeterinar
WaterpH,accordingtotheadmittedlimits,isconsideredclosetothemaximumlimit.
AveragevaluesoftheindicatorsrepresentingoxygenregimegrouparepresentedinTable4.

Table4
Averagevaluesofthechemicalindicators(OxygenrregimeOR)inMoldovawaterNeamt
County
Assessedparameters
Global
Samplingpoint

No.samples

Dissolvedoxygen

CBO5

CCOMn

(mg/l)

(mg/l)

(mg/l)

quality
class

Timieti

50

10,12

2,02

1,81

Roman

62

6,18

4,5

6,66

II

limit 7

10

II

20

III

20

50

IV

<4

>20

>50

IV

Maximum

admitted

according
toOrder161/2006

Analyzingdatafromthetableitisnoticedthat,intermsofdissolvedoxygen,biochemicaland
chemicaloxygenconsumption,waterfromTimisestisectionisincludedinclassIofqualityand
thewaterfromRomansectioninclassII.
In Roman section, Moldova River's water suffers a slight pollution caused by the alluvial
deposits,andtheeffluentsfromthetwowastewatertreatmentplantsPetrotubsiDanubina.
InTable5areshownthenutrients'averagevaluesinthe2sectionsofMoldovaRiver.
Table5
AveragevaluesofnutrientsinMoldaviaRiverwater,NeamtCounty
Assessedparameters
Global

quality
NNH4+ NNO2
NNO3
Ntotal
Ptotal
Samplingpoint
class
(mg/l)
(mg/l)
(mg/l)
(mg/l)
(mg/l)
Timieti
Roman
Maximum
admittedlimit
according
to
Order
161/2006

1,05
0,38
I 0,4
II 0,8
III 1,2
IV 3,2
V >3,2

0,03
0,007
0,01
0,03
0,06
0,3
>0,3

2,17
0,09
1
3
5,6
11,2
>11,2

5,88
1,5
1,5
7
12,0
16,0
>16,0

0,36
0,0192
0,15
0,4
0,75
1,2
>1,2

II
I
I
II
III
IV
IV

According to the data in the table, nutrients (ammonia, nitrites, nitrates, nitrogen and total
phosphorus)wererecordedvaluescorrespondingtoClassIIofqualityinTimisestisectionand
toClassIofqualityinRomansection.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Theaveragevaluesoftheindicatorsfromthesalinitygroup(fixedresidue,chrome,sulphates,
calcium,magnesium,sodium,iron,manganese)rangeMoldovaRiverwaterfrombothsections
withinclassIofquality(Table6).

Table6
TheannualaveragevaluesofphysicalchemicalparameterinMoldaviaRiver
Control
section

Final
classof
quality

Assessedparametes
Fixed
residuum
(mg/l)

Cl

SO42

Ca2+

Mg2+

Na+

Fe

Mn

(mg/l)

(mg/l)

(mg/l)

(mg/l)

(mg/l)

(mg/l)

(mg/l)

Timieti

392

20,2

28,03

22,8

1,88

18,5

0,29

0,043

Roman

480

24,04

54

35,8

5,99

19

0,24

0,028

Maximum
admitted
limit
according
to
Order
161/2006

500

25

60

50

12

25

0,3

0,05

II

750

50

120

100

50

50

0,5

0,1

III

1000

250

250

200

100

100

1,0

0,3

IV

1300

300

300

300

200

200

2,0

>1300

>300

>300

>300

>200

>200

>2,0

>1

Forthemetalschemicalindicatorsgroup,respectivelycopper,chrome,zinc,cadmium,nickel
and lead the average values are presented in Table 7. Values obtained for all parameters
determinedinthetwosectionsrangewithinClassIofquality.
Table7
AveragevaluesofthemetalsinMoldovaRiverwater,NeamtCounty
Assessedparameters
Global
Section
quality Cu
Cr
Zn
Cd
Ni
Pb
class (g/l) (g/l) (g/l) (g/l)
(g/l) (g/l)
Timieti
Roman
Maximum
admitted
limit
according
to
Order
161/2006

I
I
I
II
III
IV
V

14,02
13,62
20
30
50
100
>100

18,66
18,92
25
50
100
250
>250

55,22
82,17
100
200
500
1000
>1000

0,50
0,32
0,5
1,0
2,0
5,0
>5,0

6,55
7,22
10
25
50
100
>100

4,88
4,22
5
10
25
50
>50

From the micro pollutants group there were determined cyanides, phenols and anionic
detergents.Micropollutants'averagevaluesareprovidedinTable8.

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Lucrritiinificevol53seriaMedicinVeterinar

Table8
AnnualageragevaluesofMP(micropollutants)fromMoldovaRiver,Neamtcounty
Indicatorideterminai
Generalqualuy
Anionic
Section
Cynides
Phenols
classa
detergetns
(g/l)
(g/l)
(g/l)
Timieti
Roman
Maximum admitted
limit
according
to
Order161/2006

I
I
I
II
III
IV
IV

0,006
0,0028

0,01

0,22
0,55
1
5
20
50
>50

37,22
37,1
100
200
300
500
>500

Analyzingthedatafromthetable,itcanbenoticedthatallthreeindicatorsassessedinboth
sections,thevaluesobtainedbelongtoclassIofquality.

CONCLUSIONS

1. Average concentrations recorded for the indicators: oxygen regime, degree of


mineralization and micro pollutants of Moldova River's water Timisesti section are
appropriatetoclassIofquality,andforthenutrientstoclassIIofquality.
2.MoldovaRiver'swaterRomansectionbelongstoclassIofqualityregardingthenutrients,
degreeofmineralization and micropollutants,while regardingtheoxygenregimethewater
belongstoclassIIofquality.
3. Biological elements of quality (phytoplankton, phytobenthos and zoobenthos) frame the
generalwaterqualityforbothsectionsintoclassII.
4. Moldova River's water pollution in Roman section is based on both natural geological
conditions(alluvialdeposits)andthepollutioncamingfromRomancity,andontheeffluents
ofthetwowastewatertreatmentplantsPetrotubandDanubiana.

REFERENCES

1.
2.
3.
4.
5.

Daoud,J.R.,Amin,A.M,ResidualanalysisofsomemetalsinwaterandAnacronisniloticusfish
frompolluatedareas,VeterinarymedicaJournalGiza,47,(3),351365,Egipt,1999
Elena Mitranescu, Furnaris, F. et al., Impactul poluarii industriale asupra calitatii apelor de
suprafatadinjudetulDambovita,AlXleaCongresNationaldeMed.Vet.,sept.2007,pg.232233
Elena Mitranescu et al., Results concerning the surface water quality in Braila area, Haikou
ichukHABMimehiC.3.UKOSOtom8no,2(29),acmua4,Ucraina,2006,pp.235237
Rojanschi,V.,FlorinaBran,Diaconu,Gh.,Protectiasiingrijireamediului,Ed.Economica,Bucuresti,
1997
Teusdea,V.,ElenaMitranescu,Protectiamediului,editiaIV,EdituraOmegaPrint,2007

1087

RESEARCHESCONCERNINGFISHWELFARE
INAFARMFROMSOUTHERNAREAOFROMANIA

ELENAMITRANESCU1,F.FURNARIS1,L.TUDOR1,MAGDAGONCIAROV1,
VIOLETASIMION2,S.BUSTANI1,ADRIANAORASANU3

1
UniversityofAgronomicalSciencesandVeterinaryMedicine,FacultyofVeterinaryMedicine,
mitranescuelena@gmail.com
2
SpiruHaretUniversityBucharest,FacultyofVeterinaryMedicine
3
NationalInstituteofDiagnosisandAnimalHealth

Abstract
Inthemodernaquaculture,fishoptimumwelfareimpliesensuringrearingconditionswhichallow
the fish to maintain homeostasis, to a normal somatic development and to be protected from
stress. The present paper aims to assess fish welfare level in a fishery from southern area of
Romania populated with common, silver and grass carp based on water chemical and
microbiologicalqualityandfishbiochemicalserumprofile.Therewereharvestedwatersamples
from3pondsandtherewereestablishedthephysicalchemicalparameters(pH,dissolvedoxygen,
residual chlorine, total phosphorum, sulphates, nitrates, nitrites, phenols, copper and total
ferrum),aswellasmicrobiologicalparameters(AerobicPlateCountAPC,FungalPlateCount
FPC and total coliforms). The water physicalchemical parameters analysis were done by using
Spectroquant Nova 60 fotocolorimeter and the microbiological ones by using Compact Dry TC
(Total count), YM (Yeast and Molds) or CF (total coliforms) rapid assays. In adittion, from each
pondwereharvested2fishfromwhichtherewascollectedbloodbycaudalveinpuncture(CVP),2
ml blood/fish. The serum biochemical profile has been established by using Vettest 8008, being
analyzed 8 blood chemistry indicators: albumine, phosphatemia, uric acid, cholesterol,
triglycerides,glucose,aspartateaminotransferaseandalaninetransaminase.
The researches led to the following conclusions: concerning chemical water quality, there were
registeredforchlorinesexceedingoftheadmittedlimitsforcarprearingupto10timesandfor
phenols the admitted limits from 1146/2002 Order (water in the fifth quality class); concerning
themicrobialload,totalcoliformsare2.74.9timeshigherthanthereferencevalueforsecond
qualityclasswateraccordingto1146/2002Order;thewaterinapropriateparametersreflectsina
serumprofilemodification:noticableisanexceedingoftransaminasesactivity,mostlikelydueto
phenolstoxicity.

Keywords:fish,welfare,waterparameters,biochemicalserumprofile

Fishfarmingisanageoldactivityandinpracticefromancienttimes.Presentday,this
branchregisteredaremarkabledevelopmentrate,duetotheneedtoadoptmoreproductive
meanstofeedincreasingEarthpopulation.
Because fish capacity to feel pain was proved by many scientific researches, farming
thesespeciescouldnotbeconceivedwithoutassuringtheoptimumwelfarelevel,whichcould
beachievedonlyunderacontinuousmonitoring.
If in other species of zootechnical interest there are present nowadays practical,
numerical systems for animal a welfare assessment, which permit making comparisons
betweendifferentfieldsituationsandlivestocks,infishsuchsystemsstillarenotstructured.
However, afishwelfareassessmentsystemwillincludebothethologicalparameters(animal
based)andrearingconditionparameters(engineeringbased:waterquality,fishdensityetc.),
thelatestbeingofmainimportanceinassuringqualityoffishlife.Thefishbloodbiochemical

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Lucrritiinificevol53seriaMedicinVeterinar
profileanalysisraisesmanyproblemsandcouldnotbeyetappliedonlargescaleforwellness
screeninginfisheries.
The present study aimed to assess welfare level in a fishery from southern area of
Romania, populated with common carp, silver and grass carp, based on water chemical and
microbiologicalqualityandtorelatewaterquality,asfishlifeenvironment,withbiochemical
serumprofile.
MATERIALSANDMETHODS

Inordertoassessthefishwelfare,fromthethreepondsofthefisherywereharvested
watersamplesandtherewereanalyzedphysicalchemicalparameters:pH,dissolvedoxygen,
residual chlorine, total phosphorum, sulphates, nitrates, nitrites, phenols, copper and total
ferrum,aswellasmicrobiologicalparameters:AerobicPlateCountAPC,FungalPlateCount
FPC and total coliforms. The physical chemical parameters analysis was done using
Spectroquant Nova 60 fotocolorimeter and the results were compared with limits for carp
rearingorwithlimitsprovidedby1146/2002Orderforsurfacewatersquality.
In order to establish APC, FPC and total coliforms, there were used rapid assays,
respectivelyCompactDryTC(Totalcount),YM(YeastandMolds)orCF(totalcoliforms).The
results, namely total coliforms, because for APC and FPC are not provided limits in national
law,werecomparedwiththeprovisionsof1146/2002Order.
Thesecondpartofthestudyimpliesestablishingtheserumbiochemicalprofile.There
were harvested by angling 2 fish (silver carp individuals: Hypophthalmichthys molitrix) from
each pond and after Finquel anestezy (MS 222) 100200ppm concentration in water, they
were emersed and blood was collected by caudal vein puncture approcah (CVP): from each
fishmaximum2mlofblood.Aftersyneresis,therewasestablishedthebiochemicalprofileon
1:10dilutedsamplesbyusingVettest8008.Therewereanalyzed8bloodchemistryindicators:
albumine, phosphatemia, uric acid, cholesterol, triglycerides, glucose, aspartate
aminotransferaseandalaninetransaminase.
The results were compared with the values in the literature, although often
contradictory.

RESULTSANDDISCUSSIONS
Theresultsofwaterphysicalchemicalanalyzeareshowninthefollowingtable.

Table1.Averagevaluesofwaterphysicalchemicalparameters
Sampling
point

pH

Fishpond1
Fishpond2
Fishpond3
Maximum
admitted
limits for
carps
rearing

7.7
8.1
7.8
5.5
8

Assessedparameters
Cl2
NO3
NO2
Phenols Cu
Total
O2
P
SO42
(mg/l) (mg/l) total
(mg/l) (mg/l) (mg/l) (mg/l)
(mg/l) Fe
(mg/l)
(mg/l)
9.1
0.3
1
26
20
0.035
0.27
0.13
0.19
12
0.27
1
29
20
0.022
0.26
0.14
0.13
6.7
0.2
0.8
20
19
0.021
0.39
0.09
0.16
*
Min.
0.01
0.61
N/A
50
0.2
617
0.3
2
4
0.03
ng/l

Values up to 80 mg/l framed the water quality in first class according the 1146/2002 Order
Values higher than 0.05 mg/l (50 g/l) framed the water quality in the fifth class according the
1146/2002 Order

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UniversitateadetiineAgricoleiMedicinVeterinarIai
The water meets the requirements for an optimum carp rearing for the most
parameterswithtwoexceptions:chlorineswhichexceedtheadmittedlimitsforcarprearing
upto10timesandphenolswhichregisteredexceedingevenhigher,framingthewaterwithin
thefifthqualityclassaccordingto1146/2002Order.Thehigherconcentrationsofphenolsare
infishpond2and3:0.26mg/landrespectively0.39mg/l.
Thisexceedingmostlikelyaffectsfishwelfare.Thus,alongtimeexposureto0.040.2
mg/lactivechlorineconcentrationmayresultinnervoustroubles,agitation,jumpsoutsidethe
water,convulsions,lateralposition,spasmodicmouthmovements,finsuncoordinatedmoves,
respirator troubles and eventually death. The skin and gills are covered with a thick mucus
layer,couldappearbranchialcongestionorhaemorragies.
A nervous toxic to fish, phenols presence in ecosystem is due to the activity of
chemical, petrol or pharmaceutical industries also could be related with the production and
degradation of numerous pesticides. Phenol toxicity is related with two main processes:
unspecifiedtoxicityrelatedwithhydrophobocityoftheindividualcompoundandformationof
freeradicals.

Concerning the water microbiological parameters (table2), there wasnoticed a 2.7


4.9 times exceeding of the reference value for second quality class water according to
1146/2002Order.
Table2
Averagevaluesofwatermicrobiologicalparameters
Samplingpoint
Assessedparameters
Totalcoliforms
APC
FPC
(no./ml)
(colonies/ml) (colonies/ml)
Fishpond1
490
500
51
Fishpond2
270
495
21
Fishpond3
315
525
25
Admitted
limits 500/100ml:QualityclassI
N/A
N/A
according
the 10000/100 ml: Quality class
1146/2002Order
II
Not provided: Quality class
IIIV

Itwasalsobeingrecordedahighnumberofcoloniesforaerobicplatecountandfungal
platecount.Thehighmicrobialloadmustberelatedtoanimaloriginpollution.

Microorganisms find in water appropriate conditions to develop. They can have


autochthonorigin,formingspecificbiocenoses(planktonical,neustonical,benthal,epibiotical)
and allochthon origin (those which come accidentally in the water from soil, carried by the
wind or from precipitations' water, generated by men, animals or industrial activities or
ubiquitary living everywhere: in air, on soil or in water). From the autochthon
microorganisms,couldbementionedbacteria,fungi,aswellasprotozoansandalgae.Inwater
are present all the physiological categories of bacteria (heterotrophic, autotrophic,
chemoautotrophicetc.).Theyeastsandmoldsfromthewatercouldresultfromsoilorthe
pathogeniconesfromillorganisms(plantsoranimals).

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Lucrritiinificevol53seriaMedicinVeterinar
Table3.Serumprofileofthefishsampledintheassessedfishery

Bloodparameter

Pond
Fishpond1

ALB
Albumineg/dl

Fishpond2
Fishpond3
Fishpond1

PHOS
Phosphatemia
mg/dl

Fishpond2
Fishpond3
Fishpond1

URIC
Uicacidmg/dl

Fishpond2
Fishpond3
Fishpond1

CHOL
Cholesterolmg/dl

Fishpond2
Fishpond3
Fishpond1

TRIG
Triglyceridesmg/dl

Fishpond2
Fishpond3
Fishpond1

GLU
Glucosemg/dl

Fishpond2
Fishpond3
Fishpond1

AST
Apartate
aminotransferase
U/l

Fishpond2
Fishpond3
Fishpond1

ALT
Aanine
TransaminaseU/l

Fishpond2
Fishpond3

Sample
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample2
Sampe1
Sample 2
Sampe1
Sample2
Sampe1
Sample2

1091

Obtainedvalue
1.
0.
2.
2.
1.0
1.1
19
15.6
22.1
20.7
16.1
23.9
0.6
0.8
2.1
2.3
0.9
0.4
278
100
242
243
86
63
317
92
236
233
94
98
237
113
190
206
250
153
29
55
25
29
49
129
393
473

590
620

Referencelevel

0.450.85

6.29.3

N/A

150240

N/A

70130

2342

130350

UniversitateadetiineAgricoleiMedicinVeterinarIai
In table 3 there is shown the serum biochemical profile of silver carp individuals
sampled in the three ponds of the fishery. We used as reference values the ones from the
controlbatchesintheliterature,althoughoftencontradictory.
Atthefirsthand,couldbenoticedahighvariabilityoftheresults,thisfactsuggesting
thatindividualshavedifferentbodyconditions,somehavingsurelypoorbodyconditionand
welfare.
Iftheserumphosphateandalbuminemodificationsismostlikelycausedbytheintense
musculareffortassociatedwithfishcollecting(byangling)andrestraining,doubledbyamild
hemoconcentration caused by maintaining the fish emersed, the transaminases and glucose
increasing are caused by phenols toxicosis, having in view that the higher transaminases
values: 620 UI/l ALT and 89 U/l AST mean value are registered for fish collected from third
pond,themostcontaminatendwithphenols(0,39mg/l).
These modifications of serum profile shows hepatopancreatic disorder, hence a poor
fishwelfare.

CONCLUSIONS

1. Concerningchemicalwaterquality,therewereregisteredforchlorinesexceedingof
theadmittedlimitsforcarprearingupto10timesandforphenolstheadmittedlimits
from1146/2002Order(waterinthefifthqualityclass).
2. Concerning the microbial load, total coliforms are 2.7 4.9 times higher than the
referencevalueforsecondqualityclasswateraccordingto1146/2002Order.
3. The water inappropriate parameters reflects in a serum profile modification:
noticeable is an exceeding of transaminases activity, most likely due to phenols
toxicity.
4. Thefishwelfarelevelispoor,soimmediateactionmustbetakeninordertoimprove
it:waterdecontaminationfromphenolsisessential.

ACKNOWLEDGMENTS

This study has been financed and is a part of ID_285 grant CNCSIS UEFISCSU, Idei
290/2007contract:Researchesconcerningthewelfareoffishinfarms,duringtransportation
andinslaughteringunits

REFERENCES

1. ManCornel,ManAdrian(2006)Igienapiscicola,EdituraRisoprintClujNapoca
2. Michaowicz J., W. Duda Phenols: Sources and Toxicity, Polish J. of Environ. Stud.
Vol.16,No.3(2007),pp.347362
3. Mitranescu Elena et al. WATER QUALITY AS FISH WELFARE INDICATOR IN POTOCI
FISHFARM,ScientificWorksCSeriesVet.Medicine,LV(1),2009,ISSN12225304,pp.
161165
4. RadovanKoppetal.ModulationofBiochemicalandHaematologicalIndicesofSilver
Carp (Hypophthalmichthys molitrix Val.) Exposed to Toxic Cyanobacterial Water
Bloom,ACTAVET.BRNO2010,79:135146
5. TeusdeaV.Bunastareasiprotectiaanimalelor,EdituraOmegaPrint,Bucuresti,2005
6. *** http://news.bbc.co.uk/1/hi/sci/tech/ 2983045.htm Kirby A. (2003) Fish do
feelpain,scientistssay

1092

RESEARCHESREGARDINGTHESENSITIVITYTOANTIBIOTICSOF
SOMEBACTERIALSTRAINSISOLATEDFROMMASTITISCOWMILK

G.C.Nad,N.Fi,F.Chiril,S.Rpuntean,V.Rus
UniversityofAgriculturalSciencesandVeterinaryMedicineClujNapoca
35MnturStreet,FacultyofVeterinaryMedicine,gnadas@usamvcluj.ro

Abstract:Thisstudyconsidered22mastitiscowmilksamplesthatwereexaminedusing
bacterioscopic, bacteriologic and biochemical tests regarding the bacterial genera
involved in mastitis etiology. For each isolate, the sensitivity to antibiotics such as
Enrofloxacin, Oxitetracicline, Amoxicillin, Ceftiofur, Erythromycin, Amoxicillin
Clavulanate and Tetra Delta using the diffusimethric standardized method were also
determined. Bacteria genus most frequently isolated were represented by
Staphylococcusspp.in11(50%)samples,followedbyStreptococcusspp.in9(40,9%),
Escherichiacoliin5(22,7%),Bacillusspp.in4(18,18%),Micrococcusspp.in1(4,54%)
and Candida spp. also in 1 sample. Regarding the sensitivity to antibiotics, the most
efficientwasAmoxicillinClavulanatewithanaverageoftheinhibitionareadiameterof
21 mmfollowedbyEnrofloxacinwith17,90mmdiameter,Amoxicillinwith17,68mm,
TetraDeltawith17,18mm,Erythromycinwith13,77mm.Attheoppositepolesituated
Oxitetraciclinewithanaverageof10,5mmdiameterandCeftiofurwith6,5mm.

Keywords:mastitis,cow,bacteria,antibiotics.

INTRODUCTION

Antimicrobialagentsarewidelyusedforthetreatmentofbovinemastitis,respiratory
tract infections and diarrhea in cattle. During acute infections and outbreaks of infectious
diseaseingroupsorherdsitisimportanttouseaneffectiveantimicrobialtreatmentasearly
aspossible.Thisempiricaltreatmentisgenerallybasedonknowledgeoftheresistancepattern
of the different bacterial pathogens to antimicrobial agents used in the particular animal
species.Antimicrobialresistanceisanincreasinglyimportantproblemamongseveralbacterial
species causing infection in animals and humans in recent years. The problem for some
bacterialspeciesissocriticalthatthereisfewtreatmentoptionsleft.
Theinitialtreatmentofanimalsiscommonlybasedontheexperienceregardingthe
expected resistance of the infectious agent. The fact that occurrence of antimicrobial
resistancevariesbetweencountriesandregionshasthepotentialtocomplicatethatmatter.
Furthermore,knowledgeofexpectedresistanceislimitedbythesmallproportionofdifferent
bacterialpathogensfrominfectedanimalsthatactuallyareinvestigatedfortheirantimicrobial
resistancepattern.
The intensive use of antibiotics in both public health and animal husbandry has
selectedforantibioticresistantbacteria.Underantibioticselectivepressure,bacteriahavethe
ability to develop and exchange resistance genes, making them nonsusceptible to the
antimicrobial substances deployed. While antibiotic resistance has emerged in some
important animal and human grampositive pathogens, such as Staphylococcus and
Streptococcusspp.andClostridiumperfringens,others,suchasBacillusanthracis,arecurrently
stillsensitivetoantibiotics.

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Lucrritiinificevol53seriaMedicinVeterinar
Theaimofthestudywastheidentificationofthemostefficientantibioticthatisto
be used in case of acute mastitis, prior to the time elapsed for the isolation and sensibility
testsareavailable.

MATERIALSANDMETHODS

The researches took place between March 2009 and April 2010 within the
Microbiology Laboratory of the Faculty of Veterinary Medicine ClujNapoca. 22 mastitis cow
milksamplescollectedinsteriletubeswereculturedonglucoseagarandbloodagar,andfor
eachsampletheidentificationofthebacterialspecieinvolvedinmastitisetiologywasmade
by microscopical, cultural and biochemicalexamination.For eachbacteria isolated, the tests
regarding its sensibility to antibiotics was realized using the diffusimethric method. The
suspensionfromthetestedgermat1tubeofMcFarlandscaleturbiditywassowedonaplate
with glucose agar, then the surface dried, followed by the antibiotics positioning by the
standardized method. The antibiotics used were represented by Enrofloxacin (ENR),
Oxitetracicline (OXT), Amoxicillin (AML), Ceftiofur (CFT), Erythromycin (ERY), Amoxicillin
Clavulanate (AMC) and Tetra Delta (NPSN). After an incubation of 24 hours at 37C the
interpretationwasmadebymeasuringtheinhibitionareasaroundeachmicrodisc.

Results and discussions: The bacterial species isolated from the samples taken into
studyandtheirsensibilitiestoantibioticsarepresentedintable1.

Table1.Mastitisetiologyandgermssensibilitytoantibiotics
Sample
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

Mastitisetiology
Streptococcusspp.+B.cereus
Streptococcusspp.
Staphylococcusspp.
Streptococcusspp.
Staphylococcusspp.
Streptococcusspp.
Escherichiacoli
E.coli+Staphylococcusspp.
E.coli+Streptococcusspp.
Streptococcus+
Staphylococcus
Streptococcus+
Staphylococcus
Escherichiacoli
Staphylococcusspp.
Staphylococcusspp.
Staphylococcusspp.
E.coli+Candidaspp.
Staphylococcusspp.
Bacillusspp.+Micrococcus
spp.
Staphylococcus+Bacillusspp.
Streptococcusspp.
Staphylococcus+Bacillusspp.
Streptococcusspp.

Sensibilitytoantibiotics(inhibitionareadiameterinmm)
ENR
OXT
AML
CFT
ERY
AMC
NPSN
16
28
R
R
18
20
12
12
13
18
R
R
20
R
27
R
11
16
20
16
20
16
16
20
R
18
22
25
18
19
30
18
24
31
32
18
R
20
14
28
24
28
25
R
15
R
R
17
R
18
15
14
16
17
18
R
13
R
16
R
R
18
12
13
13
20
16
20
25
16
23

18

26

19

22

30

24

24
15
15
17
R
17
27

18
R
R
R
R
14
22

11
15
19
16
18
24
R

R
R
14
R
R
14
R

10
R
14
R
16
R
18

16
18
28
18
22
21
R

12
15
25
20
24
16
26

25
15
20
20

18
R
15
22

33
20
11
32

R
R
R
16

23
16
20
19

35
17
16
30

22
R
16
23

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UniversitateadetiineAgricoleiMedicinVeterinarIai

BacteriagenusmostfrequentlyisolatedwererepresentedbyStaphylococcusspp.in
11 (50%) samples, followed by Streptococcus spp. in 9 (40,9%), Escherichia coli in 5 (22,7%),
Bacillusspp.in4(18,18%),Micrococcusspp.in1(4,54%)andCandidaspp.alsoin1sample.
TheisolationofStaphylococcusspp.in50%ofthesamplesisnotasurprisesincetheliterature
dataispresentingthesameisolationpercentage.Thereisalsotheissueofisolatingmostlyin
persistent bovine mastitis of small colonies of Staphylococcus spp., so called dwarf colonies,
important contributor to the prolonged survival of Staphylococcus spp. in some cases of
mastitis.

Chart1.Averageoftheinhibitionareadiameterfortheantibioticstested
25
20

21
17.9

17.68

ENR
17.18

13.77

15
10.5
10

OXT
AML
CFT

6.5

ERY
AMC

NP-SN

0
Average of the inhibition area diameter

Aspresentedinchart1,themostefficientantibioticwasAmoxicillinClavulanatewith
anaverageoftheinhibitionareadiameterof21mm,followedbyEnrofloxacinwith17,90mm
diameter, Amoxicillin with 17,68mm, Tetra Delta with 17,18mm, and Erythromycin with
13,77mm.AttheoppositepolesituatedOxitetraciclinewithanaverageof10,5mmdiameter
andCeftiofurwith6,5mm.

CONCLUSIONS
1. Bacteria genus most frequently isolated from the samples of mastitis cow milk was
Staphylococcusspp.in11(50%),followedbyStreptococcusspp.in40,9%,Escherichia
coliin22,7%,Bacillusspp.in18,18%,Micrococcusspp.andCandidaspp.in1(4,54%).
2. The best sensibility to antibioticswas observed for Amoxicillin Clavulanate followed
byEnrofloxacin,AmoxicillinandTetraDelta.

BIBLIOGRAPHY
1.
2.
3.
4.

AttalaH.,etall,2008,CharacterizationofaStaphylococcusaureussmallcolonyvariant(SCV)
associatedwithpersistentbovinemastitis;FoodbornePathog.Dis.;%(6):785799.
BengtssonB.,etall,2009,Antimicrobialsusceptibilityofudderpathogensfromcasesofacute
clinicalmastitisindairycows;VetMicrobiol.;136(12):1429.
Perreten V. et all, 2005, MicroarrayBased Detection of 90 Antibiotic Resistance Genes of
GramPositiveBacteria;JClinMicrobiol.;43(5):22912302.
Roy JP. et all, 2009, Efficacy of a 5day extended therapy program during lactation with
cephapirin sodium in dairy cows chronically infected with Staphylococcus aureus, Can Vet J.
50(12):12571262.

1095

EVALUATIONOFPHOSPHORUSLEVELSINLAMBBY
LUCERNEHAYFEEDINGTESTS

AritinaNEAGU,V.CRIVINEANU,G.V.GORAN
F acu ltat ead e Med ic in Veterinar B uc u re t i,
S p l a iu l Indepen d e n e i ,n r . 105, sect or 5, 050097, Buc ure t i,
ggoran@fmvb. ro

ABSTRACT
Themaingoalofthisstudywasmonitoringthephosphoruslevelsinlambbyvariouslucernehay
feedlotfeedingtrials,asaresultoffertilizingornotthesoil.Fertilizationthesoilwithphosphate
influence the feeding value of the forage, determining improved performance of animals fed
fertilized hay compared to unfertilized hay of lower phosphorus content and sometimes toxic
accidents.
Hayproducedonvirginsandydesertedsoilwhichcontained0.10%phosphoruswasunpalatable
andproducedpooranimalgains,feedutilizationanddepressedserumphosphoruscontent.The
additionofdisodiumphosphatetothishayimprovedanimalgains,feedutilizationandincreased
thebloodserumphosphorus.Haycontaining0.15%phosphoruswasapparentlyequalinfeeding
value to hay containinggreater amounts of phosphorus. An increased retention of phosphorus
wasobtainedbyaddingdisodiumphosphatetoalowphosphorushayinonetest,inanother,the
addition of disodium phosphate to unfertilized hay resulted in increasing the phosphorus
retentionuptothatofheavilyfertilizedhay.

Keywords:phosphorus,fertilizer,poisoning,sheep

Phosphorus deficiency in soils and the forage produced there from and a
phosphorosisingrazinganimalshasbeenwidelyrecognizedinmanypartsoftheworld.The
use of phosphate fertilizers as a means of correcting soils low in phosphorus is a common
practice. Some authors have summarized the literature on the effects of fertilization with
phosphate on the feeding value of the forage, and they reported improved performance of
animals fed fertilized hay compared to unfertilized hay of lower phosphorus content, and
sometimestoxicaccidents.
Fertilizertrialsdemonstratemarkedyieldincreaseswhenlucernewasfertilizedwith
phosphoruscontaining fertilizers. Farmers generally believe that the feeding value of the
lucerneisimprovedaswellastheyieldasaresultoftheapplicationoffertilizer.Thispaper
reportsaseriesoftestsconductedataprivatefarmtoexploredifferencesinfeedingvalueof
lucerneproducedonsoilswithvariousfertilizationhistory.

MATERIALANDMETHODS

Chemicalanalysesandfeedlotfeedingtrialswereusedtotestthevariousfeeds.To
maintain continuity the various trials are reported chronologically, however, certain
techniques were common to all the work and will be reported first to avoid repetition. In
feeding trials all animal weights were obtained after 24 hours without feed and 12 hours
withoutwater.Allanimalswererandomlyassignedtotreatments.Sevendaypreliminaryand
7daycollectionperiodswereused.Phosphorusdeterminationsinallmaterialwerebasedon
ICPOES method. Other chemical analyses were made by usual methods. Blood samples

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Lucrritiinificevol53seriaMedicinVeterinar
collected from animals on test were refrigerated, the serum collected and transmitted for
analysis.

FirstSheepFeedingTest.Inthefirstsheeptestthehaysthatwerehighphosphorus
hay produced at the farm from fertilized soil and the low phosphorus hay produced on the
virgin deserted soil were ground and pelleted for the test. In addition, the low phosphorus
unfertilizedhaywassupplementedwithdisodiumphosphatetobringthephosphoruscontent
uptothelevelfoundinthefertilizedhay.Anadditionaltwolotswerefedhaygrownonaplot
whichhadpreviouslyshowna phosphorusfertilizerresponsebuthadnotbeenfertilizedfor
fiveyears.Onelotwasfedunsupplementedpelletedhaycontaining0.15%phosphorus.The
other lot was fed the same hay but with sufficient disodium phosphate added to bring the
phosphorusleveluptothefertilizedfarmhay(23%P).Phosphorusandcalciumlevelsofthe
pelletsfedinthisfirstsheeptestareshownintable1.
Second Sheep Feeding Test. As the results of the above trial were not conclusive another
attempt was made to obtain hay from the area where the original deserted soil hay was
grown.Inthemeantimetwogrowingseasonshadpassedandtheownershipofthelandhad
changed.Thenewownerwasmostcooperativeandsetasideanareaforexperimentaluse.He
understoodthat45kgofliquidphosphate(52%P)hadbeenappliedtotheareatwoseasons
beforeandknewthat90kghadbeenappliedthepreviousgrowingseason.

Table1
Compositionoffeedsusedinfirstsheepfeedingtest(drybasis)

Unfertilized
Unfertilized
Unfertilized desertedsoil
Fertilized
Unfertilized
farmhay
desertedsoil
hay
farmhay
farmhay
+
hay
+
phosphorus
phosphorus
P content of
0.23
0.15
0.22
0.10
0.23
hay,%
Calcium,%
1.57
1.66
1.57
5.37
5.26

Toobtainforagefromthisareaofvaryingphosphoruscontents,theareawasdivided
into3plots.Abouthalftheareawasleftunfertilized;onefourthwasfertilizedwith270kgof
treble super phosphate per acre and the remaining onefourth with 105 kg of treble super
phosphate.Thethirdcuttingoflucernefromtheareawasusedforthesecondsheepfeeding
trial.
All plots were cut at the same time when the lucerne was at abrupt the onetenth
bloomstage.Thehaywasrakedcarefullysoastopickupas little sand aspossible. Thehay
was then baled, ground and pelleted. After grinding, the hay from the unfertilized area was
divided into two lots. One lot received added disodium phosphate to bring the phophorus
contentuptothatofthehaygrownontheplotreceivingthelargestamountoffertilizer.The
compositionofthepelletedfeedisshownintable7.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Table2
Compositionoffeedsusedinsecondsheepfeedingtest(drybasis)

Heavily**
fertilized
desertedsoil
hay

Lightly*
fertilized
desertedsoil
hay

P content of
0.22
0.17
hay,%
Calcium,%
1.40
1.30
*105kgtreblesuperphosphateperacre.
**270kgtreblesuperphosphateperacre.

Unfertilized
desertedsoil
hay

Unfertilized
desertedsoil
hay
+
phosphorus

0.15

0.25

1.20

1.10

RESULTSANDDISCUSSIONS

As pointed out in other publications, evaluation of forages from soils receiving


differing treatments involves many complications. While phosphorus was the most obvious
differenceinthecompositionofthehaysinvolvedinthisseriesoftrials,otherconstituentsof
the feed also varied. The extreme unpalatability of this hay cannot be explained with the
informationavailable.
The addition of disodium phosphate to this hay before pelleting improved its
palatability only slightly. The increased gains, feed efficiency and blood serum phosphorus
levelsresultingfromtheadditionofthedisodiumphosphateclearlyindicatethatphosphorus
wasatleastoneofthereasonsforthepooranimalresponsetotheoriginalhay.
Thelackofanimalresponsetotheadditionofdisodiumphosphatetotheunfertilized
farmhayandthedesertedsoilhayinthesecondlambfeedingtrialappearstoindicatethat
0.15% phosphorus is not limiting for growing lambs. The response to the disodium in the
originaldesertedsoilhay(firstlambfeedingtrial)suggeststhat0.10%phosphorusislimiting.
Thedailyintakeofphosphorusonthedesertedsoilhaywasmuchlowerbecauseofthelower
feedintake.Inthefirstlambfeedingtrialsthelambsonthefeedcontaining0.15%phosphorus
ate1,53kgoffeedperdaytogetapproximately2.31gPdailyorabout2.75gper45kgbody
weight.Inthesecondlambfeedingtrialthelambsate1,77kgoffeedperdaytoget2.67gP
dailyor4.09gper45kgliveweight.Ontheunfertilizeddeserthayinthefirsttrialwhenthe
lambsonlyconsumed1kgofthehaycontaining0.10%phosphorustheirdailyintakewasonly
1.02 g or 1.46 g per 45 kg body weight. Some authors state that 0.15% is the minimum
phosphoruslevelforfatteninglambsandthat2.17gdailyper45kgliveweightwillmeettheir
phosphorus requirement. It is doubtful if the depressed intake was due to a phosphorus
deficiency in the animals, however, because the sheep showed an aversion to this hay
immediatelyratherthanafterbeingfedonitlongenoughtodevelopadeficiency.
First Sheep Feeding Test. In addition to the differences in phosphorus content the
unfertilized hay from the deserted soil was much higher in calcium (See also table 2). The
resultsofthelambfeedingtrialareshownintable3.There wasessentiallyno differencein
theperformanceofthelambsonthetwofarmhaysevenwithdisodiumphosphateaddedto
the hay which was somewhat lower in phosphorus. This appears to indicate that under the
conditionsofthistrial0.15%phosphoruswasadequateforthelambs.
Theunfertilizeddesertedsoilhaywasunpalatableeveninpelletedform.Theaddition
of disodium phosphate increased its palatability only slightly as judged by the average daily

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Lucrritiinificevol53seriaMedicinVeterinar
feedintake.Lambsfedthishayusedtheirfeedmoreefficientlythanthelambsfedthesame
haywithoutphosphorus.
Theaveragedailygainwassignificantlyimprovedbytheadditionalphosphorus,yet
bothlotsreceivingthedesertedsoilhaystillgainedverysignificantlylessthanthelambsfed
thevariousformsoffarmhay.Thefeedrequiredper45kgofgainismarkedlyinfavorofthe
lambsfedthefarmhays,however,whenthefeedefficiencyisexpressedonthebasisofthe
organic matter required per 45 kg of gain the deserted soil hay with the supplemental
phosphoruscomparesfavorablywiththeefficiencyofthefarmhay.
Table3
Resultsoffirstsheepfeedingtest(112days)

Fertilized
farmhay

Unfertilized
farmhay
+
phosphorus

Unfertilized
farmhay

Unfertilized
desertedsoil
hay

Numberof
9
10
10
9
lambs
Av.initial
29.2
28.2
27.9
28.44
weight,kg
Av.final
51.57
47.47
47.83
34,56
weight,kg
Av.dailygain,
0.19
0.17
0.18
0.05a
kg
Av.dailyfeed,
1.55
1.53
1.56
1.01
kg
Feedper45kg
351
400
396
837
gain,kg
Salt
consumption
0.45
0.9
0.27
2.2
perlamb,kg
Bloodserum
phosphorus

(mg/100mlsg)
Initial
6.48
6.45
6.09
6.20
Final
6.11
6.67
7.11
4.41c
a
Gainsignificantlyless(P<0.05)thanunfertilizeddesertedsoilhayplusphosphate
b
Gainsignificantlyless(P<0.01)thanonfarmhays
c
Serumphosphorussignificantlylower(P<0.01)thanotherlots.

Unfertilized
desertedsoil
hay
+
phosphorus
10
28.35
39.46
0.09b
1.04
476
1.35

5.67
7.86

The final blood serum phosphorus values for the lot fed the unfertilized desert hay
were significantly lower than the values for the other lots. The addition of the disodium
phosphatetothishaypreventedthisdropinbloodphosphorus.Thesaltconsumptionofthe
variouslotsinthistrialwasmeasured.Thesaltconsumptionperlambwasmarkedlyhighin
the lot fed the unsupplementedunfertilized deserted soil hay. The addition of the disodium
phosphatereducedthesaltintakeappreciablybutitwasstill higherthaninthe lotsfed the
farmhay.Itisinterestingtonotethattheadditionofdisodiumphosphatetothefarmhayalso
reduced the salt intake compared to the lambs fed the same hay without the additional
disodiumphosphate.Perhapstheadditionalsodiuminthedisodiumphosphatedecreasedthe
saltneedofthesheep.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
All sheep completed the trial on the farm hays without incident, but trouble was
encounteredingettingthesheeptoeataconstantintakeofthedesertedsoilhayeitherwith
orwithouttheaddedphosphorus.
After repeated efforts two sheep finished on the unfertilized deserted soil hay and three
finished on the same hay plus the disodium phosphate. The phosphorus retention by the
lambs fed the deserted soil hay plus sodium phosphate was significantly higher than the
retention by the lambs fed the other rations. The calcium retention was very significantly
higher by the lambs fed the deserted soil unfertilized hay both with and without the
supplementalphosphorus.
SecondSheepFeedingTest.Thephosphoruscontentofthisunfertilizedhaywasnot
as low as that obtained for the earlier experiment. Evidently the fertilizer applied the two
seasonspreviouslycarriedoverenoughtocorrecttheverylowlevelfoundintheoriginalhay.
The lower level of fertilizer only improved the phosphorus content slightly while the heavy
application of fertilizer markedly increased the phosphorus content. In other respects the
compositionofthehayproducedonthethreeplotsvariedonlyslightly.Thecalciumwasmuch
lowerthaninthehayfromthesameareausedintheearliertest.Thepelletedhayswerefed
to feeder lambs with the results shown in table 4. There was little, if any, difference in the
responseofthelambstothefourlotsoffeed.
Although the data show a slight advantage in favor of the unfertilized hay with the
added phosphorus, the difference was not significant statistically. Much of the difference in
gaincouldbeaccountedforbyonelambwhichgainedpoorly.

Table4.Resultsofsecondsheepfeedingtest(64days)

Numberof
lambs
Av.initial
weight,kg
Av.finalweight,
kg
Av.dailygain,
kg
Av.dailyfeed,
kg
Feedper45kg
gain,kg
Bloodserum
phosphorus
(mg/100mlsg)
Initial
Final

Heavily**
fertilized
desertedsoil
hay

Lightly*
fertilized
desertedsoil
hay

Unfertilized
desertedsoil
hay

Unfertilized
desertedsoil
hay
+
phosphorus

10

10

10

34,4

33.1

33.1

32.6

45.3

44.9

43.6

45.5

0.17

0.18

0.16

0.2

1.89

1.93

1.77

1.9

503

471

488

425

7.3
7.7

7.5
7.5

7.5
7.5

7.4
7.2

The phosphorus retention was again high in the lambs fed the feed supplemented
withdisodiumphosphate.Inthistrialtheretentionbythelambsfedtheheavilyfertilizedhay
was also high, but very low in the lambs fed the hay from the plot that received a light

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Lucrritiinificevol53seriaMedicinVeterinar
application of fertilizer. The calcium retention was very different from that found in the
previoustest.
In the first test large amounts of calcium were retained from the unfertilized hay with and
withoutphosphorusaddedbutinthesecondtrialtheunfertilizedhaybothwithandwithout
addedphosphorushadnegativebalances.

CONCLUSIONS

1. Feeding trials with sheep were conducted on lucerne hay of varying phosphorus
contenttostudyitsnutritivevalue.
2. Hayproducedonvirginsandydesertedsoilwhichcontained0.10%phosphoruswas
unpalatable and produced poor animal gains, feed utilization and depressed serum
phosphoruscontent.
3. The addition of disodium phosphate to this hay improved animal gains, feed
utilizationandincreasedthebloodserumphosphorus.
4. Hay containing 0.15% phosphorus was apparently equal in feeding value to hay
containinggreateramountsofphosphorus.
5. Anincreasedretentionofphosphoruswasobtainedbyaddingdisodiumphosphateto
alowphosphorushayinonetest,inanother,theadditionofdisodiumphosphateto
unfertilizedhayresultedinincreasingthephosphorusretentionuptothatofheavily
fertilizedhay.

BIBLIOGRAPHY

1.
2.
3.
4.

5.

6.

Crivineanu,V.,Studiulunorfactoritoxicocarenialinetiopatogeniainfecunditiilataurine,
Tezdedoctorat,Bucureti,1995.
Crivineanu V. Crivineanu M.,Rpeanu, M., Toxicologie sanitarveterinar, Bucureti, Ed. Coral
Sanivet,1996
LoganathanP,HedleyMJ,GraceND.,Pasturesoilscontaminatedwithfertilizerderived
cadmiumandfluorine:livestockeffects.RevEnvironContamToxicol.2008;192:2966.Review.
SiguaGC,WilliamsMJ,ColemanSW,StarksR.,Nitrogenandphosphorusstatusofsoilsand
trophicstateoflakesassociatedwithforagebasedbeefcattleoperationsinFlorida.JEnviron
Qual.2006Jan5;35(1):24052.Print2006JanFeb.
Sigua GC, Hubbard RK, Coleman SW, Quantifying phosphorus levels in soils, plants, surface
water,andshallowgroundwaterassociatedwithbahiagrassbasedpastures.EnvironSciPollut
ResInt.2010Jan;17(1):2109.Epub2009Jul30.
*** EFSA GMO Panel Working Group on Animal Feeding Trials Safety and nutritional
assessment of GM plants and derived food and feed: the role of animal feeding trials. Food
ChemToxicol.2008Mar;46Suppl1:S270.Epub2008Feb13.

1101

SOMEINVESTIGATIONSINCONSUMPTIONSEAFISH
(HERRING,MACKEREL)
1

O.Negrea1,CameliaRaducu1,L.Oana2,VioaraMiresan1,Z.Marchis1,
Gh.Mihai1,F.Chirila2,AncuaRotar1
U.S.A.M.V.Cluj,FacultyAnimalSciencesandBiotehnology,35Manasturstreet,400372Cluj
Napoca,Romaniaonegrea50@yahoo.com
2
U.S.A.M.V.Cluj,FacultyofVeterinaryMedicine,35Manasturstreet,400372
ClujNapoca,Romania
Abstract:Thestudywaseffectedonasampleof43seafish,unevisceratedandfreeze,destined
tohumanconsumption(25fishofmackerelspeciesand18ofherringspecies),proceededfrom
Markets, concerning incidence and intensity of acanthocephalosis in some morphological
determinations for parasite species establishment and also lesions table, and puts in evidence
some aspects. The incidence of acanthocephalosis in sea fish, on fish sample taken in study,
presents a high percent in both species (76% mackerel and 83.3% herring). Under report of
parasitismintensity,dominantarethereducedinfestations,inbothseaspecies(73%mackerel
and 60% herring) followed by medium infestations (15.7% mackerel and 26.6% herring) and
massive infestations (10.5% mackerel and 13.5% herring). Morphological determinations
obtained for acanthocephal species establishment, regarding following parameters: body mean
length 18.7 mm, cephalic tube mean length 1.93 mm, hook ranges 14, hook number on each
range 20 25, also taken in view the body colorization pink to reddish and compared with
speciality literature, establishes as acanthocephal parasite species Rhadinorhynchus pristis.
Morphopathologic table puts in evidence lesions of haemorrhagic enteritis and haemorrhagic
necrosis in pesthole to parasite fixation place and nodular lesions in muscle as a result of
penetrationofintestinewallbymigrationlarva(acanthales).
Keywords:acanthocephal,incidence,cephalictube,hooks

MATERIALANDMETHODS

Investigationswereeffectedonasampleof43consumptionseafish,uneviscerated
and freeze in polyethylene bags 3 5 pieces / bag mackerel 25 pieces, herring 18 pieces),
proceededfromMarkettypestoresnetworkinClujNapoca.Afterthawing,with thehelpof
scalpel general cavity is opened and it is dissected the intestine in length following by
macroscopic exam and exam with magnifying glass intestine content and intestinal wall
modifications, following extensivity level and intensity of parasitism with acanthocephales,
lesionstableandestablishmentofparasitespecies,withthehelpofanatomicclamp,parasite
aresamplingwhichareintroducedinPetriplatesforpreservationinformolsolution10%.
For species establishment were effected morphologic determinations on a sample of 15
parasitestakenfromintestine,havinginview:bodycolor,parasite andcephalictubelength
(biometers with millimeter ruler), number of hook ranges and total hook number on range
(microscope,ob.3.2x).
Inparasitismintensityappreciationwithacanthophilesitwastakenintoaccountthe
numberofparasitesfoundinintestine,considering:
Massiveinfestations:above10parasites/fish
Mediuminfestation:between510parasites/fish
Reducedinfestation:under5parasites/fish

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Lucrritiinificevol53seriaMedicinVeterinar
RESULTSANDDISCUSSIONS

Investigations done on a 43 consumption sea fish sample, uneviscerated and freeze ,


concerning the incidence and intestinal parasite intensity with species of acanthocephales,
and also morphologic determinations for parasite species identification, respectively lesions
tablebroughtabout,putsinevidencethefollowingaspects:
A.
Parasitespreadingwithacanthocephalespresentsvalueswhichareshownin
table1andgraphic1.

Table1.Parasitespreadingwithacanthocephalesinconsumptionseafish.
Species
Numberoffish
Fromwhich
examinated
positive
%
Mackerel
25
19
76.0
Herring
18
15
83.3
total
43
34
79.0

Datapresentedintable1.putinevidenceahighlevelofacanthocephalosisincidencein
sea fish, 76% in mackerel and 83.3% in herring, with an average of 79.0%. It must be
mentioned the fact that high level of parasitosis in sea fish (mackerel, herring, hake,
Thrachurus med., etc) is the result of biologic cycle of parasite. It is known the fact that
acanthocephalesarewormsthatintheircycleofdevelopmentneed2hosts(dixencycle):one
whichisintermediaterepresentedbycrustaceae,aquaticsamphipodesandisopodesandone
which is definitive, represented by vertebrates, as are the fish, ichtyophages birds and
mammals. Crustaceae takes from water the parasite eggs and into their body is formed the
infestantlarvaacanthela.
Asthesecrustaceaerepresentthesourceoffoodforpredatoryfish,quantityconsumed
andespeciallythenumberofacanthelespresented,favourthediseaseincidence.Inthepicture
nearlywepresenttheintestinalinfestationvariationwithacanthocephalesinconsumptionsea
fish,functionofspecies.
Mackerel
Herring
76,01

83,3

Picture1.Infestationvariationwithacanthocephalesinconsumptionseafish,onspecies.
B.
Regardinginfestationlevelwithacanthocephalesinconsumptionseafish,thevalues
obtainedarepresentedintable2.
Table2.Infestationintensitywithacanthocephalesinseafish,onspecies

Number
Fromwhich
Species
of
Lowinfestation
Mediuminfestation
Massiveinfestation
positive
positive
%
positive
%
positive
%
cases
Mackerel
19
16
73.6
3
15.7
2
10.5
Herring
15
9
60.0
4
26.6
2
13.5
total
34
23
67.6
7
20.5
4
11.9
Datapresentedintable2putinevidencedifferentvaluesofacanthocephalosisintensitylevel
functionofspecies,asshown:

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UniversitateadetiineAgricoleiMedicinVeterinarIai
In mackerel species, on total fish taken in study are dominant low infestations
(73.6%),followedbymediuminfestations(15.7%)andmorereducedthemassiveinfestations
(10.5%)
In herring species, on fish lot taken in study predominant are the low infestations
(60.0%),followedbythosemedium(26.6%)andlessthemassiveinfestations(13.5%)
In graphic 2, are presented percent values of parasitism intensity with acanthocephales in
speciesofseafish.
100%

90%

60,01

80%

13,5
70%

26,6

60%

Herring
Mackerel

50%

40%

73,6

30%

10,5
20%

15,7

10%

0%

Massive infestations

Medium infestation

Reduced infestation

Picture2.Intensitylevelwithacanthocephalesinspeciesofseafish
C.
Morphologic determinations to identify acanthocephales species parasites in
intestine level(bodybiometers, color, number ofhook ranges, etc), on15 parasites sample,
putinevidencethefollowingaspects:
Table3.
Morphologicdeterminationsofacanthocephalesspecies
withintestinallocalizationinseafish
Numberof
Bodysize
Numberof Numb Parasitecolor
sample
hook
erof
Body
Cephalictube
ranges
hooks
length
length(mm)
on
(mm)
range
1
15.7
2.1
14
22
Pinkreddish
2
18.2
1.8
14
20
Pinkreddish
3
19.0
2.0
14
24
Pinkreddish
4
19.4
2.1
14
20
Pinkreddish
5
20.0
2.1
14
24
Pinkreddish
6
16.6
1.9
14
25
Pinkreddish
7
17.0
1.9
14
24
Pinkreddish
8
19.2
2.0
14
25
Pinkreddish
9
18.6
1.7
14
22
Pinkreddish
10
19.2
2.1
14
25
Pinkreddish
11
20.2
2.0
14
20
Pinkreddish
12
17.4
1.9
14
24
Pinkreddish
13
19.2
2.2
14
23
Pinkreddish
14
20.4
1.8
14
23
Pinkreddish
15
20.2
1.9
14
25
Pinkreddish
total
X18.7
X1.93
14
X23

By data obtained and presented in table 3 are evaluated aspects regarding


morphology of parasites found, with the purpose of a correct specification of parasite

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Lucrritiinificevol53seriaMedicinVeterinar
acanthocephales species. Doing comparison of data obtained (average values), body length
(18.7mm),cephalictubelength(1.93mm),numberofhookranges(14),numberofhookson
range(23),color(pinkreddish),withthosefromspecialityliterature(GabrielaMunteanuand
Dumitru Bogatu, 2003), we consider that acanthocephales species found in sea fish lot
examinedisRhadinorhynchuspristis.(picture3).

Picture3.Rhadinorhynchuspristiscephalictube(ob.3.2X)

Concerningthemorphopathologictable,majorityoflesionsarepresenttothelevel
ofintestinalmucousmembrane,consequenttoadultparasitefixation.Pathogenicroleisdue,
in a great measure, to cephalic tube armed with cuticle thorns, which, through a mechanic
action irritates, produces inflammations and ulcerates, sometimes, perforations of intestinal
wall. It appears lesions of haemorrhagic enteritis and haemorrhagicnecrosis in focus,
respectivelylesionsofhyperthrophicenteritiswithathicknessofintestinalmucous,especially
inpyloricappendiceszone.

REFERENCES
1.
2.

Bogatu,D.,1987Ihtiopatologievol.IUniversitateaGalai.
Bud I., M. Bura, A. Bud, D. Ladoi, A. Totoian 2001 Petii i tainele umbrelor subacvatice
EdituraCeres,Bucureti.
3. BudI.,V.Vldu,2004GhiddelucrripracticenpisciculturEdituraRisoprint,ClujNapoca.
4. ChiriacE.,1975ParazitologiegeneralEdituradidacticipedagogic,Bucureti.
5. CosmaC.,O.Negrea,C.Gherman,1998DiagnosticulbolilorparazitarelaanimaleEditura
Genesis,ClujNapoca.
6. Ghitino P., 1985 Technologia e patologia in aquacoltura vol. II Patologia Emilio Bono,
Torino.
7. Golvan,Y.J.1994NomenclatureoftheAcanthocephala.ResearchandReviewsinParasitology
54,135205.
8. MunteanuG.,BogatuD.,2003TratatdeIhtiopatologieEdituraExcelsiorArt,Timioara.
9. NegreaO.,2007BolilepetilorEdituraAcademicPres,ClujNapoca.
10. OneE.,ConstantinescuV.,1978DiagnosticuldelaboratornmedicinaveterinarEditura
Bucureti.
11. SclotfeldH.J.,AldermanD.J.,1995WhatShouldIDo.Apracticalguideforthefreshwaterfish
farmer.TheEuropeanofFishPatologists.
12. Taraschewski, H 2000. Hostparasite interactions in Acanthocephala: a morphological
approach.AdvacesinParasitology46,1179

1105

ASPECTSREGARDINGEPIZOOTICMETALINGUATULOSAINMAIN
DOMESTICRUMINANTSPECIES

O.Negrea ,CameliaRaducu ,L.Oana ,VioaraMiresan ,Z.Marchis ,


V.Miclaus1,F.Chirila2,AncuaRotar1
1
U.S.A.M.V.Cluj,FacultyAnimalSciencesandBiotehnology,35Manasturstreet,400372
ClujNapoca,Romaniaonegrea50@yahoo.com
2
U.S.A.M.V.Cluj,FacultyofVeterinaryMedicine,35Manasturstreet,400372
ClujNapoca,Romania

Abstracts.Research results on the incidence epizootiological mesenteric limphonodular


linguatulosa, performed on 274 cattle, 43 buffaloes, 54 sheep and 38 goats from 4 counties of
Transylvania(Cluj,Brasov,Sibiu,Mures),slaughteredinslaughterhousesandcuttingpointarea,
put in evidence a visceral linguatulosa incidence in cattle, with different values. The most
increased percentage of infestation was reported in cattle in Cluj county (65.2 %), followed
descendingorderbythoseinMurescounty(58.8%)andBrasovcounty(42.5%),andtheaverage
ofinfestationisof47.4%.Inbuffaloes,theincidenceofvisceralelinguatulozaismuchlowerthan
forcattle:fromthe43controledbuffaloes,25.5%werepositive,thehigherpercentagebeingin
Cluj(40.0%)andtheminimalinBrasov(17.8%).Extensivitatyofparasitisminsheepandgoats
show significantvariationsdepending on species and area of origin. Thusthesheep infestation
levelwashighinpopulationsinSibiu(45.4%)andlowinthoseinCluj(33.3%)andmediumlevel
was40.7%.Incontrasttogoatsishigherthanthelevelofinfestationofsheepwithanaverageof
60.5%, being increased in goats in Sibiu (63.6%) than in Cluj (56.2%). Epizootiological data
obtainedrevealsthatthemaximumsensitivityinmetalinguatulozaisingoats(60.5%),followed
bycattle(47.4%),sheep(40.7%)andbuffaloes(25.5%).Parasitismintensivityinlargeruminants,
with preimaginale stages of L.serrata, checked on 141 positive mesenteric limphonodular
masses,presentslargedifferencesdependingonthespecies.Maximuminfestationlevelis47.8%
to 18.1% in cattle over buffaloes Reducedinfestations had mean values of cattle and 37.6% to
36.5%inbuffaloesInfestationswereonaverage14.6%percentofthecattle.Buffaloespresents
bothextensivityandintensivityofparasitism,preimaginaleformsofL.serrata,lowerthanother
ruminants.

Keywords:metalinguatulosis,incidens,lymphnodes,ruminants,larvastages.

INTRODUCTION

Linguatuloza epizootic disease caused by adult forms of L serrata in carnivores


located in the nasal passages and sinuses and larval forms from herbivores and some
omnivores limphonodules located mainly in mesenteric, is less studied and researched the
world,includinginourcountry.
Todate,nosystematicresearchhasbeenconductedontheepizooticparazitoze,consequently
missing information on sources of infestation, the mechanisms of transmission and features
state of receptivity or resistance of intermediate and final hosts. This prompted us to
undertake a comprehensive study on the incidence metalinguatuloza in the main species of
domesticruminantsandtheirresponsiveness.

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Lucrritiinificevol53seriaMedicinVeterinar
MATERIALANDMETHOD

Investigations regarding the incidence of domestic ruminants metaliguatuloza have


been conducted on 274 cattle, 43 buffaloes, 54 sheep and 38 goats from the 4 counties of
Transylvania(Cluj,Brasov,Sibiu,Mures),whowereslaughteredinslaughterhousesandcutting
points.
SteerswereslaughteredHolsteinbreeds,RomanianSpottedandPinzgau,aged1to12years
by sex and state of maintenance different, bubalinele were common breed, aged 5 to 15
years,sheep,theraceTurcanandPan,aged4years,andgoats,thebreedcommonwithage
410years.
Inordertoclarifythediagnosisofviscerallinguatulozawereharvestedafterslaughter
limphonodularemesentericmasses,usingmethodsofcuttingintoslicesofwatermelonandto
assessthelevelofinfestationwithlimfonodularalarvalandnymphstagesofthetaurineandL
serratabuffaloessettledthreedegreesofinfestation:
lowinfestation:110larva/mesentericlymphonodularchain
mediuminfestation:1125larva/mesentericlymphonodularchain
masiveinfestation:above26larva/mesentericlymphonodularchain

RESULTSANDDISCUSSIONS

Epizooticstudyresultsonmetalinguatulozainruminants,revealdifferentaspects
relatedtotheareasinvestigatedandaregiveninTable1.
Table1.
Incidenceincattlemetaliguatulozalymphonodulare

Centralzone
Animalsexamined Fromwhichpositive
Infestation
(n)
percentage%
Cluj
23
15
65.2
Brasov
200
85
42.5
Mures
51
30
58.8
TOTAL
274
130
47.4

Different analysis of data submitted is found that the incidence of mesenteric


linguatulozaoncattlelymphonodulareshowdifferentvaluesdependingontheorigin.
So,thehighestfrequencyofinfestationof62.5%isinClujcounty,andthesmallest,from
42.5%inBrasovcounty.ExtensivityinfestationslevelinMurescountyis58.8%andtheoverall
mean infestation is 47.4% The level extensivity metalinguatuloza limfonodulare mesenteric
may belargelyinfluenced bythethermal variation.Thus,inthecountiesofClujandMures,
thermalregimeismoderatewithsmallvariationsintemperature,withmildwinters,inBrasov
county thermal regime presents special variations, harsh winters with frosts and repeated
thaws, that influence different eggs resistance capacity of L. serrata. Linguatuloza
lymphonodulareextensivityvariationincattleincorrelationwiththeareaoforiginisshownin
Fig.1

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UniversitateadetiineAgricoleiMedicinVeterinarIai

65,2

58,8

Cluj
Brasov
Mures

42,5

Fig.1.Viscerallinguatulosavariationincattle

Lymphonodularemesentericlinguatulosaincidenceinbuffaloesinslaughterhousesof
the2countiesispresentedinTable2.
Table2.
Viscerallinguatulosaextensivetyinbuffaloes

Countycontrolled
Animalsexamined
Fromwhich
%infestation
positive
Cluj
15
6
40.0
Brasov
28
5
17.8
TOTAL
43
11
25.5

Benotedthatthebuffaloeskilledlinguatulosalymphonodularepresentsmuchlower
valuescomparedtocattle,averageextensivitybeing25.5%.
Howevertherearesignificantdifferencesaccordingtoareaoforigin,withvariationsfrom
40.0%extensivityinClujcounty,upfrom17.8%inBrasovcounty.
Suppose that the climate factor, already noted, with variations of temperature and
humiditydifferent,pastureareaswithmuchmoisture(Oltmeadow),zoninggrowthinbuffalo
ofFagaras,inthevillagessurroundingOltRiverfloodplains,arereducingmuchtheresistance
capacity of L.serrata eggs and implicit, the intermediate host contamination. Linguatulosa
lymphonodulareextensivityvariationinbuffaloisshowninFig.2.

17,8
40,01

Cluj
Brasov

Fig.2.Lymphonodularelinguatulosaincidenceinbuffalo

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Lucrritiinificevol53seriaMedicinVeterinar
Insheepandgoats,resultsofinvestigationscarriedoutonactualslaughteredinabattoirs
inthecountiesofClujandSibiu,theincidencelinguatulozavisceralmesenteric,arepresented
inTable3.

Table3.
Lymphonodularelinguatulosaincidenceinsheepandgoats

Sheeps
Goats

County
Numberof
From
Numberof
From
controlled
samples
which
%
samples
which
%
positive
positive
Cluj
21
7
33.3
16
9
56.2
Sibiu
33
15
45.4
22
14
63.6
TOTAL
54
22
40.7
38
23
60.5

Thedatapresentedarefoundwithimportantdifferencesrelatedtotheincidenceof
parasitic,dependingonspeciesandarea.Thus,theaveragevaluesofinfestationinsheepare
40.7%,slightlyhigherinSibiucounty(45.4%)andlowestinClujcounty(33.3%).Theaverage
goat infestation is higher than the sheep, of 60.5%; there are significant value differences
between the 2 county: 63.6% to 56.2% in Sibiu and respective in Cluj. In temperate climate
conditionsinourcountry,whensheepandgoatsaregrazedalmostthroughouttheyear,there
isaconstantriskofcontaminationwitheggsL.serrata.Withthisoccursalsothesensitivityof
species. In general, sheep and goats are more susceptible to infestation polyparasitary
comparedwithsteersandbuffaloes.Metalinguatulosaincidenceinsheepandgoatsisshown
inFig.3.

120

100

63,6
80

60

Sibiu
Cluj

45,4

40
56,2
33,3
20

Ovine

Caprine

Fig.3.Metalinguatulosaincidenceinsheepandgoats

Resultsonthedegreeofparasitismwithintensivityoflarvalandnymphstages,
examinationof141consecutivepositivemesentericmasslymphonodulareincattleand
buffalo,aregiveninTable4.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Table4.
ParasitismintensivitywithlarvalandnymphstagesL.serrataincattleandbuffaloes

Host
Number
Fromwhich
species
of
Low
%
Medium
%
High
%
samples infestation
infestation
infestation
cattle
130
49
37,6
20
14,6
61
47,8
buffalo
11
4
36,5
5
45,4
2
18,1

Following examination of the 141 masses lymphonodulare mesenteric,


metalinguatulozapositive,thetwospeciestakeninthestudy,resultsrevealedthefollowing:
of the 130 tables mesenteric limfonodulare of cattle were identified with 49 masses
lymphonodulare, with low infestation (37.6%), 20 masses lymphonodulare with moderate
infestation(14.6% )and61 masseslymphonodularewithmassiveinfestation(47.8%),whom
lymphonodules were hypertrophied of soft consistency and in section is running a creamy
purulentmassrichinviablelarvae.
Buffaloeswerefoundatalowinfestationin4cases(36.5%),amoderateinfestation
in5cases(45.4%)andmassiveinfestationin2cases(18.1%)

REFERENCES

1. CosoroabI.,DrbuGhe., OprescuI.,(1995).Compediude ParazitologieVeterinar.Ed.Mitron.


Timioara.
2. Dulceanu N., Carmen Povcolnicu, Solcan Ghe., Hritcu L., (1996).Observaii privind morfologia
specieiLinguatulaserrata.(Frchlide,1789).Rev.Rom.Med.Vet.4.467.
3. Hariri, F. 2001 The study of prevalence rate of Linguatula serrata nymph in mesenteric lymph
nodes of sheepin Urmia. DVMThesis no. 491,Islamic Azad University of Urmia ( in Persian, with
Englishabstract).PP:3748.
4. NegreaO.,(2003).Linguatulozalaierbivoreicarnivore.Ed.Academicpres.ClujNapoca.
5. Riley J., (1986) The biology of Pentostamids Advances in Parasitology, vol 25.Academic Press
Inc.Limited(London).
6. Saiyari, M., Babak Mohammadian and R.N. Sharma. 1996 Linguatula Serrata ( Frolich 1789)
nymphsinlungsofgoatsinIran.Trop.Anim.HlthProd.28,312314
7. Sukran D. (1982). A study of the incidence of Linguatula serata and public health
sigmficance.AnkaraUniv.Vet.FakDerg.29(34):324330.
8. uteuI.,V.Cozma(1998).Bolileparazitarelaanimaleledomestice.Ed.Ceres.Bucureti.
9. Valenzuela,G.,Bascunan,N.,Bayer,LandErnest,S1995L.serratainfectionsinbovineliversin
Valdivia.ChileArch.Med.Vet.27(1):2934

1110

APPLICATIONSOFBIOMEDICALINFORMATICSAND
TELEMEDICINE

T.NICA,V.CRIVINEANU
FacultyofVeterinaryMedicine,Bucureti
Str.SplaiulIndependenei,nr.105,tudornica@rdslink.ro

Abstract: Biomedical informatics is an emerging discipline underlying the acquisition,


maintenance, retrieval and application of knowledge and information in research, education,
healthrelated basic sciences, clinical disciplines with computer science, statistics, engineering,
mathematics, information technologies and management. Telemedicine is a rapidly developing
application of clinical medicine where medical information is transferred through interactive
audiovisualmediaforthepurposeofconsulting,andsometimesremotemedicalproceduresor
examinations. Biomedical and Health Informatics is its interdisciplinary nature: It connects
computerscience,medicine,biologyandhealthcare,andprovidesasynergythatgoesbeyond
anythingthatresearchersinanysingledomaincanprovide.

Keywords:biomedicalinformaticstelemedicineehealth

OBJECTIVES

Healthinformaticsormedicalinformaticsistheintersectionofinformationscience,
computerscienceandhealthcare.Itdealswiththeresources,devices,andmethodsrequired
to optimize the acquisition, storage, retrieval, and use of information in health and
biomedicine.Healthinformaticstoolsincludenotonlycomputersbutalsoclinicalguidelines,
formal medical terminologies, and information and communication systems. It is applied to
the areas of nursing, clinical care, dentistry, pharmacy, public health and (bio)medical
research.
Biomedical informatics coalesces the related fields of Medical Informatics and
Bioinformatics.HealthInformaticscontainssubsetssuchasTelemedicine,ClinicalInformatics,
and Dental Informatics, Pharmaceutical Informatics, Nursing Informatics and Public Health
Informatics.
Biomedicalinformaticsalsoprovidesthetoolsandskillsneededforthedevelopment
and application of new technology for improving patient care, medical education, health
sciencesandmanagementforhealthcare/hospitalsystems.
Centraltobothmedicalinformaticsandbioinformaticsisthecollectionandanalysis
of information. While medical informatics is more concernedwith structures and algorithms
for the manipulation of the data and how it can be applied in healthcare, bioinformatics is
moreconcernedwiththedataitselfanditsbiologicalimplications.

MATERIALSANDMETHODS

Telemedicine (ehealth) has been defined as the use of telecommunication to


providediagnosticandtherapeuticmedicalinformationbetweenpatientanddoctorwithout
either of them having to travel. It is the use of information and communication technology
(ICT)todeliverhealthservices,expertiseandinformationoverdistance.
Thebasicrequirementsfortelemedicineinclude:

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Lucrritiinificevol53seriaMedicinVeterinar

use of information and communication technology, such as the Internet, is


requiredforlongdistancehealthservicesdelivery.
expertadvicemustbeavailableatsitebeingreferredto.
sufficient description of the problem being referred or services required
fromamedicalopinion.
adequatetimeshouldbegivenforassessmentofallrequests.
The amount of time of would be required for a response from the expert from the
other side would depend on the type of telemedicine being practised. There are basically
threetypesoftelemedicine:
StoreandForward Telemedicine: This is usually used for transferring digital images
fromonelocationtoanother.Imagesarecapturedwithdigitalcamerasorstillvideo,andsent
toanotherlocation.Theyareattachedwithasummaryofthecaseintext(usuallybyemail).
Thistypeoftelemedicineisusuallynotusedinemergencysituations,asspecialistreviewcases
when convenient. There is no limit to the physical distance between the origin and the
destinationoftheimage.
Realtime video linking: This method is used when a 'facetoface' consultation is
necessary.Itisusuallybetweenthepatientandtheirproviderinonelocationandaspecialist
inanotherlocation.Specializedvideoconferencingequipmentatbothlocationsallowsa'real
time' consultation to take place. High bandwidth communication channels (ISDN lines) are
necessary,althoughthistypeoftelemedicineis nowwithinreachofthedesktopcomputers
connectedtotheInternet.
Telemedicine through Remote Network Monitoring. An RMON implementation
typicallyoperatesinaclient/servermodel.Monitoringdevices(commonlycalled"probes"in
this context) contain software agents that collect information and analyze packets. These
probesactasserversandtheNetworkManagementapplicationsthatcommunicatewiththem
actasclients.Theprobeshavemoreresponsibilityfordatacollection and processing, which
reduces SNMP traffic and the processing load of the clients and information is only
transmittedtothemanagementapplicationwhenrequired,insteadofcontinuouspolling.
Itispossibletocombineanyofthethreetosuittheneedsoftheorganization.
Thehospitalinformationsystemswerefirstdevelopedinthe1960sandhavebeen
an essential part in hospital information management and administration. Early systems
consisted of large central computers connected toby dumb terminals, which are now being
replaced by networked microcomputers. The systems were used to manage patient finance
andhospitalinventory.
Ahospitalinformationsystems(HIS)isacomputersystemthatisdesignedtomanage
allthehospitalsmedicalandadministrativeinformationinordertoenablehealthprofessional
performtheirjobseffectivelyandefficiently.
Hospitalinformationsystemsnowfocusontheintegrationofallclinical,financialand
administrative applications and thus could also be called an integrated hospital information
processingsystems(IHIPS).
Componentsofahospitalinformationsystemconsistoftwoormoreofthefollowing:
ClinicalInformationSystem
FinancialInformationSystem
LaboratoryInformationSystem
NursingInformationSystems
PharmacyInformationSystem
PictureArchivingCommunicationSystem

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UniversitateadetiineAgricoleiMedicinVeterinarIai
RadiologyInformationSystem
No hospital information system can be regarded as a success unless it has the full
participation of its users. Thus human and social factors would have to be considered in its
design,moreoftenthannot,theycanbeeasilyaddressedbyprovidingadequatetrainingand
educationaboutthesystem.
AClinicalInformationSystem(CIS)isacomputerbasedsystemthatisdesignedfor
collecting, storing, manipulating and making available clinical information important to the
healthcaredeliveryprocess.
ClinicalInformationSystemsprovideaclinicaldatarepositorythatstoresclinicaldata
suchasthepatientshistoryofillnessandtheinteractionswithcareproviders.Therepository
encodes information capable of helping physicians decide about the patients condition,
treatment options, and wellness activities as well as the status of decisions, actions
undertakenandotherrelevantinformationthatcouldhelpinperformingthoseactions.
SomeoftheareasaddressedbyClinicalInformationSystemsare:
Clinical Decision Support: This provides users with the tools to acquire,
manipulate,applyanddisplayappropriateinformationtoaidinthemaking
ofcorrect,timelyandevidencebasedclinicaldecisions.
Electronic Medical Records (EMRs): this contains information about the
patient,fromtheirpersonaldetails,suchastheirname,age,addressandsex
todetailsofeveryaspectofcaregivenbythehospital(fromroutinevisitsto
majoroperations)
Training and Research: Patient information can be made available to
physicians for the purpose of training and research. Data mining of the
information stored in databases could provide insights into disease states
andhowbesttomanagethem.
Anelectronichealthrecordisanevolvingconceptdefinedasasystematiccollection
of electronic health information about individual patients or populations. It is a record in
digital format that is capable of being shared across different health care settings, by being
embedded in networkconnected enterprisewide information systems. Such records may
include a whole range of data in comprehensive or summary form, including demographics,
medical history, medication and allergies, immunization status, laboratory test results,
radiologyimages,andbillinginformation.
Itspurposecanbeunderstoodasacompleterecordpatientencountersthatallowsto
automate and streamline workflow in health care settings and to increase safety through
evidencebaseddecisionsupport,qualitymanagement,andoutcomesreporting.
Information Technology applications are now becoming commonplacein veterinary
practicesthataremovingtowardsbecomingpaperlessastheyimplementelectronicmedical
records(EMRs)fortheirpatients.
EMRs provide veterinaries with the ability to provide standardized clinical patient
records and reports and the opportunity for providing their clients with continuous care
education. Some EMRs come with a graphical module that provides charts that clients can
takehomeandseetheimprovementintheweightoftheirpetsduetotreatmentorchangein
diet.
EMRs can come as standalone applications or part of a veterinary practice
management system, which provides veterinaries with tools for managing all aspects of the
practice, such as billing, printing out of reminders and handouts, decision support and
outcomesanalysis.

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Lucrritiinificevol53seriaMedicinVeterinar
RESULTS,DISCUSSIONSANDCONCLUSIONS

Some of the benefits of telemedicine include the fact that it can lead to improved
access to medical care for geographically or socioeconomically isolated patients. Also it
prevents unnecessary patient travel to secondary or tertiary health care centers, promotes
education, prevention, clinical trials and other programs, facilitates community education
concerningpertinenthealthcaretopics.
Telemedicine can assist preservation of the patients current providertopatient
relationship while facilitating access to specialty care and enhances continuing medical
education and support for ruralbased providers and remove the obstacle of professional
isolation. It can effectively utilize medical resources by creating an integrated network of
primary, secondary and tertiary care and may help secure the financial stability of rural
hospitalsandallowpatientstoobtainmedicalconsultationintheirowncommunity.
However,telemedicinealsomaysufferfromafewbarriersinitspath.Locallicensure
rulesmayprohibitaphysicianfromconsultinginanotherarea.Reimbursementsareslowto
come through because of complicated rules and requirements. Cultural resistance, fear of
malpractice and the cost and the exclusiveness of the appropriate telecommunications
technology are another hindrance. Also regular telephone lines do not supply adequate
bandwidth for most telemedical applications. Many rural areas do not have high bandwidth
telecommunicationsaccessrequired.
Regarding the Clinical Information Systems, research has been done to show their
valueandthesehavehighlightednotjustthebenefitsbutalsothebarriersthatmightbefaced
byhospitalswhoimplementsuchsystems.
Someofthebenefitsare:
Easy Access to Patient Data Clinical Information Systems can provide convenient
access to medical records at all points of care. This is especially beneficial at ambulatory
points, hence enhancing continuity of care. Internetbased access improves the ability to
remotelyaccesssuchdata.
Structured Information The clinical information captured in Clinical Information
Systemsiswellorganised,thusmakingiteasiertomaintainandquickertosearchthroughfor
relevantinformation.Theinformationisalsolegible,makingitlesslikelythatmistakeswould
bemadeduetoillegiblewriting.
ImprovedDrugPrescriptionandPatientSafetyClinicalInformationSystemsimprove
drugdosingandthisleadstothereductionofadversedruginteractionswhilepromotingmore
appropriatepharmaceuticalutilisation.
Despite the benefits being offered by Clinical Information Systems, they are not
withoutthebarriersthatpreventthemfrombeingrolledoutineveryhospital.Theseinclude
someofthefollowing:
Initialcostofacquisitionthehighcostofbasicinfrastructureofclinicalinformation
technologycanbeastumblingblocktomanyhealthcareorganisations.
PrivacyandSecuritytherearestillhugeconcernsinthehealthcareindustryabout
the privacy ofpatient data on computer systems and how to keep such informationsecure.
The HIPAAandDataProtectionActpassedbyrespectivegovernmentsintheUSandtheUK
wereintroducedtoaddresssomeoftheseconcerns.
ClinicianResistancecliniciansusuallyhave1020minutestoseetheirpatientsandif
theirinteractionswithaCISduringthesesessionsprovestobecounterintuitivebytakingup
moretimethanisnecessary,thereisboundtoresistancetoituse.

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UniversitateadetiineAgricoleiMedicinVeterinarIai

1.
2.
3.
4.
5.
6.
7.

REFERENCES

Eysenbach,Gunther.Whatisehealth.JournalofMedicalInternetResearch.2001
Fraser, Hamish S. F. Introduction to telemedicine and healthcare communication.
September2003.
GardnerRM,OverhageJM,SteenEB,etal.(2009)."Corecontentforthesubspecialty
ofclinicalinformatics".JournaloftheAmericanMedicalInformaticsAssociation
Holbrook,Anne,etal.Acriticalpathwayforelectronicmedicalrecordselection.April
2003
Patton GA, Gardner RM (1999). "Medical informatics education: the University of
Utahexperience".JournaloftheAmericanMedicalInformaticsAssociation
Smith, Ronald D.; Williams, Mitsuko. Applications of informatics in veterinary
medicine.BullMedLibrAssoc.2000January
***Biohealthmatics.comKnowledgeCenter

1115


BUILDINGANDMANAGINGANONLINEDATABASE
OFTHEMOSTIMPORTANTTOXICOSESINVETERINARYMEDICINE

T.NICA,V.CRIVINEANU
FacultyofVeterinaryMedicine,Bucureti
Str.SplaiulIndependenei,nr.105,tudornica@rdslink.ro

Abstract : The objective of this paper is to present a software program designed as an online
databaseconsistingofthemostimportantpoisoningsaveterinariancanencounterinhisactivity.
Ashortdescriptionofthethemaincomponentsofacomputersystemismade,followedbythe
presentationofthedatabaseandtheinterfaceitself.Theprogramcanbeaccessedonlinebyany
interestedperson,butitisaimedfortheuseofveterinarycliniciansandstudentsmostly.
Keywords:onlinedatabaseveterinariantoxicology

OBJECTIVES

Our target was to build and implement a computer program for diagnosis and
emergencytherapyinveterinarymedicinepoisonings.Tothisend,thefirststepwascompiling
a database that stores information about the main poisonings a veterinarian may face in
practice.ThisdatabaseisconstructedonlinetoreapthebenefitsofInternetcommunication,
bothintermsofaccessibilitytoinformationandopportunityfordevelopmentandcontinuous
improvement.

MATERIALSANDMETHODS

A computer system comprises of the computer itself (hardware platform), the


programsrunningonthatcomputer(softwarecomponent)andthecommunicationnetwork.
These three components must be in perfect harmony and work integrated to serve their
intendedpurpose.
Hardware platform. The computing era as we know it kicked off in 1951 with the
releaseofthefirstcommerciallysuccessfulelectroniccomputerknownasUNIVAC1.
Todayscomputersaremuchsmallerthananyoftheearliercomputersproducedand
are no longer beyond the reach of small to medium sized organisation. Till date, computers
havebeenmakingstridesinnotonlygettingsmaller(portablemicrocomputersandhandheld
computers) but also increasing in processing power. This has led in to an increase in the
sophistication of software programs and invariably an increase in the functionality of health
informationsystems.
Those systems are now used in carrying out almost all functions within a hospital
(patient appointment and staff scheduling, electronic prescriptions, decision support,
insurancebillingandelectronicmedicalrecords).
Software component. At first, computers were programmed by calculating circuits
thatweremanuallywiredtogetherformathematicalcalculationstobeperformed.
ThiswasfollowedbythereleaseofthefirstsoftwarebytheUniversityofManchester
inEngland.Seriesofcomputerprogramsfollowedsuitandcouldbeloadedontothecomputer
throughtheuseofmemorytapesandpunchcards.

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Lucrritiinificevol53seriaMedicinVeterinar
Howeveritwasnotuntilthe1990swiththeintroductionoftheInternet,asweknow
it today and network computing that the islands of information within healthcare
organisationscouldnowbeintegratedtogiveanalmostseamlessflowofinformation.
Althoughitwasnowpossibleforcomputerswithindifferentpartsofthehospitalor
even different hospitals could talk to one another, the data collected by different software
packages from different manufacturers were stored in incompatible formats. The concerns
raisedbythelackofcommunicationbetweenvariousmedicalinformationlocationpointsled
to the introduction of the Electronic Data Interchange (EDI). EDI defines how the
communications (called messages) should occur between different systems, and its primary
concern is the content of the messages and not how the message is carried across the
network.
In terms of costs, unlike the hardware that is in a continuous decline in prices of
manufacturing and purchasing, the software component of a computer is more expensive
(domestic computer software are sometimes two or even three times more expensive than
thecomputeritself,exponentiallyincreasingthepriceofsoftwaresystemsforcompaniesand
largecomputernetworks).
Thecomputernetwork.Anetworkisagroupofcomputersorcomputerlikedevices
that communicate to one another using a common transmission medium. Computers send
requestsanddatathroughthetransmissionmedium,usuallynetworkcablesoropticalfibres.
Wirelessnetworksuseradiowavesasthemodeoftransmissionandarebecomingincreasingly
common.
Resourcesmadeavailableonthenetworkareaccessiblebytheusersofthenetworks,
this include documents, images, video and audio clips. It is also possible to share software
acrossthenetworkandthiscanleadtoareductioninthemaintenanceofsoftwareoneach
computeronthenetwork.
Devicesonanetworkcommunicatetoanotherbybreakingdowndataintogroupsof
electrical pulses which are known as packets. These packets contain information about the
addressofthesourceoforiginofthepacketanditsdestination.Itisthisinformationthatis
usedbythenetworkequipmenttoensurethatpacketgetstoitsintendedrecipient.
The Internet is a global system of interconnected computer networks that use the
standardInternetProtocolSuite(TCP/IP)toservebillionsofusersworldwide.Itisanetworkof
networks that consists of millions of private, public, academic, business, and government
networks of local to global scope that are linked by a broad array of electronic and optical
networking technologies. The Internet carries a vast array of information resources and
services,mostnotablytheinterlinkedhypertextdocumentsoftheWorldWideWeb(WWW)
andtheinfrastructuretosupportelectronicmail.
Our program was developed on the WWW platform using PHP web programming
language, while database creation and management was done through MySQL relational
system.
Selection of toxicoses. The selection of the most important poisonings that were
placed in the database was made using statistical data from specialized centers in EU
countries, such as the National Center for Toxicological Veterinary Information from Lyon.
These organizations provide immediate access to any information in the field of veterinary
clinicaltoxicology,bothinFranceandinotherneighboringareas.
Accordingtothestudiestheycarriedout,afterthecentralizationofover38,000cases
have been relevant conclusions on the poisonings incidence and the main species affected.
Thusitwasfoundthat45.5%ofpoisoningcaseswereduetopesticides,industrialpollutants
followedby20.7%,medicinesby19.6%and14.2%intheproportionofplants.

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UniversitateadetiineAgricoleiMedicinVeterinarIai

19.6
14.2
20.7

Medicines
Plants
Pollutants

Regardinganimalspeciesmostaffectedwerethedogs(36.6%)andthecattle(25.1%).

Other
Fishes
Poultry
Cats
Dogs
Horses
Pigs
Sheeps
Cattle

14.7
2.7
2.8
7.4
36.6
3.8
2
4.9
25.1

Using this type of information, we could move to the proposed composition of the
database,withthementionthatitwillbeconstantlyenrichedwithnewdataandupdatedwith
thelatestresearchinthefield.

RESULTSANDDISCUSSIONS

The program can be accessed by any interested person equipped with a computer
terminal and internet connection. The link between the user and the database is made
throughafriendlygraphicalinterfaceandergonomicappearance.
The user can easily access information about any animal poisonings, which are
classifiedalphabetically,accordingtothespeciesofanimalsaffectedorthetypeofetiological
agentandtheclinicalsyndromeproduced.
Onceselectedatoxicosis,thevisitorispresentedwithadocumentthatcontainsdata
about the species most affected, etiology, pathogenesis, clinical manifestations, lesions,
prognosis and treatment of such poisoning. The datas presented are structured so they are
quickandeasytostudy.
Another important component of the program is the ability to query the database
through a similar questionnaire used in the Faculty Clinics Medical Records, which includes
data on the animal, clinical course and clinical signs observed. Thus, the user is guided to a
correctdifferentialdiagnosisandasanemergencytreatmentinstitutedasquickly.
Enteringandeditinginformationinthedatabaseismadethroughaseparateaddress
whichisrestricted,basedonanaccountpassword.

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CONCLUSIONS

Theprogramisintendedfortheuseofveterinarycliniciansandstudentswhowishto
informthemselvesintheveterinarytoxicologyfield.Italsocanaddresspetownersfortheir
ownknowledge,notingthat,incaseofemergencies,themainmedicalpriorityisstillaswift
visittoaveterinaryclinic.
In the European Community, the number of users of these types of programs is
constantly growing, thanks to the many benefits structured information source, with rapid
accessandcontinuousdataupdating.
In addition to advantages in clinical and teaching fields, the programs ability to
gatherandsubmitregularstatisticaldataonthedatabasequeriesisalsobeneficialinscientific
research,withmultipleusesintheanimalandenvironmentalsurveillance.

REFERENCES

1.
2.
3.
4.
5.
6.

Comer,Douglas(2006).TheInternetbook.PrenticeHall
Connolly,Thomas.DatabaseSystems(2nded.).AddisonWesley
CrivineanuV.,GoranG.ToxicologieVeterinara,EdPrintech,2004
DavidJ.Eck(2000).TheMostComplexMachine:ASurveyofComputersandComputing.AK
Peters,Ltd.
LorgueG.,LechenetJ.,RiviereA.ClinicalVeterinaryToxicology,EditedinEnglishbyMJ
Chapman,BlackwellScience,1996
***Biohealthmatics.comKnowledgeCenter

1119

ANTIMICROBIALRESISTANCEOFCAMPYLOBACTERJEJUNIAND
CAMPYLOBACTER
COLISTRAINSISOLATEDFROMBROILERSSKIN

IsabelaNICORESCU,MariaCRIVINEANU
FacultyofVeterinaryMedicineBucharest,105SplaiulIndependentei,
050097,Bucharest,Romania,isabela_nicorescu@yahoo.com

Abstract
The use of antimicrobial agents in animal production or for the treatment and control of
infectious diseases in animals has strong public health consequences by creating a reservoir of
resistantbacteriaandbacteriaborneresistancegenesthatcanbepassedontohumans.
In this study, 36 strains of Campylobacter jejuni and 22 strains of Campylobacter coli isolated
from broilers skin samples were tested for antimicrobial susceptibility. The samples were
collectedaspartofastudyontheprevalenceandantimicrobialresistanceofCampylobacterspp.
inbroilerscarcassesatretaillevel.Microorganismsweretestedfortheirabilitytoproducevisible
growthinmicrotiterplatewellsholding identical volumes of broth with a defined inoculum,in
the presence of antimicrobial agent solutions in incrementally twofold increasing
concentrations.Thetestswereperformedunderstandardizedconditions.
The obtained results showed that the most frequent resistance of Campylobacter strains was
detectedtotetracyclineandciprofloxacin;thus,theresistancetotetracyclinewas38.8%forC.
jejuni,whileforC.colitheresistancewas72.7%.Theresistancetociprofloxacinwas53.6%forC.
jejuniand63.6%forC.coli.Alowerlevelofresistancewasfoundtogentamicin(C.jejuni5.5%
andC.coli4.5%),streptomycin(C.jejuni2.7%)anderythromycin(C.jejuni5.5%).
TheprevalenceofantimicrobialresistancedifferedbetweenC.coliandC.jejuni,notingahigher
resistance of C. coli. The high prevalence of ciprofloxacin and tetracycline resistance found in
Campylobacter strains isolated from broilers is extremely worrying; this situation could have
importanttherapeuticimplications.

Keywords:C.coli,C.jejuni,antimicrobialresistance,broilers.

Thermophilic Campylobacter species, particularly Campylobacter jejuni and


Campylobacter coli, are recognized as one of the most frequent causes of acute diarrheal
disease in humans throughout the world. Campylobacter infections usually occur following
ingestionofimproperlyhandledorcookedfood.Campylobacteriosisisconsideredazoonotic
disease,andanimalssuchaspoultryrepresenttheprincipalreservoirforCampylobacter(5).
Antibioticsareusedinhumanandveterinarymedicinefortreatmentandprevention
ofinfectionsandasgrowthpromotersinfoodanimals.Theirincreasedusehasresultedinthe
increased incidence and levels of antibiotic resistance to many enteric bacteria and
encouraged the persistence and transfer of antibioticresistance determinants in microbial
genomes(2,4).
Antibioticresistanceisanincreasingprobleminsomepathogensandthecapabilityof
multidrugresistanceisaconcern.Someauthoritiesregardfoodanimalsastheprimarysource
ofantibioticresistancegenespresentinhumanfoodbornepathogens,whereastheothersas
the major source of the problem regard imprudent use of antibiotics in humans. In case of
zoonotic bacteria like campylobacters, the use of antimicrobials in animals may determine
drugresistant bacterial populations, which represent a potential threat to the consumer.
Whenapathogenhasalreadyacquiredantibioticresistance,thereisnoneedforgenetransfer
ordevelopmentofdrugresistanceforthepathogentobedifficulttocontrolclinically(1,7).

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Lucrritiinificevol53seriaMedicinVeterinar
Duetotheoftenselflimitingdiarrheainhumans,antibiotictreatmentisnormallynot
required. Nevertheless, antibiotic treatment is indicated for severe and prolonged enteritis,
septicemia and for persons at risk such as very young or immunocompromised patients.
Besides erythromycin,whichisanantibioticof choice, fluoroquinolonesandtetracyclineare
commonlyused.Particularlyfluoroquinolonesarefirstlinedrugsforempirictherapyofacute
diarrhea, as they are effective against most major pathogens causing bacterial enteritis.
Therefore, most cases of campylobacteriosis receiving antibiotic treatment will initially be
treated with fluoroquinolones. Worrying is the fact that in recent years a rapidly increasing
proportionofCampylobacterisolatedfromhumansandanimalsallovertheworldwerefound
toberesistanttofluoroquinolones(3).
The objective of this study was to determine the prevalence of antibiotic resistant
Campylobacterstrainsinbroilersskinsamplesatretaillevel.

MATERIALSANDMETHODS

Thefollowingantimicrobialagentsweretestedforbacterialresistance:ciprofloxacin,
erythromycin, gentamicin, nalidixic acid, streptomycin and tetracycline. The MIC
determination was done using broth dilution method, as recommended by Clinical and
Laboratory Standards Institute (CLSI) (8, 9). To accomplish this purpose it was used The
SensititresusceptibilityplatesforCampylobacterbyTrekDiagnosticSystem.Microorganisms
were tested for their ability to produce visible growth in microtitre plate wells holding
identical volumes of broth with a defined inoculum in the presence of antimicrobial agent
solutioninincrementallytwofoldincreasingconcentrations.Thetestswereperformedunder
standardizedconditionssothattheresultstobereproducible.

A total of 36 strains of Campylobacter jejuni and 22 strains of Campylobacter coli


isolated from broiler skin samples were tested for antimicrobial susceptibility. Isolation of
Camylobacterspp.wasperformedinaccordancewiththeSRENISO102721/2006.Samples
werepreparedasepticallybyweighing25gintoasterilestomacherbagandthen225mlof
Boltonbrothwasadded.Afterincubationfor48hoursat41.5Cinmicroaerobicatmosphere,
one loopful of enrichment broth was streaked on modified charcoal cefoperazone
deoxycholateagar(mCCDA)andincubatedfor48hoursat41.5Cinmicroaerobicatmosphere.
The confirmation of Campylobacter spp. and then the identification of the species were
performedbybiochemicaltests(12).
Afterspeciesdetermination,CampylobacterstrainswereincubatedonColumbiaagar
with sheep blood in a microaerophilic atmosphere for 24 hours at 41.5C. From each plate,
colonies weretaken and dissolved completely in 4 ml saline water so the read valueon the
McFarland scale to be 0.5, which represents approximately 12 x 108 cfu/ml. From this
suspension there were transferred 50 l to 10 ml of MullerHinton broth with lysed horse
blood, the mix was homogenized and then inoculated each 100l per well in the panel.
Inoculum was approximately 5 x 105 cfu/ml. After that, all wells were covered with a
perforated adhesive seal and the samples were introduced to incubation in an atmosphere
containing 10% CO2, 5% O2 and 85% N2 for 48 hours. The microaerophilic atmosphere was
obtainedusinggasgenerationkits(11).

After incubation, the results were read using the Sensititre manual. The MIC is
recorded as the lowest concentration of antimicrobial agent that inhibits visible growth.
Growthappearsasturbidityorasadepositofcellsatthebottomofawell(11).

The epidemiological cutoff values are established by EUCAST (the European


Committee on Antimicrobial Susceptibility Testing); in case of Campylobacter, the cutoff

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UniversitateadetiineAgricoleiMedicinVeterinarIai
points are different for Campylobacter jejuni and Campylobacter coli. These values and the
rangetestedforeachantimicrobialagentarepresentedintable1(11).

Table1
RangeofantimicrobialstestedforCampylobacterjejuniandCampylobactercoliandcutoff
valuesappliedfortheclassificationofstrainsasresistant

Cutoffvalue(g/ml)R>
Antimicrobial
Rangetested
Abbreviation
Campylobacter
Campylobacter
(g/ml)
agent
jejuni
coli
Tetracycline
TET
0,25 16
2
2
Erythromycin
ERY
0,5 32
4
16
Streptomycin
STR
1 16
2
4
Gentamicin
GEN
0,12 16
1
2
Ciprofloxacin
CIP
0,06 4
1
1
Nalidixicacid
NAL
2 64
16
32

RESULTSANDDISCUSSIONS

Intotal,58Campylobacterstrainsweretestedforantimicrobialresistance;20strains
(34.48%)weresensitivetoalltestedantibiotics,while38strains(65.52%)wereresistanttoat
least one of the antibiotics. Four strains were resistant to four or more of the tested
antibiotics;theseisolateswereconsideredmultiresistant.Noneoftheisolateswasresistantto
allsixagentstested.TheresultsofantimicrobialsusceptibilitytestingforC.jejuniandC.coli
strainsisolatedfrombroilersskinareshownintables2and3.

Table2
AntimicrobialresistanceinCampylobacterjejuni(n=36)frombroilerskin
Distribution(%)ofMICvalues(g/ml)

Distribution(%)ofMICvalues(g/ml)
Resistance
Substance
0.2
(%)
0.064 0.125
0.5
1
2
4
8
16 32 64
5
Tetracycline
38.8

27. 0.5
27.
25
8.3 5.5

6
7
Erythromyci
5.5

83. 11.
5.5

n
3
1
Streptomyci
2.7

88. 8.3
2.7

n
8
Gentamicin
5.5

47.2 25
22.
2.7

2.7

2
Ciprofloxaci
58.3

19. 22. 8.3


50

n
4
2
Nalidixic
5.5

44. 30. 16. 2.7

5.5
acid
4
5
6
Boldverticallinesdenotemicrobiologicalcutoffvalues.Whitefieldsdenoterangeofdilution
testedforeachantimicrobialagent.

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Lucrritiinificevol53seriaMedicinVeterinar
Table3
AntimicrobialresistanceinCampylobactercoli(n=22)frombroilerskin
Distribution(%)ofMICvalues(g/ml)

Distribution(%)ofMICvalues(g/ml)
Resistance
Substance
0.2
(%)
0.064 0.125
0.5
1
2
4
8
16 32 64
5
Tetracycline
72.7

13.
13.
18. 13. 40.
6
6
1
6
9
Erythromycin 22.7

54.
22.

9.0 13.
6
5
7
9
Streptomycin 13.6

31. 36. 18.

13.
6
8
3
1
Gentamicin
4.5

45.
36. 13.
4.5

4
3
6
Ciprofloxacin
63.6
13.6
22.
9.0 13. 40.

9
7
9
6
Nalidixicacid
9.09

18. 31. 40.

9.0
1
8
9
9
Boldverticallinesdenotemicrobiologicalcutoffvalues.Whitefieldsdenoterangeofdilution
testedforeachantimicrobialagent.

Inourstudy,mostfrequencyofresistanceofCampylobacterstrainswasdetectedto
tetracyclineandciprofloxacin;thustheresistancetotetracyclinewas38.8%forC.jejuni,while
forC. colitheresistancewas72.7%.Theresistance to ciprofloxacinwas58.3% for C.jejuni
and63.6%forC.coli.Bycomparison,thesevaluesaresimilartothosereportedbyEuropean
FoodSafetyAuthority(EFSA)(10).Itisinterestingthatresistanceisquitehightociprofloxacin,
considering that studies carried out up to 1987 had shown that strains resistant to
fluoroquinoloneswerepracticallynonexistent.Theuseofquinolones,mainlyenrofloxacin,in
veterinarypractice,hasbeencorrelatedwiththeincreaseresistanceinCampylobacterstrains
(6).
DifferenceswereobservedinthefrequencyofresistanceamongC.colicomparedto
C. jejuni: C. coli strains were more likely to be erythromycinresistant compared to C. jejuni
(22.7%comparedwith 5.5 %).C. coliwerealsomorelikelytobetetracycline, streptomycin
and ciprofloxacinresistant compared to C. jejuni (72.7 % compared with 38.8 % for
tetracycline,13.6%compared2.7%forstreptomycinand63.6%comparedwith58.3%for
ciprofloxacin).Thelowestlevelsofresistancewerefoundtogentamicin(C.jejuni5.5%andC.
coli4.5%),streptomycin(C.jejuni2.7%)anderythromycin(C.jejuni5.5%).

CONCLUSIONS

1. Tetracycline, ciprofloxacin and erythromycin resistance was present in


Campylobacterstrainsisolatedfrombroilersskin.
2. The prevalence of resistance differed between C. coli and C. jejuni, noting a
higherresistanceofC.coli.
3. The high prevalence of ciprofloxacin and tetracycline resistance found in
Campylobacter strains isolated from broilers is extremely worrisome; this
situationcouldhaveimportanttherapeuticimplications.

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UniversitateadetiineAgricoleiMedicinVeterinarIai

REFERENCES

1.

Engberg J., Aarestrup F.M., Taylor D.E.., GernerSmidt P., Nachamkin I. Quinolone and
macrolideresistanceinCampylobacterjejuniandC.coli:resistancemechanismsandtrendsin
humanisolates.Emerg.Infect.Dis.,7,pp.2434,2001.
2. HaradaK.,AsaiT.,KajimaA.etal.CharacterizationofmacrolideresistantCampylobactercoli
isolatesfromfoodproducinganimalsonfarmacrossJapanduring2004.J.Med.Vet.Sci.,68,
pp.11091111,2006.
3. Ishihara K., Yano S., Nishimura M. et al. The dynamics of antimicrobialresistant
Campylobacterjejunionbroilerfarms.J.Med.Vet.Sci.,68,pp.515518,2006.
4. Luangtongkum T., Morishita Teresa, ElTayeb Amna, Ison A.J., Zhang Q. Comparison of
antimicrobialsusceptibilitytestingofCampylobacterspp.bytheagardilutionandtheagardisk
diffusionmethods.J.Clin.Microb.,45,pp.590594,2007.
5. ParisiA.,LanzilottaS.G.,AddanteN.,NormanoG.,ModugnoG.,DambrosioA.,MontagnaC.O.
Prevalence, molecular characterization and antimicrobial resistance of thermophilic
Campylobacter isolates from cattle, hens, broilers and broiler meat in Southeastern Italy.
SpringerNetherlands,311,2007.
6. Saenz Yolanda, Zaragaza Myriam, Lantero Marta, Torres Carmen Antibiotic resistance in
Campylobacterstrainsisolatedfromanimals, foodsandhumansinSpain.Antimicrob.Agents
andChemother.,442,pp.267271,2000.
7. SorumH.Antibioticresistanceinfoodrelatedbacteriaaresultofinterferingwiththeglobal
webofbacterialgenetics.Int.J.FoodMicrobiol.,78,pp.4356,2002.
8. *** Clinical and Laboratory Standards Institute. Methods for antimicrobial dilution and disk
susceptibilitytestingofinfrequentlyisolatedorfastidiousbacteria;approvedguideline.M45A.
ClinicalandLaboratoryStandardsInstitute,Wayne,PA,2006.
9. *** Clinical and Laboratory Standards Institute. Performance standards for antimicrobial
susceptibility testing; 16th informational supplement. M100S16. Clinical and Laboratory
StandardsInstitute,Wayne,PA,2006.
10. *** EFSA The community summary report on trends and sources of zoonoses, zoonotic
agents, antimicrobial resistance and foodborne outbreaks in the European Union in 2005, p.
83102,2007.
11. ***SensititresusceptibilityplatesforCampylobacter.2009
12. ***SRENISO102721Foodsandfeedsmicrobiology.Horizontalmethodsfordetectionand
enumerationofCampylobacterspp.,p.48,2006.

1124

INCIDENCEOFSALMONELLASPP.
INAPOULTRYSLAUGHTERINGUNIT

MIHAIOBAD,ALINAVLADSABIE,MIHAICARPCARARE
UniversityofAgriculturalSciencesandVeterinaryMedicineIonIonescu
delaBradIai
FacultyofVeterinaryMedicine

Abstract
Salmonellaspp.onpoultry carcasses was identified usingstandard ISO SR EN 6579/AC/2006.A
numberof60sampleswereanalyzedinthe20092010period.Thesampleswerecollectedfrom
thepoultrycarcassesandtestedbyclassicalbacteriologicalmethods.Theproportionforpoultry
carcassescontaminatedwithSalmonellaspp.was5%.
Keywords:slaughteringunit,Salmonellaspp.,poultry

INTRODUCTION

The diseases originating in bacteria from genera Salmonella represent for the
majority of countries with high animal raising industry one of the most important sanitary
veterinaryproblemsduetofinanciallosesandalsoduetotheimplicationinhumanhealthof
foodbornediseasesbecauseoftheconsumptionofcontaminatedanimalproducts.
Progress in the last decades in the technology of processing, preservation and
manipulation have only partially removed the possibility of contamination with pathogenic
microorganisms, and consumer claims about the microbiological quality of poultry carcasses
grewincreasingly.Consumersaremorecarefulwhentheyarebuyingpoultrycarcasses,more
sensitive to smell, flavor and color. It could say that, currently the microbiological quality of
poultrycarcasseshasbecomeamajorfactorinitsmarketing.
Poultry carcasses is an important source of human contamination with Salmonella
spp.SalmonellaenteritidisandSalmonellatyphimuriumareserovarsmostfrequentlyisolated
frompoultrycarcassesandincasesoffoodpoisoninginhumans(EFSAJournal,2004).

MATERIALSANDMETHODS

In20092010werecollectedatotalof60samplesinaslaughteringunit.Thesamples
were obtained under sterile conditions by taking neck skin, weighing 25 grams for each
sample.
Carcasemicrobiologicalassessmentwasmadeaccordingtoprogramsofthe
National Agency for Sanitary Veterinary and Food Safety : ISO SR EN 6579/AC/2006, which
statesthatidentificationofmicroorganismslikeSalmonellaspp.frompoultrycarcasseswillbe
madeon25gramssamplesofneckskin.Accordingtothisstandardin25gsampleSalmonella
spp.hastobeabsent.

Inordernottosufferfurthercontamination,skin
fragments were collected with sterile scissors and were lodged in special sterile bag
(Stomacher305/175mm)keptonicesincesamplinguntiltothelaboratory,wheretheywere
processed.
1.Preenrichment phase consisted of inoculation of samples in a neselective liquid
medium(BPWbufferedpeptonewater)andincubationat37Cfor18h.

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Lucrritiinificevol53seriaMedicinVeterinar
Procedure:wereseededin225mlprewarmedBPW25gproduct.(KoyuncuSevincet
al.,2009).
2.Enrichment phase was made on two liquid selective enrichment mediums.
Enrichmentconsistedoftwoselectivemediainoculatedwiththeculturefrompreenrichment
medium;thususedasselectiveenrichmentmedium,RappaportVassilidiswithsoybroth(RVS
broth), incubation was done at 40.5 to 42.5C for 24 h and for medium MullerKaufmann
tetrathionatebrothwithNovobiocin(MKTTn),incubationwasdoneat37Cfor24hours.
3.Isolation phase consisted of the sowing of a selective solid mediu with cultures
obtainedinenrichmentphase.
It was used the following medium : dezoxicolat xylose lysine agar (XLD agar)
incubationat37Candexaminedafter24h.
Only the characteristic colonies developed on the selective isolation media were
takenintoconsidered.ThecharacteristiccoloniesofSalmonellaspp.grownonXLDagarhavea
blackcenterandatransparentlightareaofredcolourduetocolorchangeindicator.
4.ConfirmationphaseconsistedinelectionofthepresumptivecoloniesofSalmonella
spp. and determination of serological and biochemical characteristics, being tested one
characteristiccolonyfromeachplate.
Toobtainafasterresult,suspiciouscoloniesfromselectivemediawereinoculatedon
mediumspolitrope:TSI(triplesugariron),MIU(mobility,indole,urea),MILF.
TSIagarhavinginregardthewayhowthemediumispouredintothetube(slope
andcolumn)theslopingsurfaceofagarwasseededbystriateandcolumnbypuncture.Itwas
incubatedat37Cfor24handwereinterpretedmediumchangesasfollows:
a) column.
yellowglucozopositive(glucosefermentation)
redorunchangedglucozonegative(notfermentglucose)
blacktheformationofhydrogensulfide;
bubbleorcrackgasformationfromglucose,
b) inclinedsurfacemedium
yellowlactozo/zaharozopositive;
redorunchangedlactozo/zaharozonegative;

Characteristic cultures of Salmonella spp. showed that inclined surface medium is


alkaline(red)andtheaveragemediumisacid(yellow)withgas(bubbles)andhydrogensulfide
formation.
Ureaagarwasseededtheinclinedsurfaceofmediumbystriate.Wasincubatedat
37Cfor24h.Ifthereactionispositive,breakingureamoleculereleaseammonia,whichturns
thecolorofphenolredtopinkandlatertodarkred.
Llysinedecarboxylationmediumliquidmediumwereinoculatedimmediatelybelow
thesurface.Wasincubatedat37Cfor24h.Turbidityandpurplecolorationafterincubation
showedapositivereaction(yellownegative).
Medium for indole reaction was inoculated a tube containing 5 ml of medium
tryptone/tryptophan with suspected colony. It was incubated at 37C for 24 h. After
incubation1mlKovacs'reagentwasadded;formingaredringindicatesapositivereaction.
Researchregardindthepresenceofantigens"O"Salmonellawasperformedbyslide
agglutinationwithappropriateserafrompureculturesandafterremovalofautoagglutinated
strains.(ApostuSorin,2006,VolIII)
SerologicalconfirmationwasmadeusingserumantiSalmonella"O"polyvalentC,F,
G,H.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Indications: Serological identification of Salmonella strains belonging to serogroups
CO,FO,GO,HOismadeusingKauffmannWhitescheme.Identificationofasuspectedstrain
ofSalmonellawasmadeusingtheslideagglutinationreaction.
Slide agglutination reaction : agglutinative serum was diluted in sterile isotonic
solution of saline (SPS) as indicated on vial strength. On microscope slide is put a drop of
diluted immune serum and beside him a drop of sterile saline. Bacteriological loop is
performedusingansuspensionofabacterialcolonyidentifiedbiochemicalold18to20hours
on 2% agar medium in a drop of sterile saline. Were excluded from serotyping Salmonella
strainsform
R
whichagglutinatespontaneouslywithsterilesaline.
Ifthesuspensionishomogeneoustheprocedureisthesameincaseofdropofserum
agglutinative.Ifagglutinationispositive,whitegranulesappearin13minutes.

Faster detection method was made on chromogen Rambach medium, on


whichthecharacteristiccoloniesofSalmonellaspeciesarered.
FasterdetectionofSalmonellasppinfoodwasmadewithSinglepathSalmonellaused
forqualitativedetermination.
SinglepathimmunochromatographicSalmonellaisatestbasedonantibodieslabeled
withgold.Testdevicehasancircularareawhereisputtheculturesuspensionandanovaltest
(T)andancontrolwindow(C).
TestsamplewassubjecttoanenrichmentbrothmediumRappaportVassiliadisfor
24hoursat37C,thenthetubewithmediumwassubjectedtoboilingwaterbathfor15min.
Itwaslefttocoolatroomtemperature,andtransferredusingapipette150mlinthecircular
test.Readingwasdonein20minutesandtestpositivitywasindicatedbytheemergenceofa
distinctredlines,similartothatincontrol.

RESULTSANDDISCUSSION

Although Salmonella spp is recognized as the most important pathogens associated


to poultry is not known precisely the disease incidence in humans due to poultry. (D'Aoust,
1989)
TheincidenceofbacteriaofthegenusSalmonellainandonbirdsrangingfromone
geographicalareatoanother,thedatesrangingbetween4100%.Theincidenceisinfluenced
byfactorssuchasgrowingconditionsandclimate.TherearecountrieslikeSwedenwherethe
birdsarefreefromSalmonellaspp,situationreachedafterapplicationofgovernmentcontrol
programsandmeasuresbypoultryfarmersandmeatprocessors.
Forselectiveisolationwereusedtwosolidmedia,onehighlyselective(XLD)24hours
at37Candonelessselective(IstratiMeitert),onwhichcoloniesappeargreen.

BiochemicalconfirmationwasdoneonT.S.I.,M.I.L.F.andAPI10S.
SerologicalconfirmationwasmadeusingserumantiSalmonella"O"polyvalentC,F,
G,H.
In slaughterhouses which slaughter large numbers of birds are frequent cross
contaminationduetothesmallspacebetweencarcassesplacedonconveerandthebalance
ofcarcassesallowingthecontactofeachother.(CreuCarmenandcol.,2008)
Faster isolation from food was made using Glisa test and Rambach chromogen
medium,andtheidentifiedwaseasytodoheavinginregardthatchromogenmediumallowto
obtaincharacteristiccolonies.
The proportion for poultry carcasses contaminated with Salmonella spp. was 5%.
(Table1)

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Lucrritiinificevol53seriaMedicinVeterinar
Table1

Samples

Salmonellaspp.values/25gneckskinfrompoultrycarcasses

Nr.of
Positivesamples
Negativesamples
samples
Nr.
%
Nr.
%

Poultrycarcasses

60

5%

57

95%

After Committee WHO / FAO data, were identified based on antigenic study over
2400 (2435) of serotypes. Their spread in the world does not recognize regional boundaries
andwithfewexceptions,havelittlehostspecificity.
Ontheotherhand,iswidelyacceptedthatnolongercandrawaclearlinebetween
Salmonellapathogenicforhumansandthosefoundinanimals.Thereforetheissuesraisedby
Salmonellainfectionsinanimalsandhumanscanbetreatedonlyasawhole.(CasonJ.A.,1997,
DAoustJY.,1989)
BetweendifferenttypesofSalmonellaandorganismsinwhichtheyhavepenetrated,
differentrelationshipsareestablished.Itseemsdifficulttoexplainwhy,quitefrequently,even
incasesofhighlypathogenicserotypes,therelationshipbetweenorganismandgerms,remain
instateofstrictlyepiphyte,moreorlesslong.
Salmonella enterica serovar enteritidis was the predominant serovar in outbreaks
with Salmonella in 2007. Registration percentage was 60.2% in case of outbreaks of
Salmonellaentericaandserovartyphimuriumshowedarateof10.7%.(EFSAJournal,2009)
In2008,salmonellosiswasagainthesecondmostoftenreportedzoonoticdiseasein
humansaccountingfor131,468confirmedhumancases.Thestatisticallysignificantdecreasing
trendinthenotificationrateofthesalmonellosiscasescontinuedintheEuropeanUnionfor
the fifth consecutive year. In particular, the human cases caused by S. Enteritidis decreased
markedlyin2008,whileanincreaseinS.Typhimuriumcaseswasobserved.
Infoodstuffs,Salmonellawasmostoftendetectedinfreshbroiler,turkeyandpig
meat,onaverageatlevelsof5.1%,5.6%and0.7%,respectively.
Overall,intheEU,S.EnteritidisandS.Typhimuriumaretheserovarsmostfrequently
associatedwithhumanillness.HumanS.Enteritidiscasesaremostcommonlyassociatedwith
the consumption of contaminated eggs and poultry meat, while S. Typhimurium cases are
mostlyassociatedwiththeconsumptionofcontaminatedpig,poultryandbovinemeat.(EFSA
Journal,2010)

CONCLUSIONS

1. The 60 samples were examined under sterile conditions, taking neck skin, weighing
25 grams for each sample on poultry carcasses, determining the incidence of
Salmonellaspp.
2. Inpoultrycarcases,Salmonellaspp.wasdetectedatlevelof5%.
3. ThepercentageissimilartothatfoundinEU.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
REFERENCES

1.
2.
3.

4.
5.

6.
7.
8.
9.

ApostuS.,2006FoodMicrobiology,VolIII,Ed.Risoprint,ClujNapoca.
CasonJ.A.,BaileyJ.S.andcol.,1997Relationshipbetweenaerobicbacteria,Salmonellaeand
Campylobacteronbroilercarcasses,PoultryScience.,vol.76,p.10371041.
Creu Carmen, CarpCarare M., Floritean V., Isan Elena, Brdan Ghe., 2008 Researches
regardingSalmonellaspp.incidenceinpoultrycarcassesdestinedtohumanconsume,Lucr.t.
U.S.A.M.V.ClujNapoca,seriaMedicinVeterinar,vol.65,pag.316322.
DAoustJY.,1989SalmonellainDoyle.M.P.(Ed.),FoodborneBacterialPathogens,Chapter9,
EdituraMarcelDekkerInc.,NewYork.
EFSA Journal, 2004 EFSA opinion of the scientific panel on biological hazards on a request
from the commission related to the use of antimicrobials for the control of salmonella in
poultry.
EFSAJournal,2009TheCommunitysummaryreportonfoodborneoutbreaksintheeuropean
unionin2007.
EFSAJournal,2010The communitysummary reportontrends andsourcesof zoonosesand
zoonoticagentsandfoodborneoutbreaksintheeuropeanunionin2008
KoyuncuSevincandcol.,2009Acomparativestudyofculturalmethodsforthedetectionof
Salmonellainfeedandfeedingredients,BMCVeterinaryResearch.
SR ISO 6579/AC/2006 Microbiology of food and feed. Horizontal method for detecting
bacteriaofthegenusSalmonella.ASROStandardRomn.

1129

THEPROTECTINGEFFECTOFAPOLYPHENOLICEXTRACT
OBTAINEDFROMSEABUCKTHORN(HIPPOPHAERHAMNOIDES)
FRUITSAGAINSTPHOTOOXIDATIONOFDIFFERENTVEGETALOILS

CameliaPAPUC,V.NICORESCU,CorinaDURDUN,
Gh.GORAN,CarmenCRIVINEANU

FacultyofVeterinaryMedicineBucharest,105SplaiulIndependentei,
050097,Bucharest,Romania,cami_papuc@yahoo.com

Abstract
The stability of oils is commonly affected by photooxidation processes, which yields both
primary and secondary oxidation compounds. In this study, it was investigated the ability of
alcoholic extracts obtained from sea buckthorn fruits (Hippophae rhamnoides) to act as
antioxidantsinphotooxidationprocessofsomecommercialvegetaloils.Theexperimentswere
carriedonrafinatedsunfloweroil,virginoliveoilandcoldpressedmaizegermsoil.Theoxidative
stabilityofoilsaddedwithseabuckthornextractwasdeterminedafterexposure30minutesat
UV light. Peroxide values, the formation of conjugated dienes and malondialdehyde and free
fatty acids assay were studied in order to estimate the oxidation process. Peroxide values and
free fatty acids were investigated by volumetric assays; conjugated dienes were evaluated by
spectrophotometry at 232 nm, while the formation of malondialdehyde was investigated by
thiobarbituricacidassay.Theexperimentsshowedthatseabuckthornalcoholicextractprotected
the oils against photooxidation process. The obtained results indicate the fact that sea
buckthorn extracts could be used in food industry as natural antioxidants in order to replace
partiallyortotallysyntheticantioxidants.

Key words: sea buckthorn, photooxidation, peroxide value, conjugated dienes,


malondialdehyde,acidvalue.

Lipid oxidation caused by UV radiation is named photooxidation, and involves the


interaction of unsaturated lipids with singlet oxygen. Oxygen excited state (1O2), singlet
oxygen, is produced when light interacts with oxygen in the presence of pigments such as
chlorophyllandriboflavin,namedsensitizers:

light
senssens*
sens*+3O2sens+1O2

Singletoxygenspeciesgenerates photooxidationofunsaturatedlipidsthatinvolves
theattackatdoublebondofasingletoxygen(5).
Incaseofphotooxidation,ithasbeenpostulatedthatthedoublebondwithinafatty
acidmoleculemaybecapableofcapturingoutsidesourceofenergy,suchasheatandlight,to
reachacriticalexcitationlevel(4).Uponreachingthecriticalexcitationlevel,thedoublebond
may break, thus giving rise to a free radical species, which may in turn generate more free
radicals.
Photooxidation,whichisanaturaloxidationandchemicaldegradationprocessesof
edibleoils,alsoresultinrancidityoffooditems,wherebytheseprocessesconvertfattyacid
estersofoilsintofreefattyacids.Furthermore,thefreefattyacidsalsogiverisetosmellthat
isobservedinmanyvegetableoilsovertime(1).

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Lucrritiinificevol53seriaMedicinVeterinar
Photooxidation cannot be stopped by the synthetic antioxidants such butylated
hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) or the natural antioxidant
tocopherols(vitaminE).Photooxidationmaybeinterruptedbyintroducingmoleculeswhich
reactsmorequicklywithsingletoxygenthanunsaturatedlipids.
Plant polyphenols are toxicityfree antioxidants that have the capacity to annihilate
freeradicalswhichinitiateandpropagatelipidperoxidationprocessesand,fromthisreason,it
isthoughtthatpolyphenolscanpartiallyortotallyreplacesyntheticantioxidants(2,3,6,7,8).
The aim of this study was to demonstrate photooxidation protecting effects of a
polyphenolicextractobtainedfromseabuckthornfruitsondifferentcommercialoils.

MATERIALSANDMETHODS

Obtainingofpolyphenolicextract.Inordertoobtainvegetalextract,thedryvegetal
materialwasgroundandthensubduedtoasolidliquidextractionwithethanol60%(v:v).
Oils.Theexperimentswerecarriedoncommercialrafinatedsunfloweroil,virginolive
oil and cold pressed maize germs oil. Oils were treated with vegetal alcoholic extracts in
relation of 100:1 (v:v). The oxidative stability of oils added with sea buckthorn extract was
determinedafterexposureatUVlight(254nm)for30min.Allsamplesandcontrolsystems
wereevaluatedintriplicate.
Determination of peroxide value. Peroxide values were determined according to
Official American Oil Chemists Society (AOCS) methods (AOCS, 1985). The values were
expressedasmeqofperoxideO2/kgoil.
Determination of conjugated dienes. Conjugated dienes formed through photo
oxidation of oil samples were determined according to the analytical methods described by
IUPAC(IUPAC,1979).Thecontentofconjugateddieneswereexpressedasabsorptivityat232
nmof1%oilsin2,2,4trimethylpentane.
Determinationofmalondialdehide.Amodifiedthiobarbituricacidreactivesubstances
(TBARS) method was used to measure the antioxidant activity of alcoholic extracts. 50 l
alcoholic extract was added to 1.95 ml TBATCA (thiobarbituric acid trichloroacetic acid)
solution. The mixture was heated in a 100C water bath for 30 min and cooled at room
temperature. After the addition of 2 ml butanol, the mixture was mixed and centrifuged at
2000 rpm for 15 min. The butanol layer was separated and the absorbance at 532 nm was
measured(9).
Determination of acid value. Acid value was determined according to Official
AmericanOilChemistsSociety(AOCS)methods(AOCS,1985).Thevalueswereexpressedas
mgKOH/goil.
RESULTSANDDISCUSSIONS

Controloilsampleshaddifferentreactionstophotooxidationbyexposurefor30min
at254nmUVradiation;thisfactisduetothevariablecontentinunsaturatedlipidsandalso
to the variable contents in tocopherols of the oils. Oil samples added with sea buckthorn
alcoholicextractexhibitedphotooxidationprotectionforalltestedoils.
Peroxide value. The peroxide value was taken as a measure of primary oxidation
compoundsproducedinthephotooxidationofoilssamples.Theobtainedresultsshowedthat
seabuckthornalcoholicextractprotectedagainstphotooxidationalloilsamples(Fig.1).Sea
buckthornextractofferedthebestprotectionagainstlipidperoxidationincaseofsunflower
oil; for this oil, peroxide value was 23 times higher after UV exposure in case of control
samples,whileincaseofsunfloweroiladdedwithseabuckthornextractperoxidevaluewas

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Absorptivityat232nm

Peroxidevalue
(meqO2/kg)

8.8 times higher after UV exposure. Good results were obtained also for maize germs oil;
peroxidevaluewas14timeshigherforcontrolsamplesand10timeshigherforseabuckthorn
added samples after UV exposure. In case of virgin olive oil, peroxidation process was less
intense,probablyduetoitslowercontentinlinoleicandlinolenicacids.

25

Sunflower oil + SBT extract


20

Sunflower oil control

15
Olive oil + SBT extract

10
Control olive oil

Maize germs + SBT extract

0
Maize germs control
Initial
Photooxidation

Fig.1.Relationshipbetweenperoxidevalueandoilsexposure
at254nmUVradiationfor30minutes

Conjugated dienes. Oxidation can cause double bond position shifts in


polyunsaturated fatty acids. In this process, conjugated dienes and trienes structures are
formed, and this can be indicated by changes in UV absorption (10, 11). The changes in the
absorptivityat232nmofallsystemsarepresentedinFig.2.Theincreaseofabsorptivityat
232 nm was progressive for all control oils, proportionaly with polyunsaturated fatty acids
content.Seabuckthornalcoholicextractdeterminedadecreaseofconjugateddienesforming
ratioforalloils.Theobtainedresultsaresimilartotheonesrecordedforperoxidevalue;the
lowestconjugateddienesformationprocesswasobservedincaseofoliveoil,whilethebest
protection against photooxidation was recorded in case of sunflower oil added with sea
buckthornextract.

2,5

Sunflower oil + SBT extract


2

Sunflower oil control


1,5

Olive oil + SBT extract

1
Control olive oil

Maize germs + SBT extract

0,5
Maize germs control

Initial
Photooxidation

Fig.2.Conjugateddienesassay.Relationshipbetweenabsorptivityat232nmandoils
exposureat254nmUVradiationfor30minutes

Malondialdehyde. This dialdehyde is preferentially formed by autoxidation of fatty


acidswithtwoormoredoublebonds.Inthisstudy,malondialdehydeformingwasinhibitedby
seabuckthornalcoolicextractsinalloilsexposedatUVradiation(Fig.3).

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Absorptivityat532
nm

Lucrritiinificevol53seriaMedicinVeterinar

0,5
Sunflower oil + SBT extract

0,4

Sunflower oil control

0,3

Olive oil + SBT extract

0,2

Control olive oil

0,1

Maize germs + SBT extract

mgKOH/goil

Maize germs control


Initial
Photooxidation

Fig.3.Malondialdehydeassay.Relationshipbetweenabsorptivityat532nmandoilsexposure
at254nmUVradiationfor30minutes

Acidvalue.Photooxidationofoilsgeneratesamultitudeofcompounds,suchasacids
withshortchainmolecule,formedbytheoxidationofaldehydes.Thechangesinacidvalues
forallsamplesareshowninFig.4.

Sunfloweroil+SBTextract
1,5
Sunfloweroilcontrol

Oliveoil+SBTextract
1

Controloliveoil

Maizegerms+SBTextract
0,5

Maizegermscontrol

Initial
Photooxidation

Fig.4.Relationshipbetweenacidvalueandoilsexposureat254nmUVradiationfor30
minutes

The results obtained in this study demonstrate that sea buckthorn alcoholic extract
containscompoundsthatactasantioxidantsinphotooxidationbyquenchingsingletoxygen
ortripletsensitizerorfreeradicalsgeneratedinphotooxidation.

CONCLUSIONS

1. Seabuckthornalcoholicextractprotectsvegetaloilsagainstphotooxidation.
2. Seabuckthornalcoholicextracthadthehighestdegreeofprotectionagainstphoto
oxidationincaseofsunfloweroil.
3. Thelesssusceptibletophotooxidationisoliveoil.

ACKNOWLEDGEMENTS

ThisworkwassupportedbyCNCSISUEFISCSU,PNIIIDEIprogram,projectnumber
ID_256/2007.

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REFERENCES

1.

2.

3.
4.
5.
6.
7.
8.

9.
10.

11.
12.
13.

AnwarF.,BhangerM.I.,KaziT.G.Relationshipbetweenrancimateandactiveoxygenmethod
valuesatvaryingtemperaturesforseveraloilsandfats.J.Americ.OilChem.Soc.,80,pp.151
155,2003.
Condrat D.C., Crisan F., Szabo M.R., Chambree D.R., Lupea A.X. Flavonoids in
Angiospermatophyta and Spermatophyta species and their antioxidant activity. Rev. Chim.,
60(11),pp.11291134,2009.
DumitrescuC.,OlteanuR.L.,BumbacM.,GorghiuL.M.Antioxidanteffectofsomeflavonoids
onorganicsubstrate.Rev.Chim.,60(4),pp.329331,2009.
HowardO.T.,Leonard,A.W.Fatandfatproducts.In:FoodCompositionandAnalysis.D.Van
NostrandCompany,Inc.NewJersey,USA,1982.
LawsonH.Commonchemicalreactionsinfoodoilsandfats.In:Jain,S.K.(Eds):FoodOilsand
Fats.CBSPublishersandDistributors,NewDelhi,India,1997.
Palaghiciuc G., Baciu I., Mihai F., Lazar P. Influence of irradiation treatment on antioxidant
compoundsfromEchinaceaspecies.Rev.Chim.,60(10),pp.10001005,2009.
Palaghiciuc G., Baciu I., Oprea E., Bradu C. Comparative study on natural compounds with
antioxidantspropertiesfromtwoEchinaceaspecies.Rev.Chim.,60(5),pp.435438,2009.
PapucCamelia,DiaconescuCristiana,NicorescuV.,CrivineanuCarmenAntioxidantactivityof
polyphenols from Sea buckthorn fruits (Hippophae rhamnoides). Revista de Chimie Bucureti,
59(4),pp.392394,2008
Stoilova I., Jirovetz L., Stoyanova A. Antioxidant activity of the polyphenol mangiferin.
EJEAFChe,7(13),pp.27062716,2008.
Suleiman A.E.M., ElMakhzangy A., Ramadan M.F. Antiradical performance and
physicochemical characteristics of vegetable oils upon frying of French fries: a preliminary
comparative.EJEAFChe,5(4),pp.14291441,2006.
WarnerK.Evaluationoflipidqualityandstability.In:FoodlipidsandHealth,EdbyMcDonald
REandMinDB.MarcelDekker,NewYork,NY,1996.
***A.O.C.S.TheOfficialandTentativeMethodsofTheAmericanOilChemistsSociety,3rdEd.
AmericanOilChemistsSociety.508SouthSixthStreet,Champaign,Illinois,1985.
***IUPACStandardMethodsfortheAnalysisofoils,FatandDerivatives.Sixhed.,Pergamon
press,Oxford,1979.

1134

OBSERVATIONSONAVIANINFLUENZA

T.C.PATACHE1,M.ARSENE,2ARSENEMARINELA2

1.DSVSAGalai;2.DSVSATulcea.

Summary
Observationsonaflockof268poultryfromtwoprivatefarmsonavianflu,havefounda
morbidityrateof%2,46.
Clinically,thebirdspresentedadynamy,deepdepressionassociatedwithhoriplumation,
lossofappetite,excessivethirst,cyanosisandchinridge,dyspneaandnoisybreathing,
swellingofheadandneck,rapiddeath.
Necropsy performed showed cyanosis ridge, chins, ears, look internalbleeding and
bleedingbodiespointoftheserous,oftheproventricolandintestine.

Keywords:inquiry,flu,birds,lesions.

INTRODUCTION

FormerlyreferredtoasfevertypicalEuropeanhorse,birdplague,typhusexudation
ofchicken,bird,fishoravianinfluenzaisavirusdiseaseofbirdsknownforitshighmortality
rate.Avianinfluenzaviralnatureofthediseasewashighlightedin1955(Savuandcol,2006),
when he was involved in producing disease virus isolated. Avian influenza affects a wide
variety of domestic and wild birds, the infection evolved multiple forms, of varying severity,
from expressions subclinical or asymptomatic to serious developments, fatal, depending on
virulent strain of avian host sensitivity involved and affected. Followed this observation of
clinicalsignsandgrosslesionsinthebirdflu,accordingtotheextensivegrowth,free.

MATERIALANDMETHODS

Epidemiologicalobservations,clinicalandanatomopatologicalweremadein2009,a
totalof268 poultry,representedby130chickens,20turkeys,14 guinea fowls,40ducks, 32
geeseand46pigeons.Thebirdswererearedinextensive,free,andbelongedtotwoprivate
holdings,locatednearapond,wheretherewerediseasesandmortality.

RESULTSANDDISCUSSION

Epidemiologicalsurveysresultthatprivateholdingswhichhavebeenrecordedcases
ofillnessandsuddendeathinchickens,arelocatedclosetoponds.Thebirdsweregrownin
the extensive free access, especially ducks and geese, in the water sometimes coliving the
wild,manyofthemmigratory.Considerasourceofinfection,withahighprobabilitythatwas
represented by wild palmipedele (ducks and geese) were infected in contact directly or
indirectlywiththeflocksofthesebirds.
Ofthe268poultryweresickenedsevenchickens,representing%2,46(Table1).

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Table1

Household

1.

Theresultsoftheclinicalexamination

TotalBirds
Fromwhich
No.birds
Thatgotsick
Nr.
%.

Hens
75
4
5,33

Turkeys
11

Guineafowls
5

154
Ducks
21

Geese
17

Pigeons
25

Hens
45
3
6,33

Turkeys
9

115
Guineafowls
6

Ducks
19

Geese
15

Pigeons
21

268
268
7
2,46

Total

Mentioned that the illness and mortality were recorded only in chickens, although
thesesidebysidewithotherspecies(turkeys,guineafowl)susceptibletoinfection.
Thefirstsignsofbirdfluinflocksexaminedevolutionwastheemergenceofsudden
death in two chickens. Clinical adynamyc birds and deep depression associated with
horiplumation, loss of appetite and excessive thirst, cyanosis ridge, chins and skin, dyspnea
andnoisybreathing,swellingofheadandneck,lacrimalhypersecretionandrapiddeath.
Necropsyperformedonthesevenhensdyingridgeshowedcyanosis,chinsandears,
congestive,hemorrhagicappearanceoforgans,theproventricolhemorrhagy,congestionand
thepresenceofpatchesandintestinalmucosa.

CONCLUSIONS
Fromobservationsofbirdflufollowingconclusions:
1.
2.
3.
4.
5.

Contactbirds(ducksandgeese)todomesticmigrationfavoredthepenetration
ofthevirusinlivestockholdingsreceptivepopulation.
Ofthe268poultryexamined,sevenweresickchickens,whichis2.46%.
Registration disease only in chickens, confirming their increased sensitivity
comparedtootherpoultry.
Clinicallytheypresentedadynamy,prostate,horiplumation,lossofappetite
andswellingofconnectivetissueofheadneckregion.
The necropsy has revealed the appearance of hemorrhagic dead bird, the
proventricularandenteritalhemorrhagy.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
BIBLIOGRAPHY
1.

2.

3.

4.
5.
6.
7.

ArseneM.,ArseneMarinela,SavuaGhe.,PerianuT.(2006)Clinicalaspectsin
the avian influenza in hens, Scientific researches, USAMV Iai, Vol. 49(8),
pag.774779.
ArseneM.,ArseneMarinela,SavuaGhe.,PerianuT.(2006)Clinicalaspectsin
the avian influenza in the summer swan (Cygnus olor) Scientific researches,
USAMVIai,Vol.49(8),pag.780784.
MafteiD.,SimoncaO.,AvramM.,BriaP.,TertisM.,ArseneMarinela(2008)The
roleofthemigratorybirdsintheevolutionoftheavianinfluenzaintheTulcea
county.Workshop78February,Tulcea.
Olaru E. (2005) Instructions for recognizing the avian influenza and the
differentiationfromtheotheraffectionsofthebirds.IDSABucureti.
Perianu T. (2005) Infectious diseases of the animals. Viroses vol. II, Editura
UniversitasXXI,Iai.
Savu C., Nicolaescu Mara (2006) The influenza the same danger A new
threatEd.AcademieiRomne.
Vasiu C. (2003) Viroses and prionic diseases at animals. Editura Nereamia
NapocaeClujNapoca.

1137


MORPHOLOGICALQUALITYINFLUENCEOF
SHEEPOOCYTESONINVITROMATURATION

AnamariaPetrean,I.Groza,L.Bogdan,SimonaCiupe,
SoranaMatei,AL.Pop

UniversitateadetiineAgricoleiMedicinVeterinarClujNapoca
FacultateadeMedicinVeterinar
DisciplinadeReproducie,ObstetriciGinecologieVetrinar
CaleaMntur,nr.35,ClujNapoca,Romnia,anamariapetrean@yahoo.com

ABSTRACT
Thestudywascarriedouttoinvestigatetherelationshipbetweenthedevelopmentpotentialand
the diameter of ovine oocytes. Ovaries collected from a local abattoir were transported to the
laboratory within 13h of slaughter. The oocytes from follicles (26 mm in diameter) were
recovered by aspiration and stored in a preincubated (at 38.6 C, 5% CO2 and maximum
humidity)hepesmodifiedTCM199solution.Goodqualityoocytes(evenlygranulatedcytoplasm
withatleasttwolayersofsurroundingcumuluscells)wereselectedandsubjectedtoculturein
TCM199supplementedwith0.05IU/mlFSH,1IU/mlhCGand1g/mlE2(OCM;oocyteculture
medium). Before culturing, the selected oocytes (n = 270) were divided into three categories
basedondiameter:(a)d<70m;(b)d=7090m;and(c)d>90m.Ineachcategoryhalfofthe
oocytesweredenudedofthecumuluscells(denudedoocytesDO)andtherestremainedintact
(cumulusenclosedoocytesCEO).AlloftheoocyteswereculturedinOCMforaperiodof2627
h. After the incubation period a morphological examination was performed under
stereomicroscope along with the evaluation of the diameter under inverted microscope.The
percentage of matured oocytes was 66,66%, 77,77%, and 84,44% for the cumulus enclosed
oocyteswithdiametersof<100,100110,>110m,respectively.Thecorrespondingvaluesfor
the denuded oocytes were 62,22%, 75,55% and 77,77%. The rate of maturation in cumulus
enclosedoocyteswashigherthanthatofdenudedoocytesinallthesizecategories.Theresults
suggest that in sheep the antral follicles ranging from 2 to 6 mm contain fully grown oocytes
which, despite their varaiabilty in diameter, show good competence for in vitro nuclear
maturation.
Keywords:sheep,oocyte,diameter,invitromaturation;

INTRODUCTION
Therearemanycriteriafortheselectionofgoodqualityoocytesinordertoimprove
theinvitromaturationofoocytesindifferentfarmanimalspecies.Somecriteriaare,thesize
ofthefollicle[Pavloketal.,1992;BlondinandSirard,1995],levelofatresia[deWitandKruip,
2001] and progesterone concentration in the follicular fluid [Hazeleger et al., 1995, Groza,
1996]. It has been shown that the oocyte developmental competence is also related to the
morphologyofthecumulusoocytecomplexes(COCs)[BlondinandSirard,1995],morphology
ofthecoronaradiatacells[Laurinciketal.,1996]andsizeoftheoocytes[Raghuetal.,2002].
Oocyte size can generally be used as an indicator of oocyte growth, as there is an intensive
synthesisofRNAduringthisphasethatcausesanincreaseinsize.

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Lucrritiinificevol53seriaMedicinVeterinar
MATERIALSANDMETHODS
Sheepovaries(n=70)werecollectedduringthenonbreedingseason(ApriltoJune)
from a local slaughterhouse and transported to the laboratory in saline at 3035 C in a
thermos flask within 13 h after collection. The ovaries were washed three times with
prewarmed fresh saline (37 C), and all visible antral follicles with a 26 mm diameter
aspirated, using a 20G needle connected to a 2 ml syringe (figure 1). Prior to aspiration,
syringes were filled with 0.5 ml preincubated Hepesmodified TCM supplemented with 50
IU/mlheparin.

Figure1Oocytesrecoveryusingaspirationtechnique

The contents of syringe were transfered into a collection tube and after
sedimentation, the follicular aspirant was poured into a Petri dish and observed under
stereomicroscope and then evaluated under an inverted microscope equipped with a video
camera (Nikon). The number and the quality of oocytes was determined according to their
morphologicalcharacteristics:
theaspectandsizeofzonapellucida(integrity,thickness);
theaspectofcumuluscells(numberofcellslayers,compactness);
theaspectofcytoplasm(homogenous,heterogeneous,granular);
theaspectofperivitelinspace(uniforme,irregular).
Only oocytes surrounded by more than three layers of unexpanded cumulus cells,
withintegrityofpellucidazone,homogenouscytoplasmanduniformeperivitelinspacewere
recoveredandselectedforinvitromaturation(IVM)(figure2).

Figure2Goodqualityoocytesselectedforinvitromaturation

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Prior to maturation, the size of each oocyte was measured using the Motic Images
Plussoftwarewhichcomeswiththeinvertedmicroscope.Theselectedoocytes(n=270)were
allottedintothreecategoriesbasedondiameter:(a)d<70m,(b)d=7090m,and(c)d>90
m.Beforeculturing,cumuluscellswereremovedbypipettinghalfoftheoocytes,ineachsize
category.Thisclassificationwasperformedtoevaluatethepossibleeffectofoocytediameter
onIVMandtheinfluenceoftheoocyteandcumuluscellsinteraction.
In each category half of the oocytes were kept intact (CEO: cumulus enclosed
oocytes)andtherestweredenudedofthecumuluscells(DO:denudedoocytes).TheCEOand
DOwasrandomlydistributedinmaturationdroplets,15oocytesper50l,coveredbysterile
paraffinoil(SigmaChemical)in35mmFalcondishes.Theoocyteswereincubatedunderan
atmosphereof5%CO2,95%airwith100%humidityat38,6Cfor2627h.Theoocyteculture
medium (OCM) consisted of bicarbonatebuffered TCM 199 with lglutamine supplemented
with0,05IU/mlFSH,1IU/mlhCG,1g/mlE2,100l/mlpenicillin,100g/mlstreptomycin,
20% FCS (Sigma Chemical), and 0,2 mM NaPyrovate. To establish the degree of IVM a
morphologicalexaminationwasperformedunderstereomicroscopealongwiththeevaluation
ofthediameterunderinvertedmicroscope.
RESULTSANDDISCUSSIONS
Morphological evaluation of oocytes is often the most intimidating part of IVF
techniquebeinganinitialstep,somethingwhichstartsoff.Thisstageisnecessaryduetothe
fact that using bad quality oocytes with no chance of development for fertilization is
unjustified.
After the application of aspiration technique a total number of 394 oocytes were
obtainedfrom213follicles,withanaverageof5,63oocytes/ovary.
From 394 sheep oocytes recovered, 270 oocytes (68,53%) were included in the
cultivablequalitycategoryastheypresentedmorethan3cumuluscellslayers,homogenous
cytoplasm, intact pellucid membrane and uniforme perivitelin space. The other 124 oocytes
(31,47%) were considered noncultivable as they presented heterogeheous/granular
cytoplasm, ruptures of zona pellucid, irregular perivitelin space. Only good quality oocytes
(n=270) were used for in vitro maturation protocol, divided into 3 categories (n=90)
dependingontheirdiameter:(a)d<70m,(b)d=7090m,and(c)d>90m(figure3).

Figure 3Classificationofovineoocytesbasedondiameter:
a)d<70m,b)d=7090m,c)d>90m

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Lucrritiinificevol53seriaMedicinVeterinar
Therewasheterogeneityintheoocytediameterobtainedfromvisiblefollicleswitha
diameterbetween2and6mm.Theoocytediameterswereintherangeof65,398mforthe
smallestandlargestoocytes,respectively.
As set out in table 1, relationship was detected between oocyte size and in vitro
maturation in the cumulus enclosed and denuded oocytes. In both CEO and DO the
percentage of maturated oocytes was higher in the largest oocytes (>90 m): 84,44% and
77,77%.
Attheendofthematurationperiodtheoocytesdiameterincreasedinbothgroups:
66,66% vs. 62,22% oocytes presentedd<100; 77,77% vs. 75,55% oocytes presentedd=100
110;84,44%vs.77,77%oocytespresentedd>110.Asexpected,thepercentageofmaturated
oocytesofCEOinallsizecategorieswashigherthanDO(graphic1,graphic2).

Exp.
Group

CEO

DO

Table1Ovineoocytedistributioninsizecategoriesafterinvitromaturation
Oocytesdiameter
(m)
Cultured
Maturated
Degenerated
oocytes(n)
oocytes
oocytes
before
after
IVM
IVM
<70
<100
45
66,66%(30oocytes) 33,33%(15oocytes)
7090
100110
45
77,77%(35oocytes) 22,22%(10oocytes)
>90
>110
45
84,44%(38oocytes)
15,55%(7oocytes)
<70
<100
45
62,22%(28oocytes) 37,77%(17oocytes)
7090
100110
45
75,55%(34oocytes) 24,44%(11oocytes)
>90
>110
45
77,77%(35oocytes) 22,22%(10oocytes)

Graphic1Percentageofmaturatedoocytesdependingontheirdiameter

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Graphic2Percentageofdegeneratedoocytesdependingontheirdiameter

The quality of oocytes from follicles of different sizes may be the main source of
variation in the IVM process. The heterogeneity in oocyte diameters, regardless of follicular
size, has been reported in many species (Motlik and Fulka, 1986, Fair et al., 1995). In the
presentstudy,however,theobservedheterogeneityindiameteroftheoocytesisolatedfrom
antralfollicles,rangingfrom2to6mm,wassimilarthanthosereportedinthesameorother
species(MotlikFulka,1986,Laurinciketal.,1996,Raghuetal.,2002).
In several species, the oocyte ability to resume and complete meiosis in vitro has
beenattributedtotheoocytediameter(MotlikandFulka,1986,Fairetal.,1995).Thesmaller
oocytes tend to follow an abnormal path of meiotic maturation, resulting in disturbances in
the maturation process (Pavlok et al., 1992). In cattle, some data have indicated a clear
relationship between oocyte diameter and developmental competence. In sheep, there is a
relationship between follicular size and oocyte diameter, as oocyte diameter influences the
meioticprogression.
In the current study, despite the variation in oocyte diameter, the data showed
relationshipbetweenthesizeofoocytesanditsabilitytoreachmaturation.Itseemsthatthe
microenvironmentalconditionofsheepoocytesinperipheralfollicles(26mmindiameter)is
themainindicatorregardingtheirprogressionthroughmeiosis,regardlessoftheoocytesize.
Regarding the effect of oocyte diameter and its relationship with the presence of
cumulus cells, the data showed that in the absence of cumulus cells (DO) there was no
significantdifferenceinmaturationbetweendenudedoocytesinthedifferentsizecategories.
Theimportanceofcumuluscellsoninvitromaturationoftheoocyteemergedinthisstudy,
withresultsthatareinagreementwiththosereportedbyotherresearchers.

CONCLUSIONS
1.
2.

After the application of aspiration technique a total number of 394 oocytes were
obtainedfrom213follicles,withanaverageof5,63oocytes/ovary.
From the total of 394 sheep oocytes recovered by aspiration method 270 (68,53%)
wereconsideredcultivablewhile124(31,47%)wereconsiderednoncultivable.

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Lucrritiinificevol53seriaMedicinVeterinar
3.

4.
5.

Insheep,folliclesrangingfrom2to6mm,presentheterogeneityintheoocytesize
(diameters were in the range of 65,398 m for the smallest and largest oocytes,
respectively).
InbothCEOandDOthepercentageofmaturatedoocyteswashigherinthelargest
oocytes(>90m):84,44%and77,77%.
Attheendofthematurationperiodtheoocytesdiameterincreasedinbothgroups:
66,66%vs.62,22%oocytespresentedd<100;77,77%vs.75,55%oocytespresented
d=100110;84,44%vs.77,77%oocytespresentedd>110.

BIBLIOGRAPHY

1.

2.

3.
4.
5.

6.

7.
8.

9.

Blondin, P. and M.A. Sirard, 1995, Oocyte and follicular morphology as determining
characteristicsfordevelopmentalcompetenceinbovineoocytes,Mol.Reprod.Dev.41,pp.54
62.
de Wit, A.A. and T.A. Kruip, 2001, Bovine cumulusoocytecomplexquality is reflected in
sensitivityforalphaamanitin,oocytediameteranddevelopmentalcapacity,Anim.Reprod.Sci.
65,pp.5165.
Fair, T., P. Hyttel and T. Greve, 1995, Bovine oocyte diameter in relation to maturational
competenceandtranscriptionalactivity,Mol.Reprod.Dev.42,pp.437442.
Groza I. Actualiti i perspective n biotehnologia transferului de embrioni la specia ovin,
Ed.Ceres,1996;
Hazeleger,N.L.,D.J.Hill,R.B.StubbingandJ.S.Walton,1995,Relationshipofmorphologyand
follicular fluid environment of bovine oocyte to their developmental potential in vitro,
Theriogenology43,pp.509522.
Laurincik, J P. Hyttel, V. Baran, F. Schmoll, H. Niemann and G. Brem, 1996, Corona radiata
density as a noninvasive marker of bovine cumuluscoronacomplexes selected for in vitro
embryoproduction,Theriogenology46,pp.369377
Motlik, J. and J. Fulka, 1986, Factors affecting meiotic competence in pig oocytes,
Theriogenology25,pp.8796.
Pavlok,A.,A.LucasHahnandH.Niemann,1992,Fertilizationanddevelopmentalcompetence
ofbovineoocytesderivedfromdifferentcategoriesofantralfollicles,Mol.Reprod.Dev.31,pp.
6367.
Raghu, H.M., S. Nandi and S.M. Reddy, 2002, Follicle size and oocyte diameter in relation to
developmentalcompetenceofbuffalooocytesinvitro,Reprod.Fertil.Dev.14,pp.5561.

1143

CREATINGBESTPRACTICEGUIDESBASICREQUIREMENTFOR
ACHIEVINGSAFEANDHEALTHYFOOD

R.POPA1,MAGDAGONCIAROV2,L.TUDOR2,DOINALUPU1
1
SanitaryVeterinaryDirectionandfortheFoodSafety,
2
U.S.A.M.V.,FacultyofVeterinaryMedicineBucharest,105SplaiulIndependentei,District5,
Bucharest,050097,magdagonciarov@yahoo.com

SUMMARY
GMP/GHP sites covers the basic requirements of hygiene and processing to ensure safe and
healthyfoodproduction.
These requirements relate to infrastructure and equipment, raw materials, production control,
waste management, pest control, sanitation procedures, quality health water, maintaining the
cold chain,healthpersonnel,sanitation personneltraining. When properlyimplemented, these
rules contribute to the control .Officers possibilities of contamination, failure to comply with
hygieneprinciplesgoverningtheseareas.
Keywords:guide,EuropeanCommission,CodexAlimentarius.

Member States of the European Union encourage the establishment of national


guides to good hygiene practice and application of HACCP principles. It is encouraged the
disseminationanduseofnationalguidelinesandoftheCommunity.However,foodbusiness
operatorsmayusetheseguidesonavoluntarybasis.

National guidelines are being drawn as guides to good practice, they are made and
distributedbythefoodbusiness:
inconsultationwithrepresentativesofpartieswhoseinterestsmaybeseriouslyaffected,
suchascompetentauthoritiesandconsumerassociations;
compliancewithcodesofpracticeapplicabletotheCodexAlimentarius.

Member States shall transmit national guides and this establishes and operates a
systemofregistrationoftheseguidesandmakeitavailabletoMemberStates.

Community Guidelines, prior to completion of Community guides to good hygiene


practice and application of HACCP principles, the Commission shall consult the Standing
Committeeguides,scopeandtheme.

InestablishingtheCommunityguidelines,theCommissionshallensurethattheyare
preparedanddistributed:
by or in consultation with representatives of the resort community of food sectors,
includingSMEsandotherstakeholderssuchasconsumerassociations;
in collaboration with parties whose interests may be seriously affected, including
competentauthorities;
takinginaccountofcodesofpracticeapplicabletotheCodexAlimentarius.
CommissioninvitestheCommitteeontheFoodChainandAnimalHealthtoregularly
review all guidelines made. This analysis has the purpose to preserve practical guidelines
andtotakeaccountofscientificandtechnicalprogress.
National and Community guides shall provide guidance on good practice to combat
healthrisksinprimacyproductionandrelatedactivities.(1)

Guides to good health practice must include adequate information on the risks
involved in primacy production and related activities and measures to combat such risks,

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Lucrritiinificevol53seriaMedicinVeterinar
including the relevant set of national and Community law or national and community
programs.Theserisksandmeasuresmayinclude,forexample:
controlofmycotoxincontamination,heavymetalsandradioactivesubstances;
useofwater,organicwasteandfertilizers;
correctandappropriateuseofveterinarydrugsandfeedadditivesandtheirtraceability;
preparation,storage,useandtraceabilityandlitter;
properdisposalofdeadanimals,wasteandlitter;
protective measures designed to prevent the introduction of contagious diseases
transmissibletohumansthroughfood,andmustnotifythecompetentauthority;
procedures,practicesandmethodstoensurethatfoodsareproduced,handled,packaged,
storedandtransportedinadequatehealth,includingeffectivesanitationandpest;
hygienemeasuresforslaughterandbreedinganimals;
measuresonrecordkeeping.(3)

The official checks shall bemade on the basis of risks based on riskclassification in
theproductionoffood.

Inspectionbodieshavearesponsibilitytoclassifyunitsoffood,usingrawmaterialsof
animal origin and/or nonanimal based production activities and the actual risk associated
withexistingwork.Classificationunitsarethebasisforprogrammingthecontrolactivity.

Guides of good sanitary practice is a simple mean, but effective, to overcome


difficulties that may arise in certain food establishments to implement HACCP procedure
detailed.Representativesofvarioussectorsoffoodand,onparticular,ofthosesectorswhere
many food establishments have difficulty in developing HACCP procedures, should consider
these guidelines, and authorities should encourage sector representatives to develop such
guidelines.Neededtoassistthedevelopmentoftheseguidestogoodpracticeforthosefood
sectorsthatareinsufficientorpoorlyorganized.

Usingoftheguidestogoodpracticemayhelpfoodestablishmentstocontrolhazards
anddemonstratecompliance.Theycanbeappliedbyanyfoodsectorand,especially,where
food is handled according to procedures well known that are often part of the training of
operatorsintheusualsectors,suchas:
restaurants,includingfoodfacilitiesoftransport,suchasonships;
cateringsectorsdeliveringpreparedacentralunit;
thebakeryandconfectionery;
retailstores,includingbutchers.(5)

Forsuchunitsmaybesufficientfortheguidestogoodpracticetodescribeasimple
andpracticalmethodstocontrolhazards,notmandatorytoenterthedetailsofthenatureof
the hazards and identify critical control points. These guidelines should, however, cover all
significanthazardsinaunitandmustclearlydefinetheprocedurestocontrolthesehazards
andcorrectiveactionstobetakenincaseofoccurrenceofproblems.

Such guidelines may also emphasize the potential hazards associated with certain
foods (egg raw eggs and the occurrence of salmonella in them) and food contamination
control methods (egg purchase these raw eggs from a reliable source, and the combination
time/temperatureprocessing).

Best practice guidelines have been developed and evaluated by competent


authoritiesfor manyfoodsectors,Theyareusuallyacombinationofgoodhygienepractices
(GHP)andHACCPbasedelementsandinclude,forexample:
practiceguidelinesfortheimplementationofmandatoryrequirements;
requirementsforarawmaterials;
ahazardanalysis;

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UniversitateadetiineAgricoleiMedicinVeterinarIai
preestablishmentofcriticalcontrolpointsinthepreparation,manufactureorprocessing
offoodidentifyinghazardsandspecificcontrolrequirements;
preventivehygienemeasurestobetakenwhenhandlingsensitiveandperishableproducts
(suchas,forexample,productsreadyforconsumption);
developmentofseveralmeasures,ifpreparedfoodforgroupsofconsumerswithincreased
susceptibilitytoillness(children,elderly,etc..)
theneedfordocumentationandrecords;
protocolsfordatavalidationoflife.(3)

AspecialtypeofguidetogoodpracticeisagenericHACCPguide.

General guide may suggest common hazards and controls of certain food activities
andhelpthemanagerortheHACCPteaminmakingfoodsafetyproceduresormethodsand
appropriaterecordkeeping.

Food operators must be aware that there may be other hazards, egg. those
associated with the unit or process location that apply and that such hazards can not be
expected in a generic HACCP guide. When used generic HACCP guides exist, however, the
necessityoffurtherexaminationforthepossiblepresenceofthesehazardsandtheircontrol
methods.
Inthoseareaswherethere isasimilarity betweentheactivities,where the
manufacturingprocessislinearandwhereprevalencemaybehighhazard,genericguidelines
maybeappropriate,egg.:
forslaughterhouses,establishmentshandlingfisheryproducts,dairyunits,etc..
tounitsusingstandardfoodprocessingprocedures,suchasfoodpreservation,liquidfood
pasteurization,freezing/quickfreezingfood,etc.(4)

CONCLUSIONS
1.GMP/GHPcoverthebasicrequirementsofhygieneandprocessingtoensuresafe
andhealthyfoodproduction.
2. European Union states encourage preparing national guides to good hygiene
practice and application of HACCP principles. It is encouraged the dissemination and use of
nationalguidelinesandoftheCommunity.However,foodbusinessoperatorsmayusethese
guidesonavoluntarybasis.
3.Nationalandcommunityguidesmustprovideguidanceongoodpracticetocombat
healthrisksinprimaryproductionandrelatedactivities.
4.Generalguidemaysuggestcommonhazardsandcontrolsofcertainfoodactivities
andhelpthemanagerortheHACCPteaminmakingfoodsafetyproceduresormethodsand
appropriaterecordkeeping.

REFERENCES
1. Gonciarov Magda, veterinary and health legislation for food safety, Printech Publishing,
Bucharest,2008.
2.Savu,C.,andcol.,Healthcheck,food,CeresPublishingHouse,Bucharest,2005.
3.Tudor,L.,veterinarycontrolandanimalproducttechnology,PrintechPublishing,Bucharest,2005.
4.Regulation(EC)no.1244/2007of24October2007amendingRegulation(EC)no.2074/2005,as
regardstheimplementingrulesforproductsofanimaloriginforhumanconsumptionandlaying
downspecificrulesfortheorganizationofofficialcontrolsonmeatinspection.
th
5.TheCommissionDecisionofthe29 ofApril2004layingdownanimalhealthandpublichealth
and veterinary certificates required for entry into the Community of heattreated milk, milk
productsandrawmilkforhumanconsumption.

1146

STUDYONTHERISKANALYSIS(IDENTIFICATIONOFHAZARDS,
THEIREVALUATION,IDENTIFYINGPREVENTIVEMEASURESTO
CONTROLRISK)

RAREPOPA ,MAGDAGONCIAROV2,LAURENIUTUDOR2,DOINALUPU1
1
SanitaryVeterinaryDirectionandfortheFoodSafety,
2
U.S.A.M.V.,FacultyofVeterinaryMedicineBucharest,105SplaiulIndependentei,District5,
Bucharest,050097,magdagonciarov@yahoo.com

Summary
Therisk,accordingtoNACMF(NationalAdvisoryCommitteeonMicrobiologicalCriteria
of Foods) is any element of physical biological or chemical may pose a threat to
consumerhealth.
BasictermsusedwhendiscussingHACCPandriskassessmentare:
Hazard and risk. The terms are not used with the same meaning in the literature.
Sometimes the term hazard is defined or used to refer to the cause of an adverse,
sometimesdefinedhavingthemeaningofnegativeresult,whichisaconcern.
Keywords:danger,risk,contamination
1

Risk is something defined as the probability or possibility of occurrence of an


undesirable effect,sometimesdefinedasincluding bothelementsofprobabilityandimpact.
Sometimesitcanbedefinedtoincludeelementsofprobabilityandimpact,butisusedtorefer
onlyprobability.
It was suggested that it would be appropriate that the term hazard is used only to
designate an agent or an action that might cause adverse effects (egg. pathogen, toxins or
chemicalshavingthefoodasasource).

PerformingriskanalysisisakeystageoftheHACCPsystem,aninadequateanalysisof
hazardsthatcanleadtowrongdesignofaHACCPplan.Thisstageinvolvestechnicalexpertise
and scientific documentation in various fields to correctly identify all potential hazards. An
important role in identifying hazards has the HACCP team members with experience in
productmicrobiology,hygieneandproducttechnology.

Thecontaminantsmeanthosebodiesorelementscarryingtheroleofmicrobiological
agents or vectors, which may at the same time with achieving or penetration into food, to
contaminateitwithinfectiousagentswhichitbears.

Classificationfactorsforcontamination:
A.Bioticfactors(direct)
subjectsorcarriers(stool,excretions,secretions,hair,feathers,appendages,etc.).
Healthysubjectsrolevector(tail,horns,hair,feathers,appendages,etc...).
B.Abioticfactors(indirect)
technologicalfactors(equipment,vehicles,packages,tools,installations,etc.)
environmentalfactors(water,dust,pollen,plantdebris,etc...)

otherfactors(wood,glass,metal,plaster,etc.)(4).

Waysofcontaminatingfood:
1. Primary contamination type: animal product is contaminated before slaughter,
milking, etc... By direct transmission of the pathogen (milk obtained from an animal that
evolved a subclinical staphylococcal mastitis is a milk raw material infected with
staphylococcus);

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Lucrritiinificevol53seriaMedicinVeterinar
2.Secondarycontaminationtype:
a) through factors that depend on the sick animal (stool, hair, secretions, excretions,
appendages that come in contact with food, milk obtained from cattle that evolves
Colibacilosys and where hygiene conditions are not complied with the milking, may be
contaminatedwithcolibacillusthroughstool);
b) through factors from a healthy animal, but contaminated by a sick animal (hair,
appendages,feathers)(milkobtainedfromahealthyanimalmaybecomecontaminatedifthe
hair gets into the milk that was contaminated by sneezing or coughing by a neighboring
animal,sickoracarrierofbacillusoftuberculosis);
c) through factors that depend on the role of vector animals (rodents, birds or insects or
infected with the role of vectors that come into contact with food) (A pig carcass properly
storedfordryingmaybecontaminatedthroughoutthestoolbyarodentwhohasYersinioza).
(1)
Risksofcontamination:
primaryriskisindirectrelationshipwithprimarycontaminationfactors;
secondaryriskisindirectrelationshipwithfactorssecondarycontamination.
Theriskanalysisincludes:
a.identifyinghazardsrelatedtofoodatallstagesofmanufacture;
b.assessingseverity(consequencesofexposuretotheriskfactor)andthelikelihoodofsuch
hazards;
c.identifyingtheexistingpreventivemeasuresnecessarytocontrolthesehazards.
Risksassociatedwithfoodcanbeclassifiedintothreecategories:biological,chemical
andphysical.
Depending on the hazards involved, biological pathogens can be classified as:
bacterial,viral,parasitological,fungalandprion.
Bacteriologicalrisksareoftenassociatedwithrawmaterialsfromwhichitisproduced
meat, including animals that are major components. Bacteriological hazards can reach food
during meat processing, from those who are involved in processing; the processing
environmentismadebyotheringredientsintheproducts,orevenduetoprocessing.
Identificationofbacteriologicalhazardsthatmaybesubjecttoproductionisclearlya
difficult task, much of pathogens may be associated with meat: Salmonella, Campylobacter
jejune, Escherichia coli, Listeria monocytogenes, Clostridium botulinum, Staphylococcus
aureusandYersiniaenterocolitica.
IndevelopingtheHACCPplanisnecessarytocomplywiththerequirementofthree
biologicalhazards:
Destroying,removingorreducingrisks;
Preventrecontamination;
Haltingthedevelopmentofmicroorganismsandtoxinsproduction.

Theheattreatmentsappliedtoproducts(pasteurization,refrigeration,freezing,etc...)
Microorganismsmaybedestroyedorinhibited.
Temperature,pH,wateractivity(aw),time,substraterichinnutrients,preservatives,
thepresenceorabsenceofoxygenarefactorsthatinfluencethedevelopmentofpathogens.
Thesefactorsinfluencedifferentlydependingonthespeciesbacterialmicroorganisms.

Dangerous microorganisms and parasites were classified according to severity risk


that has three groups: severe risk, moderate risk of extensive spread, moderate risks with
limiteddistribution.
Severerisks:

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Clostridiumbotulinum,Salmonellatyphi,SalmonellaparatiphiA,B,Teniasolium,hepatitisA,
Trichinellaspiralis;
Moderaterisk:
Listeriamonocytogenes,Norwalkvirusfamily,rotaviruses,Ascarislumbricoides;
Moderatetohighriskspread:
Staphylococcusaureus,Clostridiumperfringens,Giardialambilia;
Alimentary infections are the result of eating foods that contain live pathogens,
harmfultohealth(Salmonella,Listeriamonocytogenes).
Foodorigintoxicinfectionsarediseasesthatcanbecausedbyfactorscombined,like
theconsumptionofbacterialloadedfoodand/ortheirmetabolicproducts.
Infectious diseases (tuberculosis, brucellosis, etc...) Can occur in ingesting food
containingasmallnumberofpathogens,theirmultiplicationisnotrequiredinthefood.(2)
Chemical dangers may be caused by a natural process that occurs in food or which
may occurduringprocessing.Substancesinthiscategoryare intentionallyorunintentionally
added during growth, harvest, storage, processing, and distribution and could include
elementsoffeed,water,medicines,pesticides,chemicals(lubricants,cleaningsolutions,paint,
etc...).
Chemicals used in food production can be toxic at a certain level, causing illness or
death from consumer storage (preservatives, dyes, pigments). Natural chemical hazards can
be:aflatoxins,mycotoxins,allergens,etc...Chemicalhazardsmayinvolveillnessaccumulation
inthebodyorevendeathbyingestingadangerousdose.ApplicationoftheHACCPsystemis
necessarytocontrolhazards.Allergensareproteinswhoseconsumptioncancauseanimmune
response of the body in different forms of the light phase, redness, itching to the worst,
namely anaphylaxis. Some foods can cause allergic reactions, most often involved are the
proteinsinsoy,wheat,peanuts,nuts,eggs,milk,shellfish,andsesameseeds.Sincetheallergic
reaction can be caused by very small amounts of allergen, public authorities require
manufacturerstoproperlylabeltheproductswithmandatoryindicationofapossibleallergen.

Polycyclicaromatichydrocarbons(PAHs)aresubstancesproducedduringthesmoking
process,asafoodpreservative.
Accumulationof PAHs in foods that reek of smoke depends on how the natures of the fuel,
temperature,smoke,theprocess,packagingandcoatingstructure.

Asmokingtemperatureof2025Cdoesnotsignificantlyinfluencetheproductionof
PAHs.Temperaturesof4001000CcancausealinearincreaseofPAH.

Nitratesandnitrites,sodiumorpotassium,additivesinspecificcolorprintingandthe
preservationoffoodproductsarecurrentlyusedtechnologyofmeat.Apossiblechemicalrisk
ismisuseofsodiumnitritewhichhasatoxicchemical,beingafactortobeconsideredinrisk
analysis.

Inhealthyadultbodiesnitritesandnitratesareabsorbedinthegastrointestinaltract.
Themostexposedcategorytonitritespoisoningisinfantsupto3months.

Incalculatingthecontentofnitratesandnitritesonthefinishedproduct,regulatedby
law, is considered conservation with smoke that may be another source of nitrates and
nitrites.

Maintaincontrolofchemicalriskscanbeachievedby:
Pre Reception (specifying the composition of raw materials, checking quality certificates
issuedbythesupplier,etc...)
Check before use (setting the purpose for which chemicals will be used and verify the
quantityused,ensuringproperlabelingofsubstancesused);

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Lucrritiinificevol53seriaMedicinVeterinar
Monitoringofthestorageandhandlingtoavoidconditionsfavorableforthedevelopment
oftoxicsubstances)and
Inventory of existing chemical substances, their strict records and registration of
substances,dosageandhowtousethem.

Natural Hazards refers to a natural component of food which may occur


unexpectedlyinjuryorillnessofthepersonwhoconsumedfood.Foreignmaterialslikeglass,
metal and plastic are the most familiar natural hazards which could occur if equipment or
productionprocesswasnotproperlycheckedduringprocessing.

There are manycauses that may contribute to the occurrence ofnatural hazards in
foodincludingcontaminatedmaterials,maintenanceorpoordesignofequipmentorresource
materialsusedinpackagingmaybecontaminatedornegligenceofemployees.

Naturalhazardscanbeeasilypreventedthroughgoodworkingpractices.Inthismay
takemeasurestopreventthemajorphysicalthreat.(3)

CONCLUSIONS

1.Therisk,asNACMFsays(NationalAdvisoryCommitteeonMicrobiologicalCriteriaofFoods)
isanyelementofphysicalbiologicalorchemicalmayposeathreattoconsumerhealth.
2. The risk is sometimes defined as the probability or possibility of occurrence of an
undesirable effect,sometimesdefinedasincluding bothelementsofprobabilityandimpact.
Sometimesitcanbedefinedtoincludeelementsofprobabilityandimpacthow,butisusedto
referonlyprobability.
3. Making the risk analysis is a key stage of the HACCP system, an inadequate analysis of
hazardscanleadtowrongdesignofaHACCPplan.
4. Through contaminants means those bodies or elements carry the role of microbiological
agentsorvectors,which mayoncetoachieveorpenetrationintofood,contaminatingthem
withinfectiousagentswhomitbears.

REFERENCES

1. Stancu, I. and col., Measures concerning wholesome food of animal origin, Coral Sanivet
Publishing,Bucharest,2004.
2.Tudor,L.,veterinarycontrolandanimalproducttechnology,PrintechPublishing,Bucharest,2005.
3.Regulation(EC)no.853/2004oftheEuropeanParliamentandEUCouncilof29April2004laying
downspecifichygienerulesforfoodofanimalorigin;
4.Regulation(EC)no.854/2004oftheEuropeanParliamentandEUCouncilof29April2004laying
down specific rules for the organization of official controls on products of animal origin for
humanconsumption;

1150


THEASSESSMENTOFHYGIENEINDAIRYCOWSHEDSWITHTIE
STALLSINTRANSYLVANIA

SILVANAPOPESCU,CRISTINBORDA,MARINASPINU,
R.STEFAN,CARMEND.SANDRU,EVAA.LAZAR
FacultyofVeterinaryMedicine,UniversityofAgriculturalSciencesandVeterinaryMedicine,
400372ClujNapoca,35ManasturstreetRomania;popescusilvana@yahoo.com

The aim of this study was to assess the hygiene level in dairy cowsheds with tiestall
housing, in Transylvania. We evaluated 20 tiestall barns (1376 dairy cows) for body
hygiene of cows through the method proposed by Cook. The obtained results were
statisticallyprocessed.Thedistributionofscoresof3and4differedinthethreebody
regions of the cows and among cowsheds. The mean value for the udder area was
28.15%, for upper leg and flank 44.11% and for lower leg 34.24%. The multiple
comparison TukeyKramer test showed significant differences between the mean
proportionsofscoresof3and4intheudderareaandintheupperlegsandflank(p<
0.001) and in the lower legs and the upper legs and flank (p < 0.01), respectively.
Positivecorrelationsbetweenhygienelevelsofthethreebodyregions;namelyudder
and lower legs (rs = 0.88, p < 0,0001), udder and upper legs and flank (rs = 0.85, p <
0.0001) and also lower legs and upper legs and flank (rs = 0.91, p < 0.0001) were
demonstrated.Significantnegativecorrelationsbetweenthelengthofthebedsandthe
cleanlinessoflegs(rs=0.47,p<0.01;rs=0.56,p<0.05)andudder(rs=0.62,p<0.01)
weredemonstratedtoo.Theobtainedresultsindicatepoorhygieneintheinvestigated
sheds,withnegativeinfluenceondairycattlehealthandwelfare.

Keywords:dairycow,hygienescore,cleanliness,bodyhygiene,dirtiness

Theenvironmentinwhichdairycowsarekepthavedecisiveeffectontheirhealthand
welfare.Acleanandcomfortablebarnrepresentsthekeytomaintaininghealthandlongevity
of dairy cows. The appraisal of the barns hygiene level can be done through several
assessment systems based on the quantification of the manure soiling in different body
regions of the cows (Chaplin et al., 2000; De Rosa et al., 2003; Faye and Barnouin, 1985;
Hughes, 2001). However, apart from their value in scientific research, the majority of these
systems failed as practical hygiene monitoring tools at farm level (Cook, 2002). A practical
system for quantifying the hygiene at farm level is the one proposed by Cook (2002). The
hygienescoringsystem(Cook,2002)isconsideredtobeaneffectivetoolinshowingtheneed
forremedialmeasuresfortheexistingdeficienciesinthehygienemanagement.Forascoring
system to be useful both for veterinarians and farmers, the significance of manure
contaminationindifferentbodyareasmustbeunderstoodandthedirtinesslevelcomparedto
anestablishedstandard,derivedeitherfromthecontaminationlevelofthesamefarmintime,
orfromthedataobtainedinsomesimilarfarms(Cook,2002).Forthehygienescoringtobe
taken seriously, the farmer must understand what the costs of keeping animals in a dirty
environment are. For dairy cows the outcome of low hygiene is a high risk of mastitis and
worseningoflameness.
Theconnectionsbetweencowshedhygiene,cleancowsandlownumberofsomatic
cells in mixed milk were highlighted in several studies (Barkema et al., 1999; Barkema and

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Lucrritiinificevol53seriaMedicinVeterinar
Schukken,2003;Bodohetal.,1976).Ahighhygienestandardindicatesalimitedexposureto
thepathogensofenvironmentalmastitisandconstitutesabasicaspectoffoodsafety,hygiene
protocolsandquality.
Several researches indicate also that cows housed in humid conditions or in ones
contaminatedbydejectionsaremuchmorepronetoinfectiousfootdiseases,suchasfootrot
(interdigital necrobacillosis), heel horn erosion and hairy heel warts (papillomatous digital
dermatitis)(Bergsten,1997;CookandCutler,1995).Thecleanlinessofdifferentbodyregions
(hind legs, udder) of the cows is associated with the surface of the beds and bedding type,
housingtechniquesandthelenghtofthebeds(BergstenandPettersson1992;Chaplinetal.
2000; Cook 2002). The available literature suggests that tiestall cowsheds are cleaner than
freestall ones, due to the fact that the former have no manurecovered alleys (Cook 2002,
CookandNordlund2009).
Theaimofthisworkwastoassessthegeneralhygieneofdairycowskeptintiestalls
based on cows body hygiene in cowsheds. The significance of the association between the
lengthofthebedandhindlegsanduddercleanlinesswasalsodetermined.

MATERIALSANDMETHODS

TheinvestigationsofthisstudyweremadebetweenNovemberDecember2009in
20dairycowsheds(with32113cows/shed)withtiestalls,inTransylvania.Eachcowshedwas
visitedonce.Allthecowshedswereclosed,withsolidflooring.Thecowswerekeptonshort
beds(1.61.8mlenght)in60%ofthecowshedsandonmediumsizedbeds(1.82.2mlenght)
in35%ofthecowsheds.Longbeds(2.43mlenght)werefoundinonlyonecowshed.Most
predominantbreedsweretheRomanianSpottedCattleandHolsteinFriesian,withanaverage
milkproductionof1023liters/cow/day.Alloftheassessedanimalswereadultcows,between
3 and 9 years of age, with body weights between 350 and 500 kg. In 90% of the cowsheds,
smallamountsofbedding(straw,sawdust,woodshavings)wereusedonthecowsbed.Only
one cowshed had mechanical ventilation, all the others were naturally ventilated. In 70% of
theinvestigatedcowshedsthemanureremovalwasdonemanually(onceortwiceaday).
Thehygieneofthefarmswasassessed basedonthecowsbodyhygiene,using the
system(hygienescoringsystem)proposedbyCook(2002).Threebodyregionswereassessed:
lower leg, udder and flank and upper leg, awarding points (from 1 to 4), depending on
dejectionsoilingoftheseareas.Foreachareaadifferentscorewasassigned.Attheend,the
proportion of scores of 3 and 4 (too dirty) was calculated for the three body regions of the
cowsfromeachbarnandtheirmeanpercentagewithinalloftheassessedcowsheds.Allthe
cows(1376)fromthe20cowshedsincludedinthestudywereevaluated.Ineachcowshedthe
length of beds was measured for all cows. The results obtained were statistically processed
with the software GraphPad InStat version 3 (GraphPad Software Inc. USA). TukeyKramer
Multiple Comparisons TestOneway ANOVA was used to compare the data and for
determination of the correlations, the Spearmans rank correlation coefficient (rs) was
calculated.

RESULTSANDDISCUSSIONS

The obtained results following the evaluations of the cows body hygiene in the 20
cowshedsareshowninTable1.Theproportionofscoresof3and4isdifferentinthethree
bodyareasofthecows.Thereweresignificantdifferencesamongthefarms,aswell.Thus,at
udder level, a mean value of 28.15% was recorded, ranging from 12.76% to 46.66%, at the

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UniversitateadetiineAgricoleiMedicinVeterinarIai
lowerlegleveltherewerevaluesbetween15.95%and46.66%withameanvalueof34.24%
and at upper leg and flank level the proportion varied between 25.53% and 56.81%, with a
meanvalueof44.11%.
TukeyKramerMultipleComparisonsTestshowedsignificantdifferencesbetweenthe
averageproportionofthescoresof3and4atudderlevelandupperlegandflank(p<0.001)
andbetweentheareasofthelowerlegandupperlegandflank(p<0.001)(Table2).Positive
correlations between the level of hygiene of the threebody regions, namely, the udder and
lowerleg(rs=0.88,p<0.0001),udderandupperlegandflank(rs=0.85,p<0.0001)and,also,
lowerlegareaandupperlegandflank(rs=0.91,p<0.0001)(Table3),weredemonstrated.
Thecorrelationsbetweenthelengthofstallbedsandthecleanlinessofthehindlegsandthe
udder are given in Table 4. The length of the stallbeds is negatively correlated with the
hygieneoflowerleg(rs=0.47,p<0.01),upperlegandflank(rs=0.56,p<0.05)andudder(rs
=0.62,p<0.01).

Table1.Meanproportionof3and4hygienescoresforudder,lowerlegandupperlegand
flankbodyareasin20dairycowsheds

ProportionofHygieneScores3and4
Cowsheds
Udder LowerLeg UpperLegandFlank
1
31.25
41.66
52.08
2
28.00
34.00
40.00
3
28.57
34.28
42.85
4
28.00
32.00
44.00
5
40.00
40.00
52.00
6
38.46
42.85
53.57
7
12.76
15.95
25.53
8
15.30
30.61
32.65
9
27.24
35.35
42.42
10
38.63
45.45
56.81
11
35.00
35.00
40.00
12
14.28
21.42
32.14
13
17.07
19.51
36.58
14
36.11
41.66
55.55
15
46.66
46.66
53.33
16
21.42
26.19
38.09
17
30.76
42.30
53.84
18
26.92
38.46
46.15
19
21.87
28.12
37.50
20
27.77
33.33
47.22
Mean
28.15
34.24
44.11

ThehygienescoringsystemwasdevisedbyCook(2002)inordertoquantifyhygiene
at farm level and to evaluate improvements needed in hygiene management. This system is
considered as a remedial tool for the existing deficiencies. The author believes that a
quantitative approach in the hygiene assessment of cows gives a more efficient meaning to
conveyingtothefarmerthemessagethatthecowsaretoodirty,comparedwithaqualitative
appraisal. Besides, the recording of the scores by body region allows more accurate

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Lucrritiinificevol53seriaMedicinVeterinar
recommendationsonhowtokeepthecowsclean.Becauseameanscoreofhygienelevelfor
eachbody area doesnot present any importance for the farmer, only theproportion of the
scoresindicatingthetoodirtyconditionisconsidered,namelythe3and4scores.

Table2.TukeyKramerMultipleComparisonsTest(OnewayANOVA)forsoilingdegreeofthe
bodyareasofUdder,LowerLeg,UpperLegandFlank
Comparison
Meandifference
q
Pvalue
UddervsLowerLeg
6.08
3.043
nsp>0.05
UddervsUpperLegandFlank
15.96
7.981
***p<0.001
LowerLegvsUpperLegandFlank
9.87
4.938
**p<0.01
Ifthevalueofqisgreaterthan3.405thenthepvalueislessthan0.05,p=statistical
significance,
ns=notsignificant,**=consideredverysignificant,***=consideredextremely
significant.

Table3.Correlationsbetweenthehygienedegreeinthethreebodyregions:udder,lowerleg,
upperlegandflank,in20dairycowsheds
Correlation
95%
Correlation
n
coefficient
Confidence
pvalue
(Spearmansr)
Interval
Udderandlowerleg
20
0.88
0.73to0.95
p<0.0001
Udderandupperlegandflank
20
0.85
0.66to0.94
p<0.0001
Lowerleganupperlegandflank
20
0.91
0.78to0.96
p<0.0001
p=statisticalsignificance,p<0.0001consideredextremelysignificant,n=numberof
cowsheds
investigated

Table4.Correlationsbetweenthelenghtofthestallsandthedirtinessdegreeinthethree
bodyregionsofassessedcows
Correlation
95%Confidence
Correlation
n
coefficient
pvalue
interval
(Spearmansr)
Stallslenghtandhygieneof 20 0.47
0.76to0.02
0.0096**
thelowerleg
Stallslenghtandhygieneof 20 0.56
0.81to 0.14
0.0334*
upperlegandflank
Stallslenghtandhygieneof 20 0.62
0.83to0.23
0.0035**
theudder
p=statisticalsignificance,*=p<0.05consideredsignificant,**=p<0.01consideredvery
significant,n=numberofcowshedsinvestigated

The high percentage of the 3 and 4 scores indicates a precarious, unacceptable


hygienelevelofthecowsheds,withsevereconsequencesonhealth,productionsandwelfare
ofdairycows.
Intheinvestigatedshedstheproportionof3and4scoresisdifferentforthethreebodyareas
ofthecows,thedifferencesbeingstatisticallysignificantbetweentheareasuddervsupperleg
andflanklowerlegvsupperlegandflank(Table2).Themeanproportionofthe3and4scores
inthethreebodyregions(Table1)ishigherthanCooksfindings(2002)inhisassessmentof

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UniversitateadetiineAgricoleiMedicinVeterinarIai
20 dairy cattle farms in Wisconsin, with tiestall and freestall housing. He found that, on
average the proportion of cows in tiestalls considered to be too dirty was, by zone, as
follows:20%forudder,25%forlowerlegzone,30%forupperlegandflankzone.However,
thesefiguresarehigherthanthosefoundinanotherstudywhere,whilethescoringsystems
weresimilar,thescoreswerenotidentical(Zurbriggetal.,2005).Within317studyfarms,8%
hadmorethan15%ofthecowswithsignificantlydirtyuddersand46%hadmorethan15%of
theherdwithsignificantlydirty hind limbs. Poor hygieneis a big problem in tiestallsas the
cowisbotheatingandlyinginthesamestallandtheclawsareoftenstandinginmanure.
Inourstudythedirtiestareaoftheanimalswastheupperlegandflank,followedby
theregionofthelowerlegandtheudder,respectively(Table1).Theobtainedresultsarein
agreementwiththeexistingdataintheliterature,statingthanthecowskeptintiestallshave
higher scores in the body region of the upper leg and flank than the ones in free breeding
becauseoflyingdowninthedejectionslaidinstalls(Cook2002).Thisbodyregioncanalsoget
soiledinpoorlymaintainedstallspresentingelementssplashedwithdejectionsorthroughthe
movementsofthedirtytailaroundthehindsection.In70%oftheinvestigatedcowshedsthe
manureremovalwasdonemanually,leadingtoadeficientcowshedhygieneandvitiatedair.
Similar to other researches (Schreiner and Ruegg 2003; Zurbrigg 2005), the present
studyshowsthatthecleanlinessoftheuddersandofthelegsarepositivelycorrelated(Table
3).
Thecleanlinessoftheudderandofthehindlegsisassociatedwiththestallsurface
and with the bedding materials used, with the housing techniques and the stall dimensions
(BergstenandPettersson,1992;Chaplinetal.,2000;Cook,2002).Significantcorrelationswere
demonstratedbetween the stall length and the hind legsand uddercleanliness (Table 4), in
agreementwiththeresultsofotherstudies(Anderson,2003;Zurbriggetal.,2005).Thehigh
degreeofsoilingofthecowsismainlyduetotheirhousinginshortstalls(60%oftheassessed
cowshedshadshortstalls),beingnotproperlycleaned.Thiscanbeexplainedbythefactthat
shorterstallsforcethecowstoliediagonallywhentheywanttobewithallfourlegsinsidethe
stall or makes them lay their hind legs on the manure evacuation alley. The cows lying
diagonallyonthebedshavehigherchancestourinateanddefecateonthebackpartofthe
stall. If cows lie down before the stalls are cleaned, their hind legs become dirty. The cows
lyingwith theirhindlegsonthemanureevacuationalleymaypresentlargeamountsofdirt
and dejections attached to their legs. When these cows are in decubitus, the dirt and
dejectionsaretransferredtothestall,dirtyingthebeddingtoacertainextent.Boththedirty
uddersandthedirtylegswerecorrelatedwithanincreasednumberoflamecows(Albanet
al., 1996; Zurbrigg, 2005). There are several possible explanations for these correlations
(Sprecher et al., 1997). In order to ameliorate the pain, lame cows spend more time in
decubitus and thus they have more chances to lie in dejections (Kloosterman, 1997). Cows
with severe lameness may be reluctant to rise once they are lying down and thus they can
defecateandurinateindecubitus(Herlin,1997).Bothsituationswillresultinawetanddirty
backsegmentofthestall,increasingtheriskforthosecowstohavedirtyhindlegsandudders.
Athirdpossibleexplanationcouldbelinkedtomanagementproblems.Stallcleaning,periodic
trimmingand,generally,theperiodiccareofthehoofscanbeseenbysomefarmersaslower
priorities.
Relationships between udder hygiene and outbreak of mastitis episodes were also
demonstrated.Ithasbeenlongknownthattherateofnewinfectionsisincreasingwith the
bacterial density at the level of the teats. Several researches showed correlations between
hygienichousing,cleancowsandlowernumbersofsomaticcellspresentinthemixedmilkat
farm level (Barkema and Schukken, 2003; Bodoh et al., 1976). In one of the studies an

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Lucrritiinificevol53seriaMedicinVeterinar
environmental sanitation indicator based on the amount of dejections present on the cow
body and in its environs constituted a prediction element for the occurrence of coliform
bacteria mastitis (Bartlettet al., 1992). The results of the investigation made by Cook (2002)
demonstratedasignificantcorrelationbetweentheproportionoftheuddersratedwith3and
4ineachfarm,observedovera6monthsperiod,andtheincidenceofmastitis.

CONCLUSIONS

Theresultsobtainedshowpoorhygieneintheinvestigatedcowsheds,withnegative
influence on dairy cows health and welfare. The dirtiness of the three body regions
demonstratedinthestudyismainlycausedbythedisregardingoftherecommandationsfor
dailycleaningandbeddingchangeinthecowshedsbutalsobytheimproperstalllenghtinthe
majority of the assessed cowsheds. The exposure of the cows to dirt, mud and dejections
constitutes the premise for an increased percentage of subclinical and clinical mastitis and
lameness.

ACKNOWLEDGMENT

ThisworkwassupportedbyCNCSISUEFISCSU,projectnumber1095PNIIIDEI1492/2009.

REFERENCES

1. Alban L, Agger J, Lawson L 1996: Lameness in tied Danish dairy cattle: The possible influence of
housingsystems,management,milkyieldandpriorincidentsoflameness.PrevVetMed29:135149.
2.AndersonN2003:Observationsondairycowcomfort:Diagonallunging,resting,standingandperching
infreestalls.ProcfifthInternationalConferenceonDairyHousing.ASAE,StJoseph,Michigan,pp.26
34.
3.BarkemaHW,VanderPloegJD,SchukkenYH,LamTJGM,BenedictusG,BrandA1999:Management
style and its association with bulk milk somatic cell count and incidence rate of clinical mastitis. J
DairySci82:16551663.
4.BarkemaHW,SchukkenYH2003:Theeffectofmanagementstyleonudderhealth.ProcNatlMastitis
CouncMtg,FtWorth,Texas,Madison,pp.6874
5.BartlettPC,MillerGY,Lanc SE,HeiderLE1992:Managerialdeterminantsof intramammarycoliform
andenvironmentalStreptococciinfectionsinOhiodairyherds.JDairySci75:12411252.

6. Bergsten C 1997: Infectious diseases of the digits. In: Saunders WB (Ed.): Lameness in Cattle. 3rd
Edition.Philadelphia,pp.89100
7.BergstenC,PetterssonB1992:Thecleanlinessofcowstiesinstallsandthehealthoftheirhoovesas
influencedbytheuseofelectrictrainers.PrevVetMed13:229238.
8.BlomJ1983:Traumaticinjuriesandfootdiseasesasrelatedtohousingsystems.In:BaxterSH,Baxter
MR,MacCormackJCA(Eds.):Farmanimalhousingandwelfare.ScottishfarmbuildingsInvestigation
Unit,Bucksburn,Aberdeen,UK,p.216223.
9.BloweyR1993:Cattlelamenessandhoofcare:Anillustratedguide.FarmingPressBooks,Ipswich,UK,
85p.
10.BodohGW,BattistaWJ,SchultzLH1976:VariationinsomaticcellcountsinDairyHerdImprovement
milksamples.JDairySci59:11191123.
11.ChaplinSJ,TierneyG,StockwellC,LogueDN,KellyM2000:Anevaluationofmattressesandmatsin
twodairyunits.ApplAnimBehavSci66:263272.
12.CookNB2002:Theinfluenceofbarndesignondairycowhygiene,lamenessandudderhealth.Proc
AmAssocBovinePractpp.97103.
13.CookNB,CutlerKL1995:Treatmentandoutcomeofasevereformoffoulinthefoot.VetRec136:
1920.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
14.CookNB,NordlundKV2009:Theinfluenceoftheenvironmentondairycowbehavior,clawhealth
andherdlamenessdynamics.VeterinaryJournal179:360369.
15. De Rosa G, Tripaldi C, Napolitano F, Saltalamacchia F, Grasso F, Bisegna V, Bordi A 2003:
Repeatability of some animalrelated variables in dairy cows and buffaloes. Anim Welfare 12: 625
629.
16.FayeB,BarnouinJ1985:Objectiveassessmentofthecleanlinessofdairycowsandhousingsystems
thecleanlinessindex.BullTech59:6167.
17.HerlinA1997:Comparisonofthelyingareasurfacesfordairycowsbypreference,hygieneandlying
downbehaviour.SwedJAgricRes27:189196.
18.HughesJ2001:Asystemforassessingcowcleanliness.InPractice23:517524.
19.KloostermanP1997:ClawCare.In:LamenessinCattle.SaundersWB,Toronto,Ontario,Canada,pp.
123126.
20. Schreiner D, Ruegg P 2003: Relationship between udder and leg hygiene scores and subclinical
mastitis.JDairySci86:34603465.
21.SprecherD,HostetlerD,KaneeneJ1997: Alamenessscoringsystemthatusespostureandgaitto
predictdairycattlereproductiveperformance.Theriogenology47:11791187.
22.ZurbriggK,KeltonD,AndersonN,MillmanS2005:Stalldimensionsandtheprevalenceoflameness,
injury,andcleanlinesson317tiestalldairyfarmsinOntario.CanVetJ46(10):902909.

1157


THELEVELOFMICROCLIMATEPARAMETERSINDAIRYCATTLE
BARNSWITHTIESTALLS

SILVANAPOPESCU ,CRISTINBORDA ,IULIANACHEGEDUS ,


R.STEFAN1,EVAA.LAZAR1
,1FacultyofVeterinaryMedicine,
2
FacultyofAnimalHusbandryandBiotechnologiesinAgriculture;popescusilvana@yahoo.com

Theaimofthisstudywastodeterminethemicroclimateparameterslevelintiestall
housesfordairycowsandtocompareitwiththerecommendedvaluesofourcountry.
Anumberof15dairycattletiestallbarnswereinvestigated,inTransylvania,overthe
periodofNovemberDecember2009.Themeasurementsweremadeinthemorning
andintheeveningineachofthebarns.Theobtaineddatawerestatisticallyprocessed
with the SPSS version 17 software. Excepting the ammonia concentrations, no
significant differences were demonstrated by the MannWhitney test (p>0.05),
betweenthemorningandtheeveningdeterminations.Theairstemperaturehadvery
similar mean values in the morning and in the evening, 11.82C and 11.22C,
respectively.Theairshumiditywashigherinthemorning,83.10%,comparingwithits
evening value, 79.73%, and the airflow velocity had a mean value of 0.34 m/s in the
morning and in the evening as well. All of the investigated barns were poorly
illuminated, the mean intensity of the light was 14.46 lx. The ammonias mean
concentration was higher in the morning, 24.73 ppm, than at the evening
determination, 15.13 ppm, the difference being statistically significant (p<0.05). The
carbondioxidewasof1213.3ppminthemorningand740.00ppmintheevening,and
the hydrogen sulphide was present in none of the investigated barns. The obtained
results indicate inappropriate microclimate in the majority of the investigated barns
with important divergences of the parameters from the recommended values for the
dairycows.

Keywords:airtemperature,relativehumidity,ammonia,carbondioxide

Microclimateisimportantthatdetermineairqualityindairybarns.Providinggoodairquality
infarmanimalhousingisimportantforhealthandwelfareoffarmanimalsandstaffandfor
the outdoor environment of farming enterprises (Seedorf and Hartung 1999, Radon et al.
2002).Microclimateisthelocalenvironmentaroundadairycowwheretheclimatemaydiffer
from the surrounding areas of the dairy building. The microclimate, or surrounding air,
containsoxygenforthecowsmetabolismandisthemediumforthetransportofexcessheat,
water vapors, gases emitted by the animals, gases from the decomposition of manure, and
other particulate matter. The important microclimate parameters that affect air quality in
dairybuildingsincludetemperature,relativehumidity,airvelocity,noiseandlightingaswellas
gases such as oxygen, carbon dioxide, methane, ammonia, hydrogen sulphide, and nitrous
oxide.Othersincludedustandmicroorganismsfoundinair.
Temperature is an environmental parameter that can affect the health, welfare, and
productionefficiencyofdairycows,andthustheprofitabilityofdairyproduction.Thedesign
of dairy buildings influences indoor temperature and, for that matter, the dairy cows
themselves.
1

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Dairycowscontinuouslyproduceheatandmoisture.Whenmoistureevaporatesfromtheskin
ofcows,thecowssurfacetemperaturedecreasesbecauseofevaporativeheatloss.Arelative
humidity(RH)over90%athighindoortemperaturewillinduceheatstressindairycowsdue
torestrictedevaporativeheatlosses.However,anexcessivelylowRHresultsinexcessivelydry
beddingmaterialinthedairybuildingandincreasesdustandtheincidenceoflungdiseasesin
dairy cows (Seedorf et al. 1998). Moreover, high relative humidity increases the rate of
deteriorationofbuildingmaterialsinthedairybuilding(DeBelieetal.2001ac).
Highgaseousconcentrationsinanimalbuildingsaffectthewelfareofanimals,workersandthe
life span of the buildings themselves (De Belie et al. 2001ac, Radon et al. 2002). Carbon
dioxide, methane, ammonia, hydrogen sulphide, and nitrous oxide are the most prominent
gasesfoundindairybuildings.Whengasesproducedinconcentrateddairyproductionescape
fromthebuildings, theycontributetoenvironmentalproblemssuchasglobalwarming,acid
rain,andupsettingthenutrientbalanceintheenvironment(Andersonetal.2003).
Theaimofthisstudywastodeterminethemicroclimateparameterslevelintiestallhouses
fordairycowsandtocompareitwiththerecommendedvaluesofourcountry.

MATERIALSANDMETHODS

Thisstudyinvestigated15dairycattletiestallbarns(30100dairycows/barn),inTransylvania
in the time interval November December 2009. All of the barns were closed, with solid
flooring. The manure cleaning was made by manual handling (in 80% of the barns) or
mechanically (in 20% of the barns). The cattle houses had only natural ventilation systems,
unorganized(in12ofthebarns)andorganized(in3barns).Thecowhousesusuallyhadshort
stalls(80%ofbarns).In20%ofthebarnsthereweremediumlongbeds.In80%ofthehouses
bedding was used (straw, sawdust). The cows were kept tied in the barns during the cold
season(pasturingintherestoftheyear,indaytime)orpermanently(withoutpasturing).Each
barn was visited once for the study. The temperature, the relative humidity, the air flow
velocity, the lighting intensity, the ammonia, carbon dioxide and hydrogen sulphide
concentrationsintheair,weremeasuredinthemorning(at56a.m.)andintheevening(at7
8p.m.)inthreedifferentpointsofthebarn.Themeanvaluesofthedeterminedparameters
werecalculatedforeachbarn.
Airtemperature,relativehumidityandairflowvelocityinthebarnsweredeterminedusinga
Testo400GmbH&Codevice.ThelightingintensitywasmeasuredwithanExtechelectronic
luxmeter.Ammonia,carbondioxideandhydrogensulphideconcentrationsweredetermined
byairsamplingwithDrgerMultiwarnII(DrgerSafety,Germany)device.
The obtained data were statistically processed with the SPSS version 17 software and were
compared with the maximal admitted values for dairy cattle houses in our country. The
descriptivestatisticalindicatorswerecalculated(mean,standarddeviation,median,minimum
and maximum) for the measured parameters. In order to compare values the MannWitney
nonparametrictestwasused.

RESULTSANDDISCUSSIONS

Theobtainedresultsofthemeasurementsmadeinthemorningandintheeveninginthe15
dairy cattle barns with tiestalls are shown in Tables 12. Excepting the ammonia
concentrations, no significant differences were demonstrated by the MannWhitney test
(p>0.05),betweenthemorningandtheeveningdeterminations.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Temperature had similar mean values at the two measurements (in the morning and in the
evening), in accordance with those recommended for dairy cattle barns. Yet the maximal
recorded value was higher than the recommended optimal temperature. Various
recommendations for temperature conditions for keeping dairy cows appear in literature
(CIGR 1994, Seedorf et al. 1998, Teye et al. 2008). In Romania the recommended optimal
temperaturefordairycowsrangesbetween10and14C(NSVFSA2007).Temperatureisan
environmental parameter that can affect the health, welfare, and production efficiency of
dairycows,andthustheprofitabilityofdairyproduction.Thethermalenvironmentarounda
dairycowvariesaccordingtothecomplexinteractionsbetweenenvironmentalconditionsand
animalrelated factors. Furthermore, factors such as the dairy breed and age, structural
design,floortype,stockingrate,andnutritionalsoinfluencehowthethermalconditionsinthe
building affect individual animals. Under certain optimum environmental conditions, dairy
cowsarenotonlycomfortable,butproducehigheroutputs.

Table1.Descriptivestatisticanalysisformicroclimateparametersinthe15investigateddairy
cattlebarns,inthemorning.
Parameter

Mean

SD

Median

Minimum

95%CI
FromTo

Maximum

Temperature
15
11.82 4.56 10.900
7.00
19.70
(C)
Relative
15
83.10
9.39 85.400
65.50
94.85
humidity(%)
Airflow
15
0.34
0.04 0.3400
0.29
0.400
velocity(m/s)
Light(lx)
15
14.46 6.51 12.000
7.00
28.500
Ammonia
15
24.73 7.86
28.000
9.00
35.000
(ppm)
Carbon
15 1213.3 836.55 1000.0
100.00
2000.0
dioxide(ppm)
Hydrogen
sulphide
15
0
0
0
0
0
(ppm)
n=numberofbarns,CI=confidenceinterval,SD=standarddeviation

9.2914.34
77.9088.30
0.320.36
10.8518.07
20.3729.09
750.021676
0

Themeasuredrelativehumidityhadinthemorning(83.10%)andalsointheevening(79.73%)
meanvaluesexceedingtheoptimalvaluefordairycattlebarns.InastudyrealisedbyTeyeet
al.(2008)indairycowsbarnsinFinlandandEstonia,therelativehumidityvariedfrom38%to
92%. For the relative humidity in animal buildings CIGR (1984) recommends maximum and
minimum values as a function of indoor temperature. In Romania, the recommended
optimum relative humidity for dairy cows is from 60% to 75% (NSVFSA 2007). Relative
humidityinthedairybuildingsexceededtherecommendedvalueswhentheventilationwas
inadequate.Highrelativehumidityduringthecoldseasonsisamajorprobleminmostofthe
dairy buildings. A wellinsulated roof is needed in naturally ventilated dairy buildings.
Adequate roof insulation can not only prevent the condensation of moisture at roof level,
whichleadstorustandmouldindairybuildings,butalsoimprovetheexchangeofairinthe
building.

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Lucrritiinificevol53seriaMedicinVeterinar
Thevelocityoftheairflowhadthesamemeanvaluesinthemorningandalsointheevening,
corresponding to those recommended for dairy cattle barns. The maximal recorded values
were slightly higher (0.4 m/s). The results are in conformity with those obtained by other
researchersintheirstudies(Teyeetal.2008).

Table2.Descriptivestatisticanalysisformicroclimateparametersinthe15investigateddairy
cattlebarns,intheevening.
Maximum

95%CI
FromTo

6.30

16.70

9.0813.36

82.00

61.50

94.00

74.8784.59

0.04

0.33

0.280

0.400

0.310.36

6.51

12.00

7.00

28.50

10.8518.07

Parameter

Mean

Temperature
(C)

15

11.22

3.86

10.70

15

79.73

8.77

15

0.34

15

14.46

Relative
humidity(%)
Airflow
velocity(m/s)
Light(lx)

SD

Median

Minimum

Ammonia
15 15.13
7.39
15.00
5.00
27.00
11.0319.22
(ppm)
Carbon
dioxide
15 740.00 604.51 500.00
100.00
2000.0
405.201074.8
(ppm)
Hydrogen
15
0
0
0
0
0
0
sulphide
(ppm)
n=numberofbarns,CI=confidenceinterval,SD=standarddeviation

Alloftheinvestigatedbarnswerepoorlyilluminated;themeanvalueoflightingintensitywas
14.46 lx. The light levels were about 4 times lower than the minimal recommended
illuminanceforthedairycows(5060lx).StudiesruledintheUnitedStatesrevealedincreased
milkproductionsupto16%inthecowswhichbenefittedofprolongedilluminationatahigher
light intensity (Dahl et al. 2000). The conclusion of these studies is that cows should be
exposeddailyto1618hoursoflightperday(morethan200lx),inordertomeetthebenfits
ofalongphotoperiod.
The ammonia was found in each of the investigated barns, in the morning as well as in the
evening(tables12).Itsconcentrationishigherthanthosedescribedinscientificliterature(6
10 ppm) (Groot Koerkamp et al. 1998, Seedorf and Hartung 1999, Demmers et al. 2001,
Jungbluthetal.2001,Andersonetal.2003).ClarkandMcQuitty(1987)studiedtheairquality
insixAlbertacommercialdairybarnsandfoundthattheNH3waspresentinallsixbarnsand
theoverallmeanvaluesrangedfrom7to20ppm.GrootKoerkampetal.(1998)investigated
concentrations and emissions of ammonia in different livestock buildings in England, the
Netherlands,DenmarkandGermany.Thehighestammoniaconcentrationincattlehouseswas
found in Germany (22.7 ppm), with mean values in different countries varying between 0.9
ppmand7.1ppm.Anotherinvestigationofammoniaconcentrationsinlivestockbuildingsin
Germanyfoundameanvalueof6.4ppmincowhouses(SeedorfandHartung1999).Inamore
recent study conducted in dairy cow barns in Finland and Estonia, the ammonia

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UniversitateadetiineAgricoleiMedicinVeterinarIai
concentrationsvariedbetween0and64ppm(Teyeetal.2008).Themaximalreportedvalue
washigherthanourstudyshighestvalue(27ppm).Highammoniaconcentrationsareusually
foundinclosedbuildings.Ammoniafromclosedbuildingsfordairycattleisproducedinlarge
quantitiesthroughureahydrolysisinurine(MuckandSteenhuis1981).Inthemajorityofthe
studiedbarnstheammoniaconcentrationwashigherinthemorning,theMannWhitneyTest
demonstrating very significant differences between the values in the morning and evening
measurements (p=0.0019). These results highlights the importance of ammonia
measurementsinthebarnsairearlyinthemorning,beforeaerating,inordertofindoutthe
real concentration not only of ammonia, but also of other harmful gases, which affect cows
during the night. During the day, because of the opened doors and windows, the ammonia
concentrationisusuallylowerinthedairycattlehouses.Theindoorammoniaconcentration
depends on the flooring type, bedding material, animals age, the microclimate factors, the
manure evacuating system, the frequency of cleaning and on the animals diet (Groot
Koerkampetal.1998).Inourstudy,thebarnswithhighammoniaconcentrationswerepoorly
ventilated and dirty. In 60% of the investigated barns the ammonia concentration exceeded
theadmittedtresholdvalueofourcountry(26ppm).NH3levelsinanimalhousingcanexceed
25 ppm when lower winter ventilation rates are used and can reach 40 ppm in poorly
ventilatedbuildings(GrootKoerkampetal.1998).Ammoniaisconsideredthemostsignificant
pollutant in the air of the cattle barns, due its irritating effect on respiratory epithelium
(Marschang 1973). At concentrations less than 100 ppm and in a poorly ventilated facility,
ammoniaappearstoaffectpulmonaryfunctionincattle.
Thecarbondioxidesconcentrationvariedintheinvestigatedbarns,themorningvaluesbeing
higherthanthosemeasuredintheevening,butthisdifferencewasnotstatisticallysignificant
(P>0,05).Inallofthebarnsthecarbondioxideconcentrationwasbelowthemaximaladmitted
limitvaluefordairycattlehouses.Themainsourceofcarbondioxideindairybuildingsisfrom
respiration. Minor proportions (6.1%) are produced through the degradation of manure and
urea(Kinsmanetal.1995).TheaverageconcentrationofCO2indairybuildings is1900ppm
(Phillipsetal.1998).

CONCLUSIONS

1. The air temperature and the airflow velocity registered moderate divergences from the
admittedlimitsinthemajorityoftheinvestigatedbarns.
2.Therelativehumidityshowedimportantabberancefromthevaluesrecommendedfordairy
cows.
3.Thelightingintensitywasinappropriateinallofthestudiedbarns.
4.Theammoniaconcentrationsexceededthetresholdvaluein60%oftheinvestigatedcattle
houses, indicating the need for improvement in the futures housing conditions. The values
determinedinthemorningweresignificantlyhigherthanthevaluesmeasuredintheevening.
5.Innoneofthebarnsthecarbondioxideexceededtheadmittedlimitvaluefordairycows.
6.Thehydrogensulphidewasfoundinnoneoftheinvestigateddairyhouses.
7.Exceptingtheammoniaconcentrations,nosignificantdifferenceswerefoundbetweenthe
morningandtheeveneningdeterminations.
ACKNOWLEDGMENT
ThisworkwassupportedbyCNCSISUEFISCSU,projectnumber1095PNIIIDEI1492/2009.

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REFERENCES
1. Anderson N, Strader R, Davidson C (2003) Airborne reduced nitrogen: ammonia emissions from
agricultureandothersources.EnvironInt29:277286.
2. CIGR Commission Internationale de Gnie Rural (1984) Climatisation of animal houses, Report of
workinggrouponclimatisationofanimalhouses,Aberdeen,Scotland,pp.172.
3.ClarkPC,McQuittyJB(1987)AirqualityinsixAlbertacommercialfreestalldairybarns.CanAgrEng
29(1):7780.
4.DahlGE,BuchananBA,TuckerHA(2000)Photoperiodiceffectsondairycattle:Areview.JDairySci
83:885893.
5.DeBelieN,LenehanJJ,BraamCR,SvennerstedtB,RichardsonM,SonckB(2000a)Durabilityof
BuildingmaterialsandComponentsintheAgriculturalEnvironment,PartIII:Concretestructures.J
AgricEngRes76:316.
6. De Belie N, Richardson M, Braam CR, Svennerstedt B, Lenehan JJ, Sonck B (2000b) Durability of
Building materials and Components in the Agricultural Environment: Part I, The agricultural
environmentandtimberstructures.JAgricEngRes75:225241.
7.DeBelieN,SonckB,BraamCR,SvennerstedtB,RichardsonM(2000c)DurabilityofBuildingmaterials
andComponentsin theAgriculturalEnvironment,PartII:Metalstructures.JAgricEngRes75:333
347.
8.DemmersTGM,PhillipsVR,ShortLS,BurgessLR,HoxeyRP,WathesCM(2001)Validationofventilation
rate measurement methods and the ammonia emission from naturally ventilated dairy and beef
buildingsintheUnitedKingdom.JAgricEngRes79:107116.
9. Groot KoerkampPWG, MetzJHM, UenkGH, PhillipsVR, HoldenMR, SneathRW, Short JL, WhiteRP,
HartungJ, Seedorf J, SchroderM, Linkert KH, PedersenS, TakaiH, JohnsenJO, WathesCM (1998)
Concentrations and emissions of ammonia in livestock buildings in Northern Europe. J Agric Eng
Res70:7995.
10.JungbluthT,HartungE,BroseG(2001)Greenhousegasemissionsfromanimalhousesandmanure
stores.NutrCyclAgroecosys60:133145.
11.KinsmanR,SauerFD,JacksonHA,WolynetzMS(1995)Methaneandcarbondioxideemissionsfrom
dairycowsinfulllactationmonitoredoverasixmonthperiod.JDairySci78:27602766.
12.MarschangF(1973)Review:ammonia,losses,andproductioninlargeanimalstables.DtschTierarztl
Wochenschr80:73120.
13.MuckRE,SteenhuisTS(1981)Nitrogenlossesinfreestalldairybarns.In:Anonymous(ed)Livestock
Wastes:ARenewableSource.AmericanSocietyofAgriculturalEngineers,St.Joseph,pp406409.
14.NationalSanitaryVeterinaryandFoodSafetyAuthorityofRomania(2007)Assessmentcardregarding
welfareandprotectionofcalves,pigs,layinghens,farmanimals.
15.PhillipsVR,HoldenMR,SneathRW,ShortJL,WhiteRP,HartungJ,SeedorfJ,SchroderM,LinkertKH,
PedersenS,TakaiH,JohnsenJO,GrootKoerkampPWG,UenkGH,ScholtensR,MetzJHM,Wathes
CM(1998)Thedevelopmentofrobustmethodsformeasuringconcentrationsandemissionratesof
gaseousandparticulateairpollutantsinlivestockbuildings.JAgricEngRes70:1124.
16.Radon K,Danuser B,IversenM, MonsoE, WeberC,Hartung J(2002)Aircontaminantsindifferent
Europeanfarmingenvironments.AnnAgricEnvironMed9:4148.
17.SeedorfJ,HartungJ,SchrderM,LinkertKH,PedersenS,TakaiH,JohnsenJO,MetzJHM,Groot
KoerkampPWG,UenkGH,PhillipsVR,HoldenMR,SneathRW,ShortJL,WhiteRP,WathesCM
(1998)TemperatureandmoistureconditionsinlivestockbuildingsinnorthernEurope.JAgricEng
Res70:4957.
18. Seedorf J, Hartung J (1999) Survey of ammonia concentrations in livestock buildings. J Agr Sci
133:433437.
19. Teye KF, Hautala M, Pastell M, Praks J, Veerme I, Poikalainen V, Pajumgi A, Kivinen T, Ahokas J
(2008)MicroclimateandventilationinEstonianandFinnishdairybuildings.EnergBuildings40:1194
1201.

1163

STUDIESCONCERNINGHYGIENICBODYSCORESANDTHERISKOF
INTRAMAMMARYINFECTIONSINCOW

ElenaROTARU,B.TABAC,ElenaMITRNESCU,MariaCRIVINEANU,
V.NICORESCU,MagdaGONCIAROV
FacultyofVeterinaryMedicineBucharest,105SplaiulIndependentei,050097,Bucharest,
Romania,elenrotaru@yahoo.com

Abstract
Intramammaryinfectionsincowsleadstomajoreconomicallossesduetothedecreaseofmilk
production(theconsequenceofpathologicalprocess),withdrawalperiod(drugresiduesinmilk)
and increased percentage of reformed cows (compromised mammary gland). Many authors
agreethatoneofthemajorcausesofmastitisisrepresentedbyhighchargesofmicroorganisms
in the environment. The aim of this study, performed in a farm with 270 dairy cows near
Bucharest, was to follow the correlations between the presence of mastitis (bacteriological
exams)andhygienicbodyscore(mammaryglandandposteriormembersaccordingtoCookes
evaluationkey).Theevaluationwasmadeon27cows(10%)whichshowedobviousproblemsof
mammary gland and posterior members hygiene (score 3 and 4); these cows were chosen by
clinicalinspectionattheentranceinthemilkingarea.Thestudyrevealedahighpercentageof3
and4hygienicscores,whichindicatesanunacceptablehygieneinthefarmandalsothefactthat
poor hygienic scores are positively correlated with the presence of microorganisms responsible
formastitis,especiallyArcanobacteriumpyogenes,StreptococcusagalactiaeandStaphylococcus
aureus.
Keywords:cow,environment,hygienicbodyscore,mastitis.

Intramammary infections in cows are multifactorial diseases. The relationship


between the environment as potential contamination source and the risk of mastitis was
studiedbymanyauthors(1,2,3).Theresultsdemonstratedthatenvironmentalsalubrityisan
importantfactorforthedecreaseofmastitisprevalenceandincidenceindairycowsfarms.

MATERIALSANDMETHODS

Thestudywasperformedon27dairycowsfromafarmnearBucharest(10%),chosen
aleatory at the entrance in the milking area. Using Cook system, there were evaluated
posterior members and mammary gland and there were given scores between 1 and 4
dependingontheirdegreeofdejectionsdirtiness.
Forposteriormembersitwasappreciatedthequantityofdejectionsandtheirarea,
score1correspondingtonoidentifieddejections,score2lowquantityofdejections,score3
boardsofdejections,butwithvisiblepeltandscore4boardsofdejections.
Forthemammarygland,hygienicscoreswere:score1cleanmammarygland,score
2mammaryglandalittledirtyatthesideofmamelons,score3theexistenceofdejections
boards in the inferior half of mammary gland and score 4 the presence of dry dejections
boards. Finally it was calculated the proportion of 3 and 4 scores and their average
percentage. In order to appreciate the risk of intramammary infections, there were
determined,byusualmethods,thenumberoftotalgerms(NTG)andthesomaticcellscount
(SCC) in milk samples collected before morning milking. Several authors demonstrated that
samplescollectedaftermilkingarelesssusceptibletobecontaminated,becausethemamelon
andpapillaryductarewashedanddisinfected(2).Otherauthorsadmitthat,ifthepersonnel

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Lucrritiinificevol53seriaMedicinVeterinar
are specialized and the sampling is correct, the difference between the contamination of a
samplebeforeandaftermilkingisminimal(3).

RESULTSANDDISCUSSIONS

Visual inspection of shelters and paddocks in the studied unit demonstrated the
existenceofinadequateenvironmentalconditionsforappropriatebodyhygiene.Thus,infigs.
17 are presented the main deficiencies, respectively: inadequate hygiene of posterior area
(fig.1,2)andmammarygland(fig.3,4)asaconsequenceofthecontactwithdirtysheet(fig.
5), and also inadequate paddocks hygiene, a risk factor that influences milk salubrity and
mammaryinfectionsincidence(fig.6,7).

Score3

Score4
Fig.1,2 Dirtyposteriormembers

Score3

Score4
Fig.3,4. Dirtymammarygland

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Fig.5Contactofmammaryglandwithdirtysheet

Fig6,7.Inadequatepaddocksfavortheexistenceofdirtyanimals

Theresultsobtainedfollowingtheexaminationsarepresentedintable1andhavethe
followingsignificance:
- scores1and2indicatecleananimals;
- scores3and4indicatedirtyanimalsarisksituationwhichcanbeimproved;
- numberoftotalgerms/mlhasreferencevaluesunder50000;
- somaticcellscount/mlhasreferencevaluesunder200000.

Table1
Theresultsobtainedaftertheexaminationofbodyhygiene,numberoftotalgermsand
somaticcellscountinmilk

Posteriormembers Numberof Somaticcells


Mammarygland
score
score
totalgerms
count
Nr.
Ident.nr.
(nr./ml)
(nr./ml)
1
2
3
4
1
2
3
4
1
3148

x
x
560000
290000
2
3543
x

x
45000
49000
3
2902
x

x
35000
160000
4
3007

x
x
139000
450000
5
3578
x

x
42000
45000
6
2433

x
x
293226
750000
7
2652

x
x
459350
870000
8
2699

x
x
173100
250000

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Lucrritiinificevol53seriaMedicinVeterinar
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

3932
3745
3312
9043
4405
3312
5217
3200
3301
4278
2315
1722
3387
9012
1545
7751
2346
1312
2267

x
x
x

x
x
x

x
x
x
x
x

x
x

x
x
x
x
x
x
x
x

x
x

x
x
x

x
x

x
x
x

683870
36600
30000
32500
49000
389000
967400
950000
27600
20000
14000
49000
37000
173100
368500
857300
1200000
748300
595400

450000
75000
90000
85000
120000
650000
950000
870000
49000
49000
75000
85000
49000
750000
750000
550000
980000
690000
750000

Intable1itisnoticeablethat,fromall27analyzedcows,in12cases(44.44%)there
were given scores 1 and 2 (clean posterior members and mammary gland), while 15 cows
(55.55%)obtained3and4scores(dirtyposteriormembersandmammarygland).Betweenthe
twobodyregionsthedifferencesofscoringareinsignificant;asarule,dirtyposteriormembers
are correlated with dirty mammary gland, rarely dirty posterior members being correlated
withclean mammary gland. This fact is explained by the contact of the two areaswith dirty
sheet, with consequent negative effects. The analysis of total number of germs and somatic
cellscountasindicatorsofintramammaryinfectionsriskdemonstratesthatforthecowswith
precarious posterior members and mammary gland hygiene, the values of these parameters
arehigherthanreferencevalues.
Presentstudydemonstratesthat:
- mammaryhygieneispositivelycorrelatedwithposteriormembershygiene;
- therearepositivecorrelationsbetweenmammaryhygieneandtotalnumberofgerms
andsomaticcellscountasriskfactorsformastitis(Fig.8,9).

200000
Nr. total
germs

150000
100000

Somatic cells
count

50000
0
2

10 11 12 13 17 18 19 20 21

Cow (position in table 1)

Fig.8.Thecorrelationbetweenscores1,2andNTG/SCC

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UniversitateadetiineAgricoleiMedicinVeterinarIai

1400000
1200000
1000000
800000
600000
400000
200000
0

Nr. total
germs
Somatic cells
count
1

9 14 15 16 22 23 24 25 26 27

Cow (position in table 1)

Fig.9.Thecorrelationbetweenscores3,4andNTG/SCC

CONCLUSIONS

1. Inthestudiedfarmtherearesignificantdifferencesinmilkqualitydependingonthe
hygiene,especiallymammaryglandandposteriormembershygiene.
2. Somatic cells count had different values, from an average of 56083 in case of clean
animals(scores1and2)toanaverageof660714incaseofdirtyanimals(scores3and
4).
3. Numberoftotalgermsalsohaddifferentaveragevalues,from34808incaseofclean
animalsto568725incaseofdirtyanimals.
4. The study showed the fact that there are positive correlations between mammary
hygieneandtotalnumberofgermsandsomaticcellscountasriskfactorsformastitis.

REFERENCES
1.
2.

3.

Cook,N.B.(2002).Theinfluenceofbarndesignondairycowhygiene,lamenessandudder
health.Proc.Amer.Assoc.BovinePract.,Madison,WI.,pp.97103.
Hogan, J.S., Smith K.L., Todhunter D.A., Schonberger P.S. (1988). Rate of environemental
mastitisinquartersinfectedwithCorynebacteriumbovisandStaphylococcusssp.Journalof
DairyScience,71,pp.25202525.
Zurbrigg, K. (2005). The relationships between aspects of dairy cow welfare and tie stall
design,milkproductionandmilkquality.Univ.ofGuelph,Ontario,Canada,PhDThesis.

1168


KEYBIOCHEMICALPARAMETERSCHANGESOFTHEAPIS
MELLIFERACARPATHICABEE
INSTRESSFULCONDITIONS

apcaliuAgripina1,I.Rdoi2,TudorPoliana2,TutunaruAlexandru2,MgdiciMaria3,
CuiaEliza1,N.Tudor2
1
ICDABucuresti
2
FacultateadeMedicinVeterinarBucureti
3
ICSAIai
agripinasapcaliu@yahoo.com

The study presents changes in biochemical parameters of the bee Apis mellifera Carpathica
hemolymphinstressfulconditionscomparedwithvaluesobservedinhealthybeeshemolympha.
Measurementsweremadeovertwoyearsonatotalof176familiesofhealthybeesand20bee
familiesunderstressfactors.Harvestingandprocessingofhemolymphfreshsamplesfromboth
groups were followed by analysis via classical biochemistry. In stressed bees hemolymph were
found significant increases in alkaline phosphatase (ALP), from 6070 UI/l to 110178UI/l, in
aspartataminotransferaze (AST), from 336393 UI/l to 421587 UI/l, in
gammaglutamiltranspeptidaze(GGT),from2.563.33UI/lto31262UI/l,increatininefrom6.1
6.9 UI/l to 8.1 11.48 UI/l, in total calcium from 3.1 3.28 mg/dl to 4.6 6.4 mg/dl, in
triglyceridesfrom34.936.7mg/dlto5577.7mg/dl,andsignificantreductionofmagnesium
from 3.4 3.99 mg / dl to 0.34 0.55 mg / dl, compared with healthy bees. Monitoring the
metabolic health of bees in stress conditions requires evaluation of a few biochemical
parameters(ALP,AST,GGT,totalCa,Mg,CreandTG).

Keywords:haemolymph,bee,stress,biochemicalparameters

The haemolymph is the body insects fluid comparable with the blood of the
vertebrates,butunlikehaemolymph,thebloodcarriesnotonlythenutritivesubstancestothe
cells but also a complex protein substance, called haemoglobin, which forms the colorant
materialoftheblood andtransportstheoxygen.Thehaemolymphhasonlyanutritiverole,
therespiratoryfunctionbeingnegligible[4].Thehaemolymphistransparent,uncoloured(or
lightyellowish)butthequantityrepresentsabout2530%fromthetotalweightofthebeeat
the hatching stage and decreases as the bee becomes older. The haemolymph contains
proteins, amino acids, sugars, hormones, enzymes and many other substances in different
concentrationascomparedwithvertebrateblood[1,3,4].
At the international level, most part of the already carried out researches have
focusedonbiochemicalanalysesconnectedwiththesugars(fructose,trehaloseandglucose),
proteins (vitellogen), amino acids (proline), hormones (juvenile hormone) contents and the
activityofsomeenzymes.Forexample,aseriesofdatashowsthatthetotalsugarslevelfrom
honeybees` haemolymph is the highest from all the insect species, being used as the main
energeticsupplyintheflightactivitywhichrequiresaveryhighenergeticconsumption[2].
ThemainstressfactorsfoundinnaturalconditionstoApismelliferaCarpathicabee
causing serious losses to beekeepers and country economy are traveling for long distances,
tiring, in poor conditions, at high temperature and poor ventilation, low humidity and
insufficientfood,correlatedwithpooraccesstowatersupply[5,6].

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The goal of this experimental study was to determine the variability of the main
biochemicalparametersfromhealthyhoneybeescomparedtostressedbees(exposedtohigh
temperature,over30Cfor36hours,poorventilation,afteratiringjourneyinFOTIcages).

MATERIALSANDMETHODS

The study was conducted over a two years period (20082009) and included two
distinct categories of subjects: healthy bee families (176) belonging to Research and
Development Institute of Apiculture and bee families affected by stress (20) from different
apiariesofsomeprivatebeekeepers.
Stressconditionswereachievedbyfatiguingtransportfor48hoursandmaintaining
bee families in Apiclimatron in FOTI transport cages up to 2430 hours, in high
temperature(over 30C), low humidity, poor ventilation in the internal tube, compared to
healthy bees that were kept in hives in normal microclimate conditions in the external tube
(transportationstress).
Measurements were made on samples collected from active season in April 2008
and the active season in September 2009. Each sample contains haemolymph randomly
harvestedfrom50bees.
Formation (organization) of the experimental group consisting of 176 healthy
honeybeecolonies,sanitaryveterinarysurveyedand20stressedhoneybeecolonies.

RESULTSANDDISCUSSION

TheresultsobtainedduringthestudyarepresentedinTables1,2,3,4,5and6.

Tableno.1Themainbiochemicalparametersofhaemolymphinsample1
(april2008healthybees)
N
Parameter(UM)
Value
o.
1
ALP(UI/l)alkalinephosphatase
60
2
GPT/ALT(UI/l) alanineaminotransferase
311
3
GOT/AST(UI/l) Aspartateaminotransferase
393
4
totalCa(mg/dl) calcium
3.28
5
CPK(UI/l)creatininphosphokinase
498
6
Cre(mg/dl)Creatinine
6.90
7
GGT(UI/l)Gammaglutamyltranspeptidase
2.56
8
GLU(mg/dl)glucose
340.5
9
Mg(mg/dl)magnesium
3.99
1
TPro(g/d)total protein
2.51
0
1
TG(mg/dl)triglycerides
36.7
1
1
UA(mg/dl)uric acid
2.88
2
1
BUNurea(ureanitrogen)
42.63
3

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Collection of the required quantity of fresh haemolymph (300 l/determination) by
means of a glass capillary introduced between 3 and 4 tergites and the collection of the
haemolymph from capillary in a special syringe. Biochemical analyses on the fresh
haemolymph with the Alfawassermann apparatus. Processing and interpretation of the
obtained data. For haemolymph biochemical, the samples were analyzed by wet chemistry
usingAlfawassermanndeviceaimingatassessingALP(IU/L),SGPT/ALT(IU/L),SGOT/AST(IU/L)
intotal(mg/dl),CPK(IU/L),Cre(mg/dl),GGT(IU/L),GLU(mg/dl),Mg(mg/dl),TPro(g/d),TG
(mg/dl),UA(mg/dl)andurea(mg/dl).

Tableno.2Themainbiochemicalparametersofhaemolymphinsample2
(september2009healthybees)
No.
Parameter(UM)
Value
1
ALP(UI/l)alkalinephosphatase
70
2
GPT/ALT(UI/l) alanineaminotransferase
368
3
GOT/AST(UI/l)Aspartateaminotransferase
336
4
Catotal(mg/dl)calcium
3.1
5
CPK(UI/l)creatininphosphokinase
506
6
Cre(mg/dl)Creatinine
6.1
7
GGT(UI/l)Gammaglutamyltranspeptidase
3.33
8
GLU(mg/dl)glucose
377
9
Mg(mg/dl)magnesium
3.4
10
TPro(g/d)totalprotein
2.4
11
TG(mg/dl)triglycerides
34.9
12
UA(mg/dl)uricacid
2.62
13
BUNurea(ureanitrogen)
41.2

Tableno.3Themainbiochemicalparametersofhaemolymphinsample3
(april2008healthybees)
No.
Parameter(UM)
Value
1
ALP(UI/l)alkalinephosphatase
110
2
GPT/ALT(UI/l) alanineaminotransferase
321
3
GOT/AST(UI/l) Aspartateaminotransferase
421
4
Catotal(mg/dl) calcium
4.6
5
CPK(UI/l)creatininphosphokinase
5.96
6
Cre(mg/dl)Creatinine
9.6
7
GGT(UI/l)Gammaglutamyltranspeptidase
33.3
8
GLU(mg/dl)glucose
308.5
9
Mg(mg/dl)magnesium
0.55
10
TPro(g/d)totalprotein
2.83
11
TG(mg/dl)triglycerides
61.06
12
UA(mg/dl)uricacid
1.86
13
BUNurea(ureanitrogen)
43.21

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Tableno.4Themainbiochemicalparametersofhaemolymphinsample4
(april2008healthybees)
No.
Parameter(UM)
Value
1
ALP(UI/l)alkalinephosphatase
140
2
GPT/ALT(UI/l) alanineaminotransferase
353
3
GOT/AST(UI/l) Aspartateamino transferase
496
4
Catotal(mg/dl) calcium
5.91
5
CPK(UI/l)creatininphosphokinase
526
6
Cre(mg/dl)Creatinine
8.1
7
GGT(UI/l)Gammaglutamyltranspeptidase
31
8
GLU(mg/dl)glucose
346
9
Mg(mg/dl)magnesium
0.41
10
TPro(g/d)totalprotein
3.1
11
TG(mg/dl)triglycerides
63
12
UA(mg/dl)uricacid
2.8
13
BUNurea(ureanitrogen)
49.2

Tableno.5Themainbiochemicalparametersofhaemolymphinsample5
(september2009healthybees)
No.
Parameter(UM)
Value
1
ALP(UI/l)alkalinephosphatase
139
2
GPT/ALT(UI/l) alanineaminotransferase
383
3
GOT/AST(UI/l) Aspartateaminotransferase
587
4
Catotal(mg/dl) calcium
6.40
5
CPK(UI/l)creatininphosphokinase
400
6
Cre(mg/dl)Creatinine
11.48
7
GGT(UI/l)Gammaglutamyltranspeptidase
221.34
8
GLU(mg/dl)glucose
362
9
Mg(mg/dl)magnesium
0.34
10
TPro(g/d)totalprotein
2.18
11
TG(mg/dl)triglycerides
77.7
12
UA(mg/dl)uricacid
3.56
13
BUNurea(ureanitrogen)
40.96

The results Analysis summarized in table 16 shows the existence of significant


increases in the values of biochemical parameters characterizing enzyme activity (ALP, AST,
GGT) energy metabolism (CRE) and total calcium levels in lymph. It was found that alkaline
phosphataseactivityincreasesunderstress,verysignificantly,from1960to1970IU/Lat110
178UI/L,ASTenzymeactivitysignificantlyincreasedfrom336393IU/Lat421587IU/L.
GGT enzyme activity increased under stress, very significantly from 2.563.33 IU/L,
31262IU/L.Creatininevaluesincreasedsignificantlyfrom6.1to6.9IU/Lto8.1to11.48IU/L,
values are 56 times higher than the mammal blood. n comparison, total calcium values
increasedsignificantlyfrom3.1to3.28mg/dl(beesound)from4.6to6.4mg/dl(intermsof
stress).Triglyceridesincreasedverysignificantlyfrom34.9to36.7mg/dltovaluesbetween55
to77.7mg/dlintermsofexposuretostressfulfactors.

It is noted that for monitoring the health of bees in metabolic stress


conditions,isnecessarytoevaluateasmallnumberofbiochemicalparameters:ALP,AST,GGT

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UniversitateadetiineAgricoleiMedicinVeterinarIai
enzymes, creatinine, total calcium, magnesium and total triglycerides, other biochemical
parametersofmovinginsignificanthemolymph.

Tableno.6Themainbiochemicalparametersofhaemolymphinsample6
(september2009healthybees)
No.
Parameter(UM)
Value
1
ALP(UI/l)alkalinephosphatase
178
2
GPT/ALT(UI/l) alanineaminotransferase
362
3
GOT/AST(UI/l) Aspartateaminotransferase
455
4
Catotal(mg/dl) calcium
5.98
5
CPK(UI/l)creatininphosphokinase
671
6
Cre(mg/dl)Creatinine
10.1
7
GGT(UI/l)Gammaglutamyltranspeptidase
262
8
GLU(mg/dl)glucose
312
9
Mg(mg/dl)magnesium
0.5
10
TPro(g/d)totalprotein
2.1
11
TG(mg/dl)triglycerides
55
12
UA(mg/dl)uricacid
4.5
13
BUNurea(ureanitrogen)
42.6

Compared with previous research conducted on healthy bees [6], which


werefollowedseasonalvariationsofbiochemicalparametersofbeeshemolymph,wefound
lowvariabilityinmostbiochemicalparametersstudiedinhealthybeesinthetwoyearsofthe
experiment.Glucoseandtotalproteinvalueswerecomparabletothosereportedinliterature,
and activity levels of enzymes AST, ALT, GGT, CPK, and total urea showed higher values
comparedwiththosefoundinthebloodofmammals,whilecalcium,magnesium,uricacidand
triglycerideshadvalueswithintherangeinbloodplasmaofmammals[5,6].

CONCLUSIONS

1. The values obtained for the main studied biochemical parameters in the
haemolymph of the healthy honeybees collected from honeybee colonies kept in natural
conditionsshowaslowlyvariableevolutioninthetwoconsecutiveyearsofexperiments.
2.Thevaluesrelatedtothelevelofglucoseandtotalproteinsarecomparablewith
thosereportedintheliterature.
3. The AST, ALT, GGT, CPK enzymes, glucose and ureea levels in haemolymph
registeredveryhighvaluescomparingwiththosefoundinthebloodplasmaofthemammals,
thecalcium,magnesium,uricacidandtriglyceridesvaluesareclosetothosefromtheblood
plasma and the creatinine values are 56 times higher than those detected in the blood
plasma.
4.Theparametersofthemineralmetabolismofhemolympharecharacterizedbylow
detectablevaluesforCaandsomeveryfluctuantvaluesforMg.
5.Theresultsofwetchemistryanalysisperformedonthetwogroupsofbeesshowed
significant changes in biochemical parameters of hemolymph concentration and enzymatic
activityundertheinfluenceofstress.
6. Metabolic profile investigation of bee families under stress compared with bees
families unstressed in terms Apiclimatron, based on the assessment of major changes in
biochemical parameters in lymph, shows increased enzyme activity, the amount of total

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Lucrritiinificevol53seriaMedicinVeterinar
calcium, creatinine and triglycerides, while decreasing total magnesium under conditions of
stress.
7. Assessment of metabolic profile of bees in stressful conditions shows the
importance of monitoring enzyme activity (alkaline phosphatase, aspartataminotransferaze,
gammaglutamiltranspeptidaze) and calcium and magnesium total concentration, creatinine
andtriglycerides,reducingthenumberofinvestigatedbiochemicalparameters

REFERENCES

1. Blatt,J.,F.RocesHaemolymphsugarslevelsinforaginghoneybees(A.m.carnica:dependence
on metabolic rate and in vivo measurement of maximal rates of trehalose synthesis). The
JournalofExperimentalBiology,2001,204,pp27092716.
2. Bozic,J.,J.WoodringEffectofactivityonthehaemolymphsugartitresinhoneybees.J.A.Res,
1997,36,pp3339.
3. Leta,M.,A.Gilbert,R.MorseLevelsofhaemolymphsugarsandbodyglycogenofhoneybees
(A.Mellifera)fromcoloniespreparingtoswarm,J.InsectPhisyol,1996,42,pp239245.
4. Milani, N. Propriet fisiche e composizione chimica dell'emolinfa in Apis mellifera L.: una
rassegnabibliografica.Apicoltura,1988,4.
5. Rdoi I., M. Cornil Principii generale de terapii alternative i complementare n medicina
veterinar.Ed.MaterialdidacticUSAMV,Bucureti,2001.
6. apcaliu Agripina, A. Siceanu, Cristina Mateescu, Eliza Cuia, Crengua Pavel, I. Rdoi, D.
Condur Seasonal variations of the main biochemical parameters of the hemolymph of Apis
melliferacarpathicainRomania,XLVNaukowaKonferrencjaPszczelarskaPulawy,2008,1112.

1174

THEEFFECTOFXAG2O(1X)[42B2O324CAO34P2O5]OXIDE
POWDERSONGRAMPOSITIVEBACTERIA

R.STEFAN,SilvanaPOPESCU,MarinaSPINU,
N.FIT,A.MACRI,G.NADAS
UniversityofAgriculturalScienceandVeterinaryMedicine3400,ClujNapoca,

GlassfromxAg2O(1x)[42B2O324CaO34P2O5]systemwithhighmolarsilveroxidecontent0x
11 mol%, has been obtained using undercooled method. Antibacterial properties against gram
positive bacteria were tested using diffusimetrical method for Staphylococcus aureus strains
ATCC 6538p, Arcanobacterium pyogenes, Bacillus cereus ATCC 14579. The greater effect was
observedforArcanobacteriumpyogene.
Antibacterial effect was dependent on Ag2O concentration into the glass matrix, evaluated by
measuringthediameterofinhibitionareafortheinvestigatedcompounds.
Keywords:silveroxide,glass,antibacterial.

INTRODUCTION

Theoxidicmaterialsthatcontainsilver[1]andcopper[2]havebeenusedsincethe
oldest times for the treatment of infections generated by bacteria and pathogen agents [3].
Thelongtimeinwhichthesilverwasusedforthetreatmentagainstbacterialedtotheidea
thatbacteriacannotadapttoitsaction,likemanytypesofantibioticsdo,developingspecific
resistance.Theusageonalargescaleofthistypeofmaterials,canbeachieveddependingon
theinteractionwiththebiologicalenvironmentinwhichthiswillbeinvestigatedandused.The
chemicaldurabilityleadstotheprojectionofabiodegradablematerialthatgraduallyreleases
theactivesubstance,whileamaterialwithhighchemicaldurabilitycanserveinimplantology,
havingactivesubstanceonlyonthesurface.
Recentresearch[4,5]showedthenecessitytoobtainglasswithcomponentsthatare
closest to those of the organism in order to be used in bone implants, or as mechanic
supportsonwhichbonecellstobeimplantedtodevelop.
The structural study of the boron based oxidic glass was widely investigated [6, 7]
anditwasshowedthatthestructureoftheboronbasedoxidicglassmaycontain[BO3]and
[BO4]unitsinterconnecteddependingonitscomposition.
Phosphorusoxideisacomponentthatformsbioactivestructures[8,9]andhasahigh
dissolutionrateifit isencountered intheorganism.Therewith, thebehaviorofthevitreous
componentsinbiologicalliquidsdependsonthechemicalcompositionandtheirstructure.
In the present work, we studied the antibacterial effect of the oxidic boron and
phosphorus based vitreous materials from the system xAg2O(1x)[42B2O324CaO34P2O5]
preparedwithahighcontentofsilverinthesamples(x=11mol%).

MATERIALSANDMETHODS

The oxidic glasses from the xAg2O (1x)[42B2O324CaO34P2O5] system were obtained
using as start materials P2O5, H3BO3, CaCO3, Ag2O with a chemical purity of 99+. The oxides
were mixed in appropriate proportions, homogenized and melted at 1230 C, founded and
pressedbetweenstainlesssteelplates.
For the fitting the experimental points the Origin v8.1. software was used. The
vitreous character of the samples was tested though diffraction of Xrays using a

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Lucrritiinificevol53seriaMedicinVeterinar
diffractometer. Detailed experimental descriptions should be restricted to X ray powder
diffraction data were obtained with Bruker D8 Advanced Diffractometer equipped with Si
monochromator.
The antibacterial efficiency of the studied compounds was tested on gram positive
germs (standard germs Staphylococcus aureus ATCC 6538p, Arcanobacterium pyogenes,
BacilluscereusATCC14579)throughthemethodofdiffusionin2%agargel.Theagarporedon
aPetriplateswithadiameterof12cm,wassownwiththebacterialinoculum,preparedfrom
the24hourcultureontheagarMuellerHinton,dilutedinphysiologicalserumatthedensity
of tube 0.5 on Mc Farland scale. Immediately after sowing wellswitha diameter of4,5 mm
weremade,inwhichthecompoundswereplaced,inaconcentrationof15%,inaquantityof
50 l/well. Each compound was double tested. The diameters of the inhibition areas were
readafteranincubationof24hoursat37C.

RESULTSANDDISCUSSION

The antibacterial effect of the oxidic glass in the xAg2O(1x)[42B2O324CaO34P2O5]


with0x7vitreoussystemwasevaluatedbymeasurementofthediameteroftheinhibition
area,notedd,ofthebacterialgrowthinthecaseofStaphylococcusaureusATCC6538p(Fig.
1).Theradiusoftheinhibitionzonegrowsmonotonouslyoncewiththegrowthofthemolar
contentofdeAg2Ointhevitreousmatrix.ThesamplesdopedwithAg2Oantibacterialeffect
dependantofthe molarconcentrationofthemetalinthematrixeffectalsoobtainedinthe
caseofotherboronbasedoxidicsystemsdopedwithsilverions.[10].Thus,theinhibitionzone
iscontainedintheintervald[16;21]mm.Thesamplethatdoesnotcontainsilver(x=0)also
induces an inhibitive effect and the dependence of the effect on the silver oxide molar
compositionisobviousstartingwithx=5mol%.

Figure1.ThedependenceoftheradiusoftheinhibitionzoneoftheStaphylococcus
aureusATCC6538pstrainontheA2OmolarcontentinthecaseofthexAg2O(1
x)[42B2O324CaO34P2O5]vitreoussystem.

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The Arcano Bacterium Pyogenes inhibition diameter (Fig. 2) depends on the molar
concentrationofAg2OofthevitreoussamplesofthexAg2O(1x)[42B2O324CaO34P2O5]system.
Forthe0x5compositionalleveltherearenomodificationsinthesizeoftheinhibitionzone,
thesampleswithsilverexibitingthesameantibacterialeffectasthosewithoutsilvercontent
(x=0),theeffectisduemore totheexistenceofthepowderandthemechanicalinteraction
betweentheseandtheinoculatedstrains.

Figure2.ThediameteroftheinhibitionzonevsAg2OconcentrationfortheArcano
BacteriumPyogenesstrain.

For x>5, the effect is exponential and the silver excess from the surface of the
material propagates through the nutritive substratum, inhibiting the most intensely and
rapidlythegrowthofthistypeofbacteriumofallthegrampositiveinvestigatedbacteria.The
inhibitionprocessisnotsaturatedforthex=11mol%Ag2Oinvitreousmatrixeither.
Figure3illustratesthemannerinwhichtheBacilluscereusstraininteractswiththe
vitreouspowderofthexAg2O(1x)[42B2O324CaO34P2O5]system.

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Figure3ThemodificationoftheinhibitiondiameterofthegrowthofBacilluscereus
dependingonthemolarcompositionofthexAg2O(1x)[42B2O324CaO34P2O5]system.

The antibacterial effect depends on the silver oxide molar content in the 1x6
compositionaldomain. For x7 the environment is saturated in Ag+ ions and a more intense
effectisnotproduced.
From the point of view of the inhibitive behavior Bacillus cereus behaves like
Staphiloccocus Aureus at the interaction with oxidic vitreous powders, the diameter of
saturation being dmax=21 mm. For x=7 mol% the process is saturated, the inhibition zone
remainingconstantlyforhighermolarconcentrationsofsilveroxide.

CONCLUSIONS
1. The antibacterial effect is strong, depending on the concentration of Ag2O in the
structureoftheinvestigatedstartingwithx5mol%.
2. Thesampleswhichdoesnotcontainsilverhaveaninhibitiveeffectdueespeciallyto
themechanicalinteractionfromtheseandthestudiedstrains.
3. Arcano Bacterium Pyogenes is the most sensitive at the action of the investigated
vitreoussystem.

ACKNOWLEDGEMENTS
This work was supported by CNCSIS UEFISCSU, project number 1117 PNII IDEI code
2528/2008

REFERENCES
1. Liu,H.,etal.,Agdopedantibacterialporousmaterialswithslowreleaseofsilverions
JournalofNonCrystallineSolids,2008.354:p.13141317.
2. Pickup, D.M., et al., Xray absorption spectroscopy and highenergy XRD study of the local
environmentofcooperinantibacterialcopperreleasingdegradablephosphateglasses.Journal
ofNonCrystallineSolids,2006.352:p.30803087.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
3. Rai, M., A. Yadav, and A. Gade, Silver nanoparticles as a new generation of antimicrobials.
BiotechnologyAdvances,2009.27:p.7683.
4. Kasuga, T., Bioactive calcium pyrophosphate glasses and glassceramics. Acta Biomaterialia,
2005.1:p.5564.
5. Silva,G.A.,etal.,SolublestarchandcompositestarchBioactiveGlass45S5Sparticles:Synthesis,
bioactivity, and interaction with rat bone marrow cells. Materials Science and Engineering C
2005.25:p.237246.
6. Chryssikos, G.D. and E.I. Kamitsos. Borate structures by vibrational spectroscopy. in Borate
Glasses,Crystals&Melts.1997:TheSocietyofglassTechbnologySheffield.
7. Osipov, A.A. and L.M. Osipova, Structure of Glasses and Melts in the Na2OB2O3 System from
HighTemperature Raman Spectroscopic Data: I. Influence of Temperature on the Local
StructureofGlassesandMelts.GlassPhysicsandChemistry,2009.35:p.121131.
8. Rainer,A.,etal.,Fabricationofbioactiveglassceramicfoamsmimickinghumanboneportions
forregenerativemedicine.ActaBiomaterialia,2008.4:p.362369.
9. ubik, J., et al., Structure and properties of MnO3containing zinc borophosphate glasses.
JournalofNonCrystallineSolids,2009.355:p.970975.
10. Simon,V.,M.Spinu,andR.Stefan,Structureanddissolutioninvestigationofcalciumbismuth
borateglassesandvitroceramicscontainingsilver.J.MaterSci:MaterMed,2007.18:p.507
512.

1179

THEANTIBACTERIALEFFECTOFSILVERCONTAININGB2O3CAO
P2O5GLASMATRIXONGRAMNEGATIVEBACTERIA

R.STEFAN,MarinaSPINU,SilvanaPOPESCU,
N.FIT,MariaBINDEA,FloreCHIRILA
UniversityofAgriculturalScienceandVeterinaryMedicine
3400ClujNapoca,CaleaManastur,35,Romania

Abstract
Antibacterial effect of vitreous samples from B2O3CaOP2O5 glass matix doped with silver ions
wasmeasuredbythediffusimetricalmethodinagarinoculatedwithgramnegativespecies.
Escherichia coli ATCC 10536, Salmonella typhymurium, Klebsiella pneumoniae bacteria were
investigated, the antibacterial effect being present on every investigated strain. The greater
effectwasmeasuredforSalmonellatyphymurium.
Keywords:gramnegative,antibacterialefect,silverglass

INTRODUCTION
Thenecessitytoobtainoxidicmaterialsbasedonphosphorusinordertousethemin
medical applications (implants, structures for cellular growth, etc.) appeared along with the
biocompatibilitytests[1,2],whichshowedthatasusefulasabiomaterialmightbe,themain
conditionsisnottoberejectedbytheorganismasaforeignbody.
TheP2O5 basedglasswaswidelyinvestigatedinthelastyears[3,4]andtherewere
highlighted specific relations between the internal structure and the behavior in biological
liquids.Thecalciumoxidewasintroducedintheinitialcompositionoftheglassesinorderto
realize the bioactivity of the material in relation with the bone tissue. The molar relation
between the two oxides influences the physical properties among which the chemical
durabilityinsuchawaythatthiscanbeusedinimplantologyaswellasbiodegradablestorage
foractivesubstances[5,6].Inthecaseoftheuseofimplantologyorinexternaluseinorderto
avoid the apparition of recurrent infections, it had been attempted the introduction of ions
with antibacterial properties in the first case on the surface through the method of ionic
changeandinthesecondinthestructurethroughthesolgelmethodofundercoolingofthe
melting
InthepresentworktheantibacterialeffectoftheB2O3CaOP2O5 matrixdopedwith
Ag2Owasstudiedpreparedthroughthemethodofundercoolingofthemelting,inthecaseof
gramnegativebacteria.

MATERIALSANDMETHODS

The oxidic glasses from the xAg2O(1x)[42B2O324CaO34P2O5] system were obtained


using as start materials P2O5, H3BO3, CaCO3, Ag2O with a chemical purity of 99+. The oxides
were mixed in appropriate proportions, homogenized and melted at 1230 C, founded and
pressedbetweenstainlesssteelplates.
For the fitting the experimental points the Origin v8.1. software was used. The
vitreous character of the samples was tested though diffraction of Xrays using a
diffractometer. Detailed experimental descriptions should be restricted to X ray powder

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diffraction data were obtained with Bruker D8 Advanced Diffractometer equipped with Si
monochromator.
The antibacterial efficiency of the studied compounds was tested on gram positive
germs(standardgerms
Escherichia coli ATCC 10536, Salmonella typhymurium, Klebsiella pneumoniae) through the
methodofdiffusionin2%agargel.TheagarporedonaPetriplateswithadiameterof12cm,
wassownwiththebacterialinoculum,preparedfromthe24hourcultureontheagarMueller
Hinton, diluted in physiological serum at the density of tube 0.5 on Mc Farland scale.
Immediately after sowing wells with a diameter of 4,5 mm were made, in which the
compounds were placed, in a concentration of 15%, in a quantity of 50 l/well. Each
compound was double tested. The diameters of the inhibition areas were read after an
incubationof24hoursat37C.

RESULTSANDDISCUSSION

InthecaseofEscherichiacoliATCC10536mthediameteroftheinhibitionzonehas
thevaluecontainedintheintervald[16,19]mm(Fig.1)dependingonthemolarcontentof
thesilveroxideinthestructureofthesamples.Thisbacteriumissensitivetotheactionofthe
silver ions in the structure of the samples. [79]. The samples which does not contain any
Ag2O,inhibitbacterialgrowthaswellasthesamplewithalowconcentrationofsilver(x=0,5
mol%).For0,5x4mol%theareaofinhibitionincreasesanddependsonthequantityof
Ag+ availableonthesurfaceoftheglassthatisreleasedinthedissolutionenvironment.When
x5mol%thegrowthenvironmentissaturatedwithsilverionsandnomatterhowmuchthe
molarsaturationwithsilveroxidegrowstheprocesssaturates.Forthistypeofstrainitcanbe
statedthat16mmrepresentsthediffusionareaoftheproperpowders.

Fig.1Theinhibitiondiameter(d)vstheAg2OmolarconcentrationintheB2O3CaO
P2O5matrix.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Salmonella typhymurium is most strongly inhibited by the investigated vitreous
materials(Fig.2).Inthe1x5compositionaldomainhereisnoincreaseoftheinhibitionarea,
thequantityofsilverbeingnecessarybutnotenoughinordertomanifestamoreextended
bacteriostaticeffect.Forx>5itcanbeobservedanincreaseoftheinhibitionareaalongwith
themolarcontentofAg2Ointhesamplesalineisdrawnonlyinordertohighlight,thepoints
beinglinearlyfitted.Thisstrainisthemostsensitiveontheactionofthesesamples.

Fig.2Thedependenceofthediameterofinhibitiononthemolarcontentofsilveroxidefrom
theB2O3CaOP2O5matrix.

Theleast sensitive gramnegativestraintothe actionofthevitreous samplesfrom


the B2O3CaOP2O5 matrix doped with silver ions is Klebsiella pneumoniaae (Fig. 3), whose
developmentisinhibitedonlyonanareawiththediameterofdmax=17mm.

Fig.3Thedependencebetweeninhibitiondiameterandmolarcontentofsilveroxide
forKlebsiellapneumoniaaestrain.

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Theminimumrateofinhibitionisgivenbythesamplewhichdoesnotcontainsilver
and it can be supposed that this is due to the diffusion of the sample in the nutritive
environmentofthebacterium.Forx5ml%theseisnodependenceoftheinhibitionareaon
the molar Ag2O content all the samples from this compositional area inhibiting equally the
developmentofthebacteriumculture.

CONCLUSIONS
1. Therewerepreparedvitreousstructureswithhighconcentrationofsilveroxidefrom
theAg2OB2O3CaOP2O5systemthroughthemethodofundercoolingofthemelt.
2. Theantibacterialeffectisdependentonthesilverconcentrationfromacertainmolar
concentrationforeachgramnegativestrainseparately.
3. ThemostsensitivestrainisSalmonellatyphymurium.

ACKNOWLEDGEMENTS
This work was supported by CNCSIS UEFISCSU, project number 1117 PNII IDEI code
2528/2008

REFERENCES
1. Ahmed,I.,etal.,Processing,characterisationandbiocompatibilityofironphosphateglassfibresfor
tissueengineering.Biomaterials,2004.25:p.32233232.
2. Fathi,M.H.andA.D.Mohammadi,Preparationandcharacterizationofsolgelbioactiveglass
coatingforimprovementofbiocompatibilityofhumanbodyimplant.MaterialsScienceand
EngineeringA,2008.474:p.128133.
3. Brow,R.K.,Review:thestructureofsimplephosphateglasses.JournalofNonCrystallineSolids,
2000.263&264:p.128.
4. Metwalli,E.,etal.,Propertiesandstructureofcopperultraphosphateglasses.JournalofNon
CrystallineSolids,2004.344:p.128134.
5. Zhao,D.,etal.,Mechanicalverificationofsofttissueattachmentonbioactiveglassesandtitanium
implants.ActaBiomaterialia,2008.4:p.11181122.
6. Hench,L.L.,Geneticdesignofbioactiveglass.JournaloftheEuropeanCeramicSociety,2009.29:p.
12571265.
7. Liu,H.,etal.,AgdopedantibacterialporousmaterialswithslowreleaseofsilverionsJournalof
NonCrystallineSolids,2008.354:p.13141317.
8. Simon,V.,M.Spinu,andR.Stefan,Structureanddissolutioninvestigationofcalciumbismuth
borateglassesandvitroceramicscontainingsilver.J.MaterSci:MaterMed,2007.18:p.507512.
9. I.SondiandB.SalopekSondi,Silvernanoparticlesasantimicrobialagent:acasestudyonE.coliasa
modelforGramnegativebacteria.JournalofColloidandInterfaceScience,2004.275:p.177182.

1183


MANGANESEIMPACTONMALEREPRODUCTIVESYSTEM
INTEGRITYANDPERFORMANCESBIOMARKERS

NicoletaSimonaSteliac(Munteanu),AlexandraTrif
CaleaAraduluinr.119,cod:300645,Timisoara
simonasteliac@gmail.com

Abstract
The aim of this study was to determinate manganese impact on morphological (body, genital
organs, sexual accesory glands weight; arhitecture of testis, epididym, seminiferous tubuls)
markersandbiochemical(serictestosterone,LH,FSHlevel)andspermqualitybiomarkersbased
onrecentreferences.
The study pointed out the manganes impact on some male reproductive system integrity and
performancesbiomarkers:controversiallydataregardingtheimpactonbodyweight,decreasof
sizeandweightofsexualorgansandsomeaccesorysexualglands,severearchitecturechangesin
sexual organs (testes degeneration of germinal epithelium characterized by loss of developing
spermatozoaandspermatids,degenerationofSertolicellsandLeidigcells),necroticseminiferous
tubules,epididymis damages in epithelial cells of epididymis, tubules with sperms loss, and
sexual accesory glands (prostatis degeneration),controversially opinians regarding the imact on
sexual hormones, especially testosterone decrease of sperm quality (decrease of sperm count,
density, mobility, fertilizing capacity, abnormalities). Clinical observation of impotence, loss of
libidoinexposedworkersarementioned.
Keyword:manganese,reproductivetoxicity,male

INTRODUCTION

Theoriginofthenamemanganeseiscomplex.Inancienttimes,twoblackminerals
fromMagnesiainwhatisnowmodernGreecewerebothcalledmagnes,butwerethoughtto
differ in gender. In the 16th century, manganese dioxide was called manganesum (note the
twon'sinsteadofone)byglassmakers,possiblyasacorruptionoftwowordssincealchemists
and glassmakers eventually had to differentiate a magnesia negra (the black ore) from
magnesiaalba(awhiteore,alsofromMagnesia,alsousefulinglassmaking).MicheleMercati
called magnesia negra Manganesa, and finally the metal isolated from it became known as
manganese(German:Mangan).Thenamemagnesiaeventuallywasthenusedtoreferonlyto
the white magnesia alba (magnesium oxide), which provided the name magnesium for that
freeelement,whenitwaseventuallyisolated,muchlater.(2,19)
Manganese is a chemical element, designated by the symbol Mn. It has the atomic
number 25. It is found as a free element in nature (often in combination with iron), and in
manyminerals.Asafreeelement,manganeseisametalwithimportantindustrialmetalalloy
uses,particularlyinstainlesssteels(10).
Manganese phosphate is used as a treatment for rust and corrosion prevention on
steel. Depending on their oxidation state, manganese ions have various colors and are used
industriallyaspigments.Thepermanganatesofalkaliandalkalineearthmetalsarepowerful
oxidizers.Manganesedioxideisusedasthecathode(electronacceptor)materialinstandard
andalkalinedisposabledrycellsandbatteries(10).
Manganese(II) ions function as cofactors for a number of enzymes in higher
organisms,wheretheyareessentialindetoxificationofsuperoxidefreeradicals.Theelement

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isarequiredtracemineralforallknownlivingorganisms.Inlargeramounts,andapparently
with far greater activity by inhalation, manganese can cause a poisoning syndrome in
mammals,withneurologicaldamagewhichissometimesirreversible(16).
Themostcommonoxidationstatesofmanganeseare+2,+3,+4,+6and+7,though
oxidation states from 3 to +7 are observed. Mn2+ often competes with Mg2+ in biological
systems. Manganese compounds where manganese is in oxidation state +7, which are
restricted to the oxide Mn2O7 and compounds of the intensely purple permanganate anion
MnO4, are powerful oxidizing agents. Compounds with oxidation states +5 (blue) and +6
(green)arestrongoxidizingagentsandarevulnerabletodisproportionation(10).
Themoststableoxidationstateformanganeseis+2,whichhasapinktoredcolor,
and many manganese(II) compounds are known, such as manganese(II) sulfate (MnSO4) and
manganese(II)chloride(MnCl2).Thisoxidationstateisalsoseeninthemineralrhodochrosite,
(manganese(II) carbonate). The +2 oxidation state is the state used in living organisms for
essentialfunctions;otherstatesaretoxicforhumanbody(26).
The +3 oxidation state is known, in compounds such as manganese(III) acetate, but
thesearequitepowerfuloxidizingagentsandalsodisproportionateinsolutiontoMn(II)and
Mn(IV). Solid compounds of Mn(III) are characterized by its preference for distorted
octahedralcoordinationduetotheJahnTellereffectanditsstrongpurpleredcolor(26).
The oxidation state 5+ can be obtained if manganese dioxide is dissolved in molten
sodiumnitrite(26).Manganate(IV)saltscanalsobeproducedbydissolvingMncompoundsin
alkalinemeltsinair(18).
Permanganate(+7oxidationstate)compoundsarepurple,andcangiveglassaviolet
color. Potassium permanganate, sodium permanganate and barium permanganate are all
potent oxidizers. Potassium permanganate, also called Condy's crystals, is a commonly used
laboratoryreagentbecauseofitsoxidizingpropertiesandfindsuseasatopicalmedicine(for
example,inthetreatmentoffishdiseases).Solutionsofpotassiumpermanganatewereamong
the first stains and fixatives to be used in the preparation of biological cells and tissues for
electronmicroscopy(18).

Methylcyclopentadienyl manganese tricarbonyl is used as an additive in unleaded


gasoline to boost octane rating and reduce engine knocking. The manganese in thisunusual
organometalliccompoundisinthe+1oxidationstate(8,27).
Manganese(IV) oxide (manganese dioxide, MnO2) is used as a reagent in organic
chemistryfortheoxidationofbenzylicalcohols(i.e.adjacenttoanaromaticring).Manganese
dioxide has been used since antiquity to oxidatively neutralize the greenish tinge in glass
causedbytraceamountsofironcontamination(19).MnO2isalsousedinthemanufactureof
oxygen and chlorine, and in drying black paints. In some preparations it is a brown pigment
thatcanbeusedtomakepaintandisaconstituentofnaturalumber(6).
Manganese(IV)oxidewasusedintheoriginaltypeofdrycellbatteryasanelectron
acceptor from zinc, and is the blackish material found when opening carbonzinc type
flashlight cells. The manganese dioxide is reduced to the manganese oxidehydroxide
MnO(OH) during discharging, preventing the formation of hydrogen at the anode of the
battery(6).

MATERIALSANDMETHODS

Forwritingthispaperwereused27references.
Theaimofthisstudywastodeterminatemanganeseimpactonreproductivesystem
integrity and performances biomarkers. Biomarkers are tracers that give evidence of some

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UniversitateadetiineAgricoleiMedicinVeterinarIai
eventsindifferentsamplesorbiologicalsystems.Certainbiologicalbiomarkerswereusedto
localizeinmalesphysiopatologicalmodificationproducedbytoxicsubstances.Theinterestof
this study was focused onthe morphological biomarkersand biochemical and spermquality
biomarkers(20).
Manganese impact on morphological biomarkers: (body, genital organs, sexual
accesoryglandsweight;archiotectureoftestis,epididym,seminiferoustubuls).
Manganeseimpactonbodyweight;
Theopinionsonthesubjectarecontroversially:
Administrationofmanganese(II)tetraazamacrocycliccomplexesdidnotbringabout
anysignificantchangeinthebodyweightsofthetreatedrats(25).
InhalationexposuretoMnSO4 duringgestationandlactationdidnotaffectmaternal
body weight gain or terminal maternal body weight. Combined in utero and postnatal
inhalation exposure to MnSO4 did not affect neonatal body weight gain in either female or
malepups(7).
HighdoseMnSO4exposurewasasociatedwithdecreasedbodyweightinpups(7).
Mancozebafungicideofethylenebisdithiocarbamateorallyadministeredatdosesof
500,1.000and1.500mg/kgbodyweight/dayfor30,90,180and360daysdeterminedinboth,
treated animals and control ones, increase of body weight the, percent increase beeing in
order of 133>116>85>80 of rats exposed to Mancozeb (0, 500, 1.000, 1.500 mg/kg/d),
respectivelyincontrol(12).
Manganeseimpactongenitalorgansandsexualaccesoryglandsweight;
Administration of manganese(II) tetraaza macrocyclic complexes caused changes in
genitalorgansweight.Theweightsoftestes,epididymis,seminalvesicleandventralprostate
weredecreasedsignificantlyinalltheexperimentalgroupswhencomparedwiththevehicle
treatedcontrols(25).
Male mice exposed to elevated dietary manganese showed decreased size of the
testicles,seminalvesicles,andpreputialglands(9).
Theabsoluteweightoftestesand epididymisofanimalsexposed todifferentdoses
(500, 1.000 and 1.500 mg/kg/d) of Mancozeb did not show any change except for a mild
decreaseinepididymalweight,at1.000and1.500mg/kg/dfor360days.Therelativeweight
of testes and epididymis after the exposure to different doses of Mancozeb (500,1.000 and
1.500 mg/kg/d) during 3090 days did not indicate any appreciable change. Higher doses
(1.000 and 1.500 mg/kg/d) over a period of 180 and 360 days produced a slight increase in
relativeweightoftestesandadecreaseinepididymis(12).
Manganeseimpactontestis,epididym,seminiferoustubulsarhitecture;
Microscopic examination of testes and epididymis of rats exposed to Mancozeb at
doses 500 and 1.000 mg/kg/d over a period of 360 days did not exhibited any significant
pathomorphologicalchanges.However,testesofratsdosedwithMancozeb(1.500mg/kg/d)
produced time dependent pathological changes in seminiferous tubules. The observed
changesincludednecroticseminiferoustubuleswithsloughingofgerminalcells,formationof
giant cells and accumulation of debris matter into the lumen. The clubing of spermatogonia
andspermatocytesmayberesponsiblefortheformationofgiantcells.Similarly,ratsexposed
to high doses of Mancozeb has also showed damaged epithelial cells in the tubules of
epididymiswithlossofsperms(12).
Adultratsexposedtovaryingdoses:306,612,1225,and1838mgMn/kgdissolvedin
distilledwaterfor63days,presentedsegmentaldegenerationsofgerminalepitheliumwithin
seminiferoustubulesandalsotocharacterizelossofdevelopingspermatozoaandspermatids.
Histopathologicalexaminationconfirmedtesticulardegenerationintreatedratsof612,1225,

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and1838mgMn/kggroups.Themostprominentabnormalityobservedinmaleanimalswas
testicular degeneration. Affected animals has mild to moderate segmental degeneration of
germinal epithelium within seminiferous tubules. This change was characterized by loss of
developing spermatozoa and spermatids, vacuolation and swelling of Sertoli cells,
degeneration of Sertoli cells, and widening of the tubular luminal diameter. Binucleate and
multinucleate cells were observed within tubular lumina and degenerate epithelium. Many
degeneratedtubulesalsohadadecreasedcrosssectionaldiameter.Thebasementmembrane
ofaffectedtubulesappearedintact,butoccasionallyhadawavyappearance.Oftentubules
had relatively normal areas adjacent to affected areas. These changes, collectively, were
consistentwithsegmentaltubulardegenerations(22).
One rat in high dose group, 1838 mg Mn/kg, had a lesion consistent with a sperm
granuloma.Thismassconsistedofalargecollectionofspermatozoaencasedinathicksheath
of macrophages and multinucleate giant cells surrounded by several layers of dense fibrous
connective tissue. No significant lesions were observed in the coagulation glands, seminal
vesiclesorductusdeferens(22).
Katira et al. (13) reported disturbed spermatogenesis and histopathological
observationsrevealeddegenerationofseminiferoustubules,epididymisandprostatitis.
Chandra(3)reportedacellularchangeinducedbymanganeseintherattestis.Studies
onrabbitsinstillatedwithMnO2,demonstratedthatasinglehighdoseof158mg/kg,asMnO2
couldcauseseveredegenerativechangesintheseminiferoustubulesandleadtosterility.
Testicular damage was produced in rats by subcutaneous administration of
manganesechloride(3mg/kg)foraperiodofthreeweeks(5).
Singh et al. (24) reported that in rats manganese sulphate injection for 25 days
produced degeneration in seminiferous epithelium, depleted number of spermatids and no
spermatocytesintheseminiferoustubules.
Inhumans,reproductiveeffectssuchasimpotenceandlossoflibidohavebeennoted
inmaleworkersexposedtohighlevelsofmanganesebyinhalation(1).
Manganese impact on biochemical (seric testosterone, LH, FSH level) and sperm
qualitybiomarkers.
Prestifilippo et al. (23) described that MnCl2 can cause LHRH release from the MBH
(medialbasalhipotalamus)inadultmaleratsandthispeptidecontrolsthepituitarysecretion
ofLHandFSH.InordertodeterminetheinvivoeffectofMnCl2onplasmaLHlevels,ratswere
stereotaxically implanted with a cannula into the lateral cerebral ventricle and received a
single intracerebroventricularly (i.c.v.) injection of MnCl2 (10g/5l saline). Results pointed
outthatMnCl2increasedtheplasmaLHlevelsat30minpostinjection.Thehormoneremained
elevatedat60min,thendeclinedtopreinjectionlevelsat90minpostinjection.Controlrats
injectedwithsalinedidnotshowedachangeinLHsecretionatanyofthetimepoints.
The rats that were injected with saline or the 1.0g dose of MnCl2 exhibited no
changeinLHreleasedascomparedtotheirrespectivebasallevels.Theratsthatwereinjected
withthe2.5,10and25gdosesofMnCl2 exhibitedthree,sixandsevenfoldincreasesinLH
release,respectively,comparedwiththeirbasallevels(9).
Serumgonadotropinlevelswerenotalteredat48or55daysinanimalsthatreceived
the 10 mg/kg supplemental dose of MnCl2 . The 25 mg/kg dose caused nonsignificant
elevations in LH, FSH, testosteron and spermatogenesis by 48 days. Supplementation of
animalswiththe25mg/kgdoseofMnuntil55daysofageproducedincreasedlevelsofmean
serumLH,FSHandtestosteronecomparedwiththecontrolanimals(17).
Many studies on experimental animals have reported strong evidence of harmful
effectsofexposuretomanganeseonmalefertility(11).Manganeseeffectsonfertility,serum

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UniversitateadetiineAgricoleiMedicinVeterinarIai
concentrationsoffolliclstimulatinghormone(FSH)andtestosterone,morphologicalindicesof
reproductivedevelopment,andbehaviorhavebeennotedinseveralspecies(9).Experimental
results in animals are consistent with the clinical observations of impotence and reduced
excretion of 17ketosteroids (as consequence of decreased testosterone production) in
humansfollowingexposuretoMndust(5).
Laskeyetal.(14)reportedthattherewasareductionintheserumtestosteroneof
theanimals,butnoindicationofhypothalamicorpituitarymalfunctioninLongEvanratpups
that were dosed orally with Mn3O4 for 21 days. The decreased testosterone secretion may
leadtodelayedgrowthandmaturationoftestesandotherreproductivetissues.Theseresults
suggest that the site of manganese damage that causes depression of sustained serum
testosteroneconcentrationisinthetesticularLeydigcell.
Testosteronelevelswerereducedinmaleratsgivenanoraldoseof13mgMn/kg/day
for100224days(15).
InSDratsthatweretreatedwithMnCl2atdoses7.5,15,30mg/kg/dfor40days,the
serumtestosteroneconcentrationwasdecreasedandalterationsofLeydigcellmitochondria,
suchasmitochondrialswellinganddisruptionofintramitochondrialcristaewerealsoobserved
(4).
A significant decline in the sperm motility in cauda epididymis was noticed in rats
treated with manganese (II) tetraaza macrocyclic complexes . Sperm density in testes and
cauda epididymis were also reduced. Sperm motility and fertilizing capacity of spermatozoa
were affected severely, which could be due to the androgen deficiency. Manganese(II)
tetraaza macrocyclic complexes cause reduction in the weight by the accessory sex organs
whichindicatestheatrophyofglandulartissue
andalsoreductioninthesecretionability.Thusreflectingthedecreasedlevelsoftestosterone
(25).
Abnormal sperm morphology was observed in mice treated with 23198 mg
manganese/kg body weight per day as potassium permanganate or manganese sulfate by
gavageinwaterforupto3weeks(21).

REFERENCES

1.
2.
3.
4.

5.
6.
7.

Agency for Toxic Substances and Disease Registry (ATSDR) (1997) Toxicological profile
manganese,CRCPressInc,BocaRaton,FL.P2526
Calvert,
J.B.
(2003)

Chromium
and
Manganese.
http://www.du.edu/~jcalvert/phys/chromang.htmRetrieved20090430.
Chandra, S.V. (1971) Cellular changes induced by manganese in the rat testispreliminary
results,Acta.Pharmacol.Toxicol.29:7580
Cheng,J.,Fu,J.L.,Zhou,Z.C.(2003)Theinhibitoryeffectsofmanganeseofseroidogenesisin
ratprimaryLeydigcellsbydistruptingsteroidogenicacuteregulatory(StAR)proteinexpression,
Toxicology187,139148(accesat26.02.2010)
Cheng, J., Fu, J.L., Zhou, Z.C. (2005) The mechanism of manganeseinduced inhibition of
steroidogenesisinratprimaryLeydigcells,Toxicology211,111,(accesat26.02.2010)
Dell,R.M.(2000)Batteriesfiftyyearsofmaterialsdevelopment.SolidStateIonics134:139
158.
Dorman, D.C., McElveen, A.M., Marshall, M.W., Parkinson, C.U., James, R.A., Struve, M.F.,
Wong, B.A. (2005) Tissue manganese concentrations in lactating rats and their offspring
followingcombinedinuteroandlactationexposuretoinhaledmanganesesulfate,Toxicological
Sciences84,1221

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Lucrritiinificevol53seriaMedicinVeterinar
8.

9.

10.
11.
12.
13.
14.

15.

16.
17.

18.
19.
20.
21.

22.

23.

24.

25.

26.
27.

Graham,L.A.(2005)Manganese(I)poly(mercaptoimidazolyl)boratecomplexes:spectroscopic
andstructuralcharacterizationofMnHBinteractionsinsolutionandinthesolidstate.Dalton
Trans:171180.
Gray, L.E. Jr, Laskey, J.W. (1980) Multivariate analysis of the effects manganese on the
reproductive phisiology and behavior of the male house mouse, J. Toxicol. Environ. Health
6:86186
Holleman, A. F., Wiberg, E., Wiberg, N. (1985) Mangan, (in German). Lehrbuch der
AnorganischenChemie(91100ed.).WalterdeGruyter.pp.11101117.
Imam, Z., Chandra, V.S. (1975) Histochemical alterations in rabbit testis produced by
manganesechloride,Toxicologyandappliedpharmacology,32:534544,(accesat10.03.2010)
Kackar, R., Srivastava, M.K., Raizada, R.B. (1997) Induction of gonadal toxicity to male rats
afterchronicexposuretomancozeb,IndustrialHealth,35,104111
Katira,V.,Bawa,P.(1993)Histopathologicalchangesinducedbymanganeseintheratstestes,
Uttar.Pradesh.J.ofZool.13:6062
Laskey,J.W.,Georgia,L.,RehnbergG.L.,Hein,F.J.,Laws,S.C.(1985)Assessmentofthemale
reproductive system in the preweanling rat following (Mn3O3) exposure, J. Toxicol Environ.
Health15:339350
Laskey, J.W., Rehnberg, G.L., Hein, J.F., Carter, S.D. (1982) Effects of chronic manganese
(Mn3O4)exposuronselectedreproductiveparametersinrats,J.Toxicol.Environ.Health8:677
687
Law, N., Caudle, M, Pecoraro, V (1998) Manganese Redox Enzymes and Model Systems:
Properties,Structures,andReactivity.46.p.305.
Lee, B., Pine, M., Johnson, L., Rettori, V., Hiney, J.K., Dees, W.L. (2006) Manganese acts
centrallytoactivatereproductivehormonesecretionandpubertaldevelopmentinmalesrats,
ReproductiveToxicology,22,580585,(accesat26.02.2010)
Luft,J.H.(1956)JournalofBiophysicalandBiochemicalCytology2(6):799802.
Mccray, W. P. (1998) Glassmaking in renaissance Italy: The innovation of venetian cristallo.
JournaloftheMinerals,MetalsandMaterialsSociety50:14.
National Research Council (1989) Biologic Markers in Reproductive Toxicology, National
AcademyPress,Washington,D.C.
NTP (1993) Toxicology and carcinogenesis studies of manganese (II) sulfate monohydrate
(CAS No. 10034965) in F344/N rats and B6C3F1 mice (feed studies), Research Triangle Park,
NC,NationalToxicologyProgram(NTPTechnicalReportSeriesNo.428),(accesat10.03.2010)
Ponnapakkam,T.P.,Sam,G.H.,Iszard,M.B.(2003)Histopathologicalchangesinthetestisof
the Sprague Dawley Rat following orally administered manganese, Bull. Environ. Contam.
Toxicol.71:11511157
Prestifilippo,J.P.,FernandezSIolari,J.,DeLaurentiis,A.,Mohn,C.E.,DeLaCal,C.,Reynoso,R.,
Les Dees, W., Retori, V. (2008) Acute effect of manganese on hypothalamic luteinizing
hormone releasing hormone secretion in adult male rats: Involvement of specific
neurotransmittersystems,Toxicologicalsciences105(2),295302
Singh, J., Husain, R., Tandon, S.K., Seth, P.K., Chandra, S.V. (1974) Biochemical and
histopathologicalalterationsinearlymanganesetoxicityinrats,EnvironPhysiolBiochem4:16
23
Singh, R.V., Chaudhary, A. (2004) Biologically relevant tetraazamacrocyclic complexes of
manganese: synthetic, spectral, antimicrobial, antifertility and antiinflammatory approach,
JournalofInorganicBiochemistry98,17121721,(accesat26.02.2010)
Temple, R. B., Thickett, G. W. (1972) Australian Journal of Chemistry 25: 55.
http://www.publish.csiro.au/?act=view_file&file_id=CH9720655.pdf.
WHO (1981) Manganese. Environmental Health Criteria 17, World Health Organisation,
Geneva

1189


ANATOMOPATHOLOGICALANDEPIDEMIOLOGICALSTUDYOF
VISCERALANDNONVISCERALHEMANGIOSARCOMAINDOGS

TBRANA.F.,C.CTOI,A.GAL,P.BOLF,M.TAULESCU,A.L.NAGY,COSMINACUC,G.
BORZA,R.MOUSSA
UniversityofAgriculturalSciencesandVeterinaryMedicine,DepartmentofPathology,35
MnturStreet,400372,ClujNapoca,Romania
email:flaviutabaran@gmail.com

Summary
From two hundred and thirty two canine tumors that were necropsic and histolopatologic
examined during the 3year period from May 2007 through April 2010 twelve cases of
hemangiosarcoma were diagnosed. Visceral types of hemangiosarcoma stand for up to
approximately 3% of the specimens submitted for histologic examination and the nonvisceral
hemangiosarcomafor2%.Theaverageageatthetimeofthediagnosisofhemangiosarcomawas
9 years. The primary tumors were found to be developed mainly in the spleen, with frequent
metastasis in liver and omentum. In 1/3 cases the tumors were widespread, with multiple
"cannonball"likemetastasesinliver,lungs,omentum,andmesentery,intwocasesinheartand
in one case in the kidney. The commonly affected breeds were the large ones, especially the
Shepherd and Rottweiler breeds. The tumors were equally distributed at both sexes, the
hemangiosarcomaprovingtohavenosexpredilectionintheoccurrence.

Keywords:Caninehemangiosarcoma;angiosarcoma,doghemangioendothelioma

INTRODUCTION

The hemangiosarcoma (angiosarcoma, malignant hemangioendothelioma) is the


malignant tumor of the endothelial cells, accounting for up to 7% of all dog tumors in
publishedreports(Smith2003,Baba2007).Thehemangiosarcomaoccursinfrequentlyincats,
horses,cows,andsheep(Jubb2007).Theontogenyofthemalignantcellsisthemultipotential
bone marrowderived stem cells, precursors with potential for endothelial differentiation
(AngelaR.2006).Thehemangiosarcomacanariseasaprimarytumoranywhereinthebody,
but the spleen, skin, right atrium, lungs and liver are more frequent primary sites for the
tumoraldevelopment(Brown1985,FunIn2001,Robinson,1993).
Thecaninecutaneoushemangiosarcomasareclinicallystagedaccordingtothedepth
of the involvement, in stage I the tumors being limited at the dermis layer, in stage II the
tumorsextendingthemselvesinthesubcutaneoustissue,andinstageIIIthetumoralprocess
involving the underlying muscle tissue being typically large, poorly circumscribed, with an
appearancewhichcanbemistakenforahematoma(WithrowandMacewen,2007).
The spreading from the primary sites may occur via metastasis, local invasion and
transcavitary implantation (Oksanen 1978). The average age in clinical presentation is 11
years, and at the time of the examination80% of the subjects had metastasis in thedistant
organs such as liver and lung (Gabor 2006). The hemangiosarcoma tends rapidly to
metastasize through the bloodstream, since the tumor cells have an easy access to the
vascular channels, and it presents a markedly poor prognosis even if intensive treatment is
provided(Fujita2008,Gulbahar,1997).Whenmetastasesarewidespread;theprimarytumor
sitemaybedifficulttobedetermined,themulticentricoriginofthetumorbeingapossibility.

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By immunohistochemical staining for factor VIIIrelated antigen and CD31 (marker of
endothelialcells),poorlydifferentiatedhemangiosarcomasmaybedifferentiatedfromspindle
cellsarcomasorothernonendothelialneoplasms(Withrow2007).

MATERIALSANDMETHODS

Fromthetwohundredandthirtytwocaninetumorsthatwerebroughtbeggingfrom
May2007untilApril2010attheDepartmentofAnatomicPathology,NecropsyandForensic
MedicinefromtheFacultyofVeterinaryMedicineClujNapocaforexaminationwedetermined
the total number of hemangiosarcoma cases. The biopsy and the necropsy specimens were
fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 57 m with a
microtomeLeicaRM2125RT,andstainedroutinelybyHematoxylinEosin(HE)andMasson's
trichrome (TM) methods. For the cytologic examination we used the Diff Quick panoptic
staining protocol. The slides were examined using the Olympus BX 51microscope and the
images were taken with the Olympus SP 350 digital camera and processed by a special
acquisitionandimageprocessingprogram,OlympusCellB.
After histopathological diagnosis, the number of diagnosticated cases were
statistically compared to the total amount of canine tumors examined during this 3 years
period. Then, the age, sex, breed and location of the primary and metastatic tumors were
analyzed,submittedtostatisticalinterpretationandafterwardsthenumberswerecorrelated
withthenecropsyandhistologicalfindings.

RESULTSANDDISCUSSION

Pathologicalfindings
Themaingrossfindingsofthehemangiosarcomasfoundsecondarytothenecropsic
examinationwere:softconsistence,friable,easilyremovable,reddishblacktoblackincolor,
resembling sometimes in a grade to the aspect and consistence of an organized hematoma.
The shape of these tumors was in most cases round, with the diametric size ranging from a
coupleofmillimeterstoextremesizesof1520cm.
Onthecutsurface,thetumorscharacteristicsvary,fromasarcomatouswithyellowishaspect
to reddish black to black color. In some cases, especially in large tumors necrotic and
hemorrhagicareaswereseen.
The primary localization of the visceral hemangiosarcoma was the spleen, the
hemangiosarcomabeinginourcasesthemostfrequentcauseoftumoralenlargementofthis
organ.Metastatictumor masswith"cannonball"likeaspectswerescatteredthroughoutthe
abdominalcavity,mainlyintheliver,ontheparietalandvisceralperitonealserosa(omentum)
anddiaphragm.
In the cases of widespread hemangiosarcoma the metastases were encountered in
the liver, omentum, pleura, lungs and in two cases in the heart. In one case, the metastasis
from the primary splenic hemangiosarcoma was found in the scapular region, with the
infiltrationofthesubcutaneoustissue,regionalmuscleandscapula,followedbydiffusebone
destruction.
Thefivecasesofprimarycutaneous(nonvisceral)hemangiosarcomas,werelocalized,
inthefirstcaseinthemammaryglandofa10yearGermanshepherdbitchand,inthesecond
oneinvolvingboththesoftandosseoustissuesina9yearGreatDanemale.Theotherthree
localizationswereinonecasetheprescapularregionandthesofttissuesfromthehindlimbs
intheothertwocases.

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Fig.1Multiplepulmonaryhemangiosarcoma
metastasisina6yearoldDalmatianfemale.

Fig2Cardiacmetastasisintheleftatrium

Fig.3Hepaticmetastasisofaprimarysplenic Fig.4Hemangiosarcomamultiplemetastatic
hemangiosarcomaina10years,common
tumorina10years,commonbreedmale
breedmale

These five hemangiosarcomas were clinically staged according to the depth of


involvementlikestageIItumorsinthemammary,prescapularandhindlimblocalizationsand
stage III in the facial localization because of the involvement of the underlying muscle and
bonetissue.

Microscopicexamination

Histological, analyzed hemangiosarcomas consist in vascular spaces delimitated by


anaplastic, elongated, endothelial cells. The vascular spaces were delimitated by single or
multiplelayersofcellsand containingerythrocytesin variablenumbersorthrombi.Insome
cases, endothelial cells formed solid areas or vascular spaces and cavernous channels
supportedbydiscretecollagenfibersthatweredetectedthroughMassonstrichromestaining.
The neoplastic endothelial cells were pleomorphic with variation in the nuclear diameter,
chromatin characteristics, and mitotic index. Hemorrhage and lymphocytic infiltration were
foundinsomecases.

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Fig.5,Cytologicexaminationofan
Fig.6.Cytologyofhemangiosarcoma; the
hemangiosarcoma;spindleandplumpshaped smearcontainspleomorphicneoplasticcells;ob
cells;obx100,DiffQuickpanopticstain
x100,DiffQuickpanoptic

Thesolidareasofsometumorsshowedsmallsizes,vascularspaceslinedbyasingle
layer of pleomorphic plump, shaped young endothelial cells with hyperchromatic nuclei and
pale, abundant cytoplasm that formed irregular vascular spaces. Tumors were considered
poorly differentiated, when the cells were pleomorphic forming solid sheets rather than
channels.

Fig.7.Hemangiosarcomaina10yearCocker
Spanielfemale;vascularspacesdelimitated
byanaplastic,elongated,endothelialcells
HE,obx40

Fig.8Hemangiosarcomavascularspaces
delimitatedbypleomorphic,elongatedand
plumpendothelialcellsHE,obx40

Fig.11Hemangiosarcoma,proliferationof
vascularstructuresdelimitatedby
pleomorphic,plumptumoralendothelial
cells;TMobx100

Fig.12Hemangiosarcoma;tumoral
structuresdelimitatedbypolymorphic,
elongatedandplumpendothelialcells;TM
obx100

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Cytological examination of tumoral samples reported spindle shaped cells,


sometimes quite plump. The nuclei usually contain prominent, often multiple nucleoli. The
cytoplasmislight,basophilicandsometimesvacuolated.

STATISTICALANALYZE

The age at the time of the hemangiosarcoma diagnosis, ranged from 6 to 15 years
withanvisceraltypeofhemangiosarcomawhichwasdiagnosticatedin3%ofalltumors,and
a reduced incidence of nonvisceral hemangiomas represented in approximately 2% of the
examined tumors. Three from ten hemangiosarcomas were highly metastatic, with multiple
metastasesinliver,lungs,omentum,heart(intwocases)andkidneyinonecase.

CONCLUSIONS
1. Theoccurrenceofhemangiosarcomaisagerelated,affectingespeciallythemiddle
agedandoldagedanimals.
2. In dogs, the visceral types of hemangiosarcoma are more frequent than
nonvisceral(coetaneous)ones.
3. Thehemangiosarcomaisthemostfrequentcauseoftumoralenlargementofthe
spleen.
4. Thehemangiosarcomahasnosexpredilectionintheoccurrence.
5. The frequently affected breeds are the large ones, especially the Shepherd and
Rottweilerbreeds.
6. The majority of hemangiosarcomas produce metastasis, the most frequent areas
beingtheliver,mesentery,omentumandlungs.

REFERENCES

Angela R., L.Kozickia, K. Helmb, M. Cristan, K Jubalaa, C. Cutterc, and F. Jaime. Modiano, 2006,
Caninehemangiosarcomaoriginatesfromhematopoieticprecursorswithpotentialforendothelial
differentiation,ExperimentalHematology,34,870878.
2. BabaA.I.,C.Catoi,2007,ComparativeOncology,Bucharest:ThePublishingHouseoftheRomanian
Academy,pag.228.
3. Brown, N.O., A.K. Pataik,. and E.G. MacEwen, 1985, Canine hemangiosarcoma: Retrospective
analysisof104cases,JournaloftheAmericanVeterinaryMedicalAssociation,
4. Fujita M., Y Takaishi., D. Yasuda, D. Hasegawa, K. Takahashin and H. Orima, 2008, Intranasal
HemangiosarcomainaDog,J.Vet.Med.Sci.525528
5. FunIn Wang and HueL Su, 2001, A Renal Hemangiosarcoma Causing Hematuria in a Dog, Proc.
Natl.Sci.Counc.No.3,pp.187192.
6. GaborL.J.andR.V.,2006,VanderstichelPrimaryCerebralHemangiosarcomaina6WeekOldDog
VetPathol43:782.
7. Gulbahar M. Yavuz, T.Guvenic, . Besalti, 1997, Splenic hemangiosarcoma with abdominal
disseminationinadog,Tr.J.ofVeterinaryandAnimalSicences459463.
8. JubbK.V.F.,P.C.Kennedy,N.Palmer,2007,PathologyofDomesticAnimals,FifthEdition,Saunders
Ltd.
9. MillerjM.A.,A.Ramosa,NDJ.M.Kreeeger,1992,CutaneousVascularNeoplasiain15Cats:Clinical,
Morphologic,andImmunohistochemicalStudies,VetPathol29:329336.
10. Oksanen,A.,1978,Haemangiosarcomaindogs,J.Comp.Pathol.,88:585595.
11. Patricia C. Schultheiss, 2004, A retrospective study of visceral and nonvisceral hemangiosarcoma
andhemangiomasindomesticanimalsJVetDiagnInvest16:522526.
1.

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12. Robinson, W.F. and M.G. Maxie, 1993, The cardiovascular system. In: Pathology of Domestic
Animals,Vol.3,pp.1100(Jubb,K.V.F.,Kennedy,P.C.andPalmer,N.,Eds.AcademicPress,SanDiego.
13. Smith A.N., 2003, Hemangiosarcoma in dogs and cats. Vet Clin Nth Am. Small Animal Practice.
33:533552.
14. SpanglerW.L.,M.R.Culbertson.1992.Prevalence,type,andimportanceofsplenicdiseasesindogs:
1480cases,JAVMA,200:829834.
15. Srebernik N. and E.C Appleby, 1991 Breed prevalence and sites of haemangioma and
haemangiosarcomaindogs,Vet.Rec.,129:408409.
16. Withrow and Macewen, 2007, Small Animal Clinical Oncology, SaundersElsevier, pag. 132, 433
434.

1195


ARGULUSSPP.(FISHLOUSE)INFECTIONINACOMMONCARP
(CYPRINUSCARPIO)FROMAFISHFARMINCLUJCOUNTY

M.TAULESCU,C.CTOI,G.BORZA,P.BOLF,
A.NAGY,A.GAL,F.TABARAN,COSMINACUC,R.MOUSSA,A.BARBU
Email:taulescumarian@yahoo.com
UniversityofAgriculturalSciencesandVeterinaryMedicine,ClujNapoca,Romania

Summary
Argulus species or the ,,fish louse,, parasitizes the skin of most fish species, frequently in
cyprinids. It causes skin lesions due to direct tissue damage and secondary infections. For this
study, twenty Cyprinus carpio, taken from a local private fish farm with symptoms such as
abnormal swimming and death, were examined for ectoparasites and other bacterial or viral
disease. The skin lesions were represented by multiple hemorrhages, necrosis of the skin and
scalesloss.TheparasitespresentontheskinwereidentifiedasArgulusspecies.Theeradication
hasbeenachievedwithcopper(II)sulfate,2dosesattendaysintervals.Afteradministration,all
fishwerecheckedforparasite.Noparasitewasobservedonthefish.
Keywords:Cyprinuscarpio,Argulus,ectoparasites,fishlouse,copper(II)sulfate.

INTRODUCTION

Argulids are obligate fish ectoparasites, commonly called fish louse. In wild
populations where the parasite's burden is low, it usually does not cause death. Argulus sp.
werereportedfromdifferentfishspeciesworldwide(Molnaret.al.,1995).Argulusfoliaceus
preferstoparasitizecyprinids.Thespeciesiswidelyspreadintemperatezonesandfrequently
foundinshipmentsofornamentaltropicalfish.Argulusspp.arefoundnearlyworldwidewith
about 150 species known at present. Three species documented in Europe are Argulus
foliaceus,ArgulusjaponicusandArguluscoregoni(Oge,2002).
The general bodyform of Argulus is a dorsoventrally flattened body covered by a
large chitinous carapace (Soulsby, 1982). The body can be divided into 3 distinct regions:
cephalothorax,thoraxandabdomen.ThecephalothoraxissonamedbecauseinArgulusthe
headisfusedwiththefirstsegmentofthethorax.Oneofthe mostobviousfeatures ofthis
groupofparasitesisthelargeventralsuckers.Thesearemodifiedmaxillaeandarethemain
attachmentorgansinadultargulids.Variousotherspinesandhooked/clawedappendagesare
alsobelievedtoplayamajorroleinaidingtheattachmentoftheanimal toits host.Infact
larval stages must depend on these numerous spines etc to keep them fixed to their hosts
(Walker et al., 2004). Once attached on the fish body, the parasite pierces the flesh using
stinger mouthparts and will suck the blood of the fish. Argulus inject a toxin that will kill
smaller fish and leaves reddened, inflamed lesions on larger fish. This lesion often becomes
infected.Clinicalsignsininfectedfishincludeerraticswimming,poorgrowthandflashing.The
intense irritation brought on by the Argulus parasite causes fish to rub or scrape against
objects from the water. Fish that have been infected with Argulus spp. often show several
small haemorrhagic regions. In all fish, mucous and club cells will be absent from the
epidermaltissuewithinthecraterbutmucouscellsareoftenabundantinthetissueobserved
around the wounds margins. However, the infection increases susceptibility of the fish to
bacterialpathogenssuchasFlavobacteriumcolumnare,Aeromonas,Pseudomonasandspring
viremia(Rabdoviruscarpio)ofcarpwhichcanlowersurvivalrates(Yildizetal.,2002).Argulus

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Saprolegnia infections significantly suppress carp growth and indicate that comparative
growth rates can be used as indicators of parasite stress (Singhal et al. 1990). Deaths will
occur if the number of parasite is too high. The most successful and effective treatments
againstliceareorganophosphatesandotherpesticidesorpotassiumpermanganateinwater.
OnestudyshowedgoodresultsfromtheuseatreatmentwithDDVP(dichlorvos,0,0
dimeethyl02,2dichloro vinyl phosphate) against to A. foliaceus. Trials were carried out at
water temperature of 26 C. After administration, all fish were checked for parasite. No
parasite was observed on the fish. The symptoms observed before, disappeared after
treatment(ErolToken,2006).
Inthisstudy,acaseofinfectionwithArgulusspp.inCyprinuscarpiofromfromfish
farminClujCountyisdiscussed.

MATERIALSANDMETHODS

The biological material consisted of 20 fish (Cyprinus carpio) taken from a local
private fish farm (3300 m2 with a total of 8000 fish) in Cluj County (Dej location) with
symptomssuchasabnormalswimminganddeath.Thefishwereweighed,measuredandbody
surface, body cavity and internal organs were examined for other infections. The parasites
were identified morphologically following keys characteristics given by Bykhovskaya
Pavlovskaya. The treatment has been achieved with copper (II) sulfate, one milligram per
cubicmeterofwater(1mg/m3),using2dosesattendaysintervals.

RESULTSANDDISCUSSION
The fish weighed 110170 g and were 15 20 cm in size. On the skin were observed
multiplefociwithhemorrhages,necrosisoftheskinandscalesloss.Intheseregionsasmall
quantity of mucus was identified also. The lesion appeared due to parasitic irritation and
tissuedamage.Themeannumberofparasitesperfishwas14.Theectoparasiteswere0,50,7
cm in lenght. Under the light microscope,these parasites were identified as Argulusspp. by
thelookingmorphologickeycharacteristic.

Fig.1.Cyprinuscarpioskinwithnecrosisandhemorrhagesandlossofthescales(arrow);
parasiteArgulusspp.ontheskin(seeneedletip).Affectedregionisboundedwithblack
color.

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Fig.2.Argulusspp.(arrowheadanddelimited)ontheskinofthecarp,necrosisand
hemorrhagesoftheskinandlossofthescales(arrow).Aanteriorbodyoftheparasite.

Fig.3.AnteriorregionofbodyoftheArgulusspp.Notethetwocompoundeyes
darkspots(ventralview)
Duringtreatment,theadversereactionsinfishwerenotobserved.Aftertratment,all
fish were checked for parasite. No parasite was observed on the fish. The skin lesions
disappeared after two weeks later. The eggs destroyed by flaming after were collected on
woodplanksplacedinwater.
Argulus spp. were reported from different fish species worldwide (Molnar et. al.,
1995).Argulusfoliaceuspreferstoparasitizecyprinids(Oge,2002).Inthisstudy,Argulusspp.
was reported on carp (Cyprinus carpio). Three species documented in Europe are Argulus
foliaceus, Argulus japonicus and Argulus coregoni (Oge, 2002). Based on the morphological
aspects in this case the fish were parasitized by Argulus foliaceus, it is commonly found in
Europe.

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FishthathavebeeninfectedwithArgulusspp.oftenshowseveralsmallhemorrhagic
regions and mucous are often abundant in the tissue observed around the wounds margins
(Yildizetal.,2002).Inthepresentstudythemainsymptomswererepresentedbyabnormal
swimming,scrapeagainstobjectsfromthewateranddeathofthefish.
ItisknownthatArgulusspp.infectionsleadtosecondaryparasiticinfestationofthe
skin or secondary systemic infection with Flavobacterium columnare, Aeromonas,
Pseudomonas and spring viremia (Rabdovirus carpio) of carp which can lower survival rates
(Soulsby, 1982; Yildiz et al., 2002). In thisresearch, inall fish examined, wehavenotfound
otherassociatedorsecondarydisease.
Themostsuccessfulandeffectivetreatmentsagainstliceareorganophosphatesand
otherpesticidesorpotassiumpermanganateinwater(ErolToken,2006).Thetreatmenthas
been achieved with copper (II) sulfate, one milligram per cubic meter of water (1mg / m3),
using 2 doses at ten days intervals. . After tratment, all fish were checked for parasite. No
parasitewasobservedonthefish.Theskinlesionsdisappearedaftertwoweekslater.

CONCLUSIONS

Argulus folliaceus parasites the carp (Cyprinus carpio) in fish farms from Cluj Couty
region.TheinfectedfishwithArgulusspp.presentedlackofappetite,poorgrowth,abnormal
swimming,scrapeagainstobjectsfromthewateranddeath.
Theskinlesionswererepresentedbymultiplehemorrhages,necrosisoftheskinand
scalesloss.Thetreatmenthasbeenachievedwithcopper(II)sulfate.

REFERENCES:

1.

2.
3.
4.
5.
6.
7.
8.

BykhovskayaPavlovskaya,I.E.,Gusev,A.V.,Dubinina,M.N.,Izyumova,N.A.,Smirnova,T.
S., Sokolovskaya, I. L., Shtein, G. A., Shulman, S. S. and Epstein, V. M.: Key to Parasites of
Freshwater Fish of the U.S.S.R. Leningrad, 1962. Israel Program for Scientific Translations,
Jerusalem,1964.
Erol Token, 2006 Argulus foliacesus Infestation on Oscar, Astronotus ocellatus and Its
Treatment.E.U.JournalofFisheries&AquaticSciences.Volume23,Say/Issue(12):177179.
Molnar,K.andC.Szekely,1998.OccurrenceofskrjabillanidnematodesinfishesofHungary
andintheintermediatehost,ArgulusfoliaceusL.ActaVet.Hun.46:451.
Oge,S.,2002Chemotherapyforparasitesoffreshwaterfish.T.Parazitol.Derg,26:113118.
SinghalR.N.,S.Jeet,R.W.Davies,1990Theeffectsofargulosisaprolehniasisonthegrowth
andproductionofCyprinuscarpio.Hydrobiologia.202:2731
Soulsby,E.J.L.,1982Helminths,ArthropodsandProtozoaof DomesticatedAnimals.7th Ed.
BaillireTindall,London,U.K.
Yildiz K., A. Kumantas 2002, israelian journal of veterinary medicine, Argulus foliaceus
infectioninagoldfish(carassiusauratus),Vol.57(2).
Walker, P.D., Flik, G. and Wendelaar Bonga, S.E., 2004 The biology of parasites from the
genus Argulus and a review of the interactions with its host. In HostParasite Interactions.
EditedbyG.WiegertjesandG.Flik.Garland/BIOSScientificPublishers,Abingdon,U.K.pp.107
129.

1199


LOSSOFBODYWEIGHTASANINDICATORFOREVALUATING
LAMBSWELFAREDURINGTRANSPORTATION

Marianaerbea(1),ElenaMitrnescu(2)
1
NationalSanitaryVeterinaryandFoodSafetyAuthority
2
)FacultyofVeterinaryMedicineBucharest

Abstract
Evaluation of animal welfare during transportation relates to the determination of the way in
whichareassuredtheconditionswhichtominimisetheaccomodationeffortsoftheanimalsat
physiological and psychologicalstress factors. As significant indicators of animal welfare during
transportation, easy to register, are the morbidity percent, mortality, the compulsory
slaughteringfollowingtoaccidents,bonesfractures,lesions.Mightbealsoutilizedasindicators
ofanimalwelfareduringtransportationthebehaviourandphysiolologicalindicators.
Amongstthephysiologicalindicatorsmostutilizedare:bodytemperature,heartandrespiratory
rate. For their signification in evaluation of the travel impact upon welfare, are determined in
laboratory,certainhormones,enzimesandbloodindicators.
Oneofthemostimportantconsequencesoftheanimaltransportationislosingofbodyweight.
Thisstudywasrealizedonbatchesoflambscolectedfromsmallbackyardholdingsintendedfor
intercommunity trading in Greece. The consignment was realized in different environmental
conditions related to teperature and watering. The duration of the transportation were of 11
hours,andthetravellingdistanceofaproximatelly300km.
For lambs transported at small temperature values, without watering, the weight loss was of
4,5%, comparing with those transported in the same temperature values, but with watering
duringtransport,wheretheweightlosswasonlyof2,6%.
Atanimalstransportedinconditionofhightemperaturevalues,thepercentoflosswereof5,8%
inthosewithoutwatering,andof3,1%,atthosewatered.
The loss in body weight in lambs as an indicator of appreciation of animals welfare during
transportisaffectedbyenvironmenttemperatureandbywateringofsheep.

Key words: welfare, transportation, lambs, environment teperature, water deprivation, loss in
bodyweight

INTRODUCTION

The extension of the intensive systems of rearing animals, both in Romania and
worldwide, has determined the appropriate development of animal transportation. Animals
haveto bemovedfromaplacetoanotherwhenaholdingispopulatedwithanimals,when
theyarereplacedandsoldtosloughterhouses,whentheyarepresentedinfair,marketsand
expositions,tradeorexportetc.
Theconditionsinwhichisrealizedthetransportationhasagreatimportanceunder
epidemiological aspect and strongly influences the quality of products intended for human
consumption, therefore the monitoring of animal circulation and of the transfortation
conditionswereimposedforalltheMemberStatesoftheEuropeanUnion.
Thescopeofthisworkistodeterminethelossinbodyweightoftransportedlambs,
loses determined by factors as: middle of transport used, duration of transportation and
conditions for transport, watering and environmental temperature during transport, the
preparingofthetransport.

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Lucrritiinificevol53seriaMedicinVeterinar
MATERIALANDMETHODS

The study was realized during the period July 2008 November 2009 on lambs
batches transported from backyards holdings from the NorthWeast of the country in a
collecting centre for sheep intended for intercommunity trade in Greece, from Prahova
County.

The animal consignments were realized in different environmental temperature


conditions,withtwotypeofvehicles:oneshavingfunctionalwateringsystem,inaccordance
with the provisons of the Commission Regulation no. 2005/1/CE, and other without having
functionalwateringsystems.Also,somegroupsoflambswerewateredduringtravel,others
werenot.
Alllambsbatcheswereweightedbeforeandafterembarkment.
The travel distance was of 300 km, and duration of the transportation was of
aproximately11hours,thetimeofthetravellbeingregisteredfromthefirstanimalembarked
atorigin,tothelastanimallandinginthecollectingcentreatdestination.

RESULTSANDDISCUSIONS

Intableno.1arepresented,comparatively,theaveragevaluespercentofthebody
weight loss oflambs transported at low temperatureconditions (56 0C) in bothcategory of
animals:withwatering,andinabsenceofwateringduringtravel.

Making an analyse of the obtained values, may be seen that, in batch of lambs
transportedinabsenceofwatering,theaveragepercentofbodyweightlosswasof4,5%,and
forthebatchwherethewateringwasrealized,theaverageofbodyweightlosswasof2,6%.

We have to underline that the duration of transportation and the environmental


temperatureforbothbatcheswerethesame.

Tableno.1
Theaveragevaluesofbodyweightlossduringtransportationinlowtemperature
environmentalconditons

No.of
Without
animals
watering
during
transportation

Watering
during
transportation

Average
oflossin
body
weight
(%)

9234

Body
weightof
thelamb
batches
atlanding
(kg)
8818,47

9153

8915,03

2,6

Environmental
temperature

Durationof
transportation
(hours)

Bodyweight
ofthelamb
batchesat
embarkment
(kg)

342

60C

11

339

11

5 C

4,5

The average values of the body weight loss in lambs batches transported in
conditions of raised environmental temperature conditions (24250C) are presented in the
Tableno.2.

Fromtheanalysisrealized,maybeobservedthattheaveragepercentofbodyweight
loss in lambs transported withour watering was of 5,8%, and in those transported being
watered,wasof3,1%.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Tableno.2
Theaveragevaluesofbodyweightlossduringtransportinraisedenvironmentaltemperature
conditions
Without
watering
during
transport

With
watering
during
tranportation

No.Of Environmental
Durationof
animals temperature transportation
(hours)

381

250C

11

Bodyweight
ofthe
batchesat
embarkment
(kg)
8382

392

11

9016

24 C

Body
weightof
thebatches
atlanding
(kg)
7895,85

Average
percentof
body
weightloss
(%)
5,8

9836,51

3,1

Animastransportedinrisedconditionsoftemperature,withavehiclewhichdoesnot
corespondingfromthepointofviewofthefunctionalwateringsystemduringtransportation,
andwhichwerenotwateredbeforeembarkmenthavehadanaverageofthebodyweihtloss
higherwith2,7%,comparingwithanimalswhichweretransportedatthesametemperature,
butwerewateredbeforeembarkmentandduringtravel.
A similar situation has been registered in case of animals transported in low
environmentaltemperatureconditions,withavehiclewithoutafunctionalsystemofwatering
duringtransport,andwhichwerenotwateredbeforeembarking,whichregisteredanaverage
bodyweightlosswith1,9%higher,comparingwithanimalswhichhavebeentransportedata
similartemperature,buthadbeenwateredbeforetransportationandduringtravel.

In accordance with the data found in the literature, in which is accepted a body
weightlossofaboutto3%,fromtheobtainedresultsmaybeconcludedthat,forthebatches
of lambs which were watered during travel, no matter the value of the environmental
temperatureinwhichthetraveltookplace,theaveragelossinbodyweightisjoininginthe
acceptedlimits,respective2,6%atatemperatureof50Cand3,1%atatemperatureof240C.

Overtakingsofthepercentof3%bodyweightlosswereregisteredatbatcheswhich
weretransportedinconditionswithoutwatering,respective4,5%atatemperatureof60Cand
5,8%at250C.

Thelossinbodyweight,asanindicatorofanimalwelfareduringtransportationhas
registeredadequatevalueswithdatainliterature.

CONCLUSIONS
1. Theveragebodyweightlossforanimalswithoutwateringduringtransportation
hasbeenwith1,9%higherthanforethosewatered,atlowtemperatures.
2. At higher temperatures, the average body weight loss for animals which were
notwateredduringthetransportationhasbeenwith4,7%higherthaninthose
watered.
3. The watering of animals and the environmental temperature during
transportationinfluencethelossinbodyweightoflambsandthisisanindicator
inappreciationofanimalwelfareduringtransportation.

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Lucrritiinificevol53seriaMedicinVeterinar
RECOMANDATIONS

In oder to register a level in average of body weight as small as posible, we


reccomendthat,beforeembarking,animalstobewatered,andthesystemforwateringtobe
functionalduringtravel.

During the seasons with high environmental temperatures, animals need to be


embarked and transported in the very early morning in order to avoid the highest
temperaturesofthedaywhichmightinfluencedehidrationand,implicitelly,thebodyweight
lossoftheanimalsinanimportantpercent.

BIBLIOGRAFY:
1.
2.

3.
4.
5.
6.

Knowles, T.G., Brown, S.N., Warriss, P.D., Phillips, A.J., Doland, S.K., Hunt, P., Ford, J.E.,
Edwards,J.E.andWatkins,P.E.(1995).VeterinaryRecord136,431438.
Knowles,T.G.;Wares,P.D.;Brown,S.N.;Kerstin,S.C;Edwards,J.E.;Perry,A.M.;Watkins,P.
E. and Phillips, A. J. (1996). Effects of feeding, watering and resting intervals on lambs
transportedbyroadandferrytoFrance.VeterinaryRecord139:335339.
Morris Dareen, Queensland Livestock and Meat Authority (1995). Handling and transport of
sheepandlambs:Areview,56.
MihaiDecun(2004),Ethology,welfareandanimalsprotection,350351.
MolebelediHDMareko(2005).Effectsofpreslaughteroncarcass/meatquality:Implicationfor
Botswana,JournalofAnimalandVeterinaryAdvances4(9):761767.
ValerTeudea(2005).Welfareandanimalsprotection,9798

1203


COMPARATIONOFTWOMETHODSFORDETERMINING
SALMONELLAspp.

I.IBRU,SORINAMARIAIRIMIE
FacultyofVeterinaryMedicineTimioara
CaleaAraduluiNo.119,300645,Timioara,Romania
Email:tibruioan@yahoo.com

Abstract
ThisworkpresentanalysisresultsoftwomethodsfordetectingbacteriaofthegenusSalmonella
on the surface of pig carcass and environmental samples from the production of foods and
movingthem.Thefirstmethodofdetectionishorizontal(ISO6579:2002)andthesecondisan
indirectmethodthatanalyzesimpedancedeviceusingTrac4200.

Keywords:Salmonellaspp.detection,analysismethods.

The determination of total germ counts and germ counts from selected micro
organisms on the basis of their indicators and/or index characters in, respectively, raw
materials,interimandendproductsaswellasonsurfacethatcomeintocontactwhitthose
products, including whitin the contex of the HACCP programs, has become increasingly
significantasanindicatorofgoodmanufacturingpracticeinthefoodordrinksindustry(1).
The most effective of these are controls of the product during manufacture,
treatmentandintroductionintothesupplychain(processcontrols)(1,11).
Especially microbiological process controls require instrumentation which delivers
resultsquickly,sothattheprocesscanbeadjustedwherenecessary(1,8,11).
Genus Salmonella belongs to the family Enterobacteriaceae and include species S.
Bongor,S.Enteridis,S.Typhi,S.typhimuriumandS.choleraesuis,withsubspeciescholeraesuis,
arizonae,diarizonae,hautenae,showandsalamae(3).
Salmonellaspp.isanimportantcauseoffoodborneillnessinhumans.Farmanimals
andfoodsofanimaloriginformanimportantsourceofhumanSalmonellainfections(2,3,11
).
The most common species involved in salmonelossis foodborne in humans are S.
typhimurium,S.enteridisandS.infantis(11).
From statistics recently acquired salmonelossis is placed first among zoonoses.
Salmonellosisin2007wassecondzoonosisaftercamphilobacteriossis,accoundingfor151,995
confirmedhumancauseinE.U.(11).
HorizontalmethodforthedetectionofSalmonellaspp(7).
Sampling and their preparation for analysis will be specifically dealing with
international standard product. If there is not an international standard specifically
recommendedthanpartiescanconcludeanagreementregardingthissubject.
Detection of Salmonella spp. is in compliance with ISO 6579:2002 and has four
successivestages:
The first step is preenrichment in nonselective liquid medium, peptonebuffered
water,butifthesamplesarethecocoaorcocoaproductsasenvironmentpreenrichmentwill
use waterbuffered peptone casein or milk powder and Green Diamond . If acid or acidified
foods using wholebuffered peptone water, but the pH of the sample with preenrichment
environmentshouldnotexceedpH4.5(4,5,6).

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Lucrritiinificevol53seriaMedicinVeterinar
Thesecondstepisselectiveenrichmentinliquidmedium.Selectiveliquidmediaare
recommended: RappaportVassiliadis medium with soya (RVS broth) and MullerKauffmann
brothtetraionat/novobiocyn(MKTTn)(7,9).
Isolation and identification is the third stage. This stage involves the use of solid
culture media xyloselysinedezoxycolat agar (XLD) and Brilliant Green agar (BGA).
Confirmationofidentityisthefinalstepandisthebiochemicalandserologicaltests(7,9).
The media used in this stage are: agar TSI (Triple sugar / iron agar), urea agar
(Christensen),theaverageLlysinedecarboxylation(9).
Also used for identity confirmation reagents for detection galactosydase,
Reagents VogesProskauer reaction (VP), reagents for indole reaction and a mono or
polyvalentseraagainstSalmonella(9).
Asweseeissueoftheanalysistimerequiredisfivedaysofsampletaking.
In economic terms this method has the following price/ sample. The price of each
mediaisshownintheTable1(10).Foreachsampleitwasconsideredthatwillbeused10ml
ofliquidmediumand12mlofsolidmedium.
Table1.
Thepriceofeachmediaandofonesample

Medium
Mediumprice(RON)
Sampleprice(RON)
Peptonebuffered
145,77
0,05
water
MKTTn
535,85
1,43
RVS
171,36
0,18
XLD
145,77
0,22
BGA
245,61
0,41
Api20E
800
40
Nutritiveagar
428,28
0,24
Total
2472,64
42,53

MicrobiologicalanalyzerdeviceusingTrac4200,whichhasthefollowingprinciple.
TheTrac4200isbasedontheprinciplesofimpedanceanalysis.
Impedanceanalysisisadynamicprocess,whichdeterminesthemetaboliccapacities
ofproliferatingmicroorganisms.
Microbialmetabolism utilisesnutrientsubstrateswhichforthemost partconsistof
higher molecularcompounds,producinglowermolecular,chargeddescompositionproducts.
These newly produced, charged compounds and/or their dissociation products alter the
conductivity of liquid nutrient and lower their resistence. This reduction can be techniclly
measuredusingaminimumof2electrodesinthenutrientsolution.IfanelectricalACvoltage
is applied to the electrodes then according to definition the reduction of this socalled
impedanceintheACcurrentfieldcanbemeasured(8).
The typical Trac measurement curve can be divided into 3 charecteristic ranges:
initial or adaptation phase (the microorganism adapt their metabolism to the substrate
present; the result ions cannot be mesured tehnically), exponential phase and stationary
phase(8).
The higher the contamination rate of the sample, the shorten the length of time
beforetheformationofthecharacteristicmeasuringcurve.
Qualitativemethodspresence/absenceofSalmonella

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Sample preparation is made like this: 25g of sample are preenriched in 225 ml
Buffered Peptone Water at37 for 16 18 hours; for red meatpreenrichement timecan be
reducedto68hoursbutthenprewarmed(40C)BufferedPeptoneWatershouldbeused(4
,5,6).In9,9mlofsterilBiMedia205Aareinoculeted0,1mloftheenrichedculture.
Trac 4200 analyser must be setting: temperature at 37C, duration 22,5 hours, warm up
time1hour,threshold10%,scale1%to25%M,classificationred22,5hours,nameofanalysis
SalmonellaPA.Positivesamples(redcolour)areindicatingthepresenceofSalmonellainthe
sample and must be confirmed by subculture on selective agars or by immunological or
molecularbiologicalmethods(1,8).Suchanalysisreportisissuedwithin3days.Thecostofa
pack of 120 measuring cell for determining Salmonella spp. is 2277.66 RON and a package
measuring120cellsofSalmonellasppand12confirmatorytestsis2466.16RON(10).
Advantagesofimpedancemicrobiology
Using dynamic detection of metabolic processes to measure microorganisms levels
eliminatestheneedfordilutionorseparationofmicroorganisms,whichconsiderablyreduces
thesamplepreparationtime.Theliquidmediausedinimpedanceanalysisisfarbettersuited
to the proliferation demands of mocroorganisms then the semisolid agar media in the
traditionalprocess.Theproliferationrateisacceleratedandtheanalyticaltimesarereduced.
Afurtherreductionoftheanalysistimeisachievedviathehigerdetectionsensitivity.
Asasignalchangescanbedeterminetedviaelectricalmeasurementtechinquesonce
the microorganisms have increased to 106107 CFU/ml in comparation to the plate method
(108109CFUforavisiblecolony),thedetectionsensitivityexceedsthatoftheplatemethodby
afactorof1000(8).
In all, the total analytical time for impedance analysis rarely exceeds 24 hours. In
mostcasestheresultsareavailablewithinonlyafewhourswhitthegreatadvantagethatthe
resultsaregenerallyavaibleerlier,themorecontaminetedasampleis.

REFERENCES

1. da Cunha, R.,G.,T., Innovation in pathogen detection and quantification, Meat International,


vol.18,no.3,2008,2627.
2. MARIANA LUPU, Evaluarea economic a sntii efectivelor de animale, Tez de doctorat,
FacultateadeMedicinVeterinarTimioara,2008
3.MogaMnzat,R.,Boliinfecioasealeanimalelorbacterioze,Ed.Brumar,Timioara,2001.
.
4.***ISO68672,MicrobiologyoffoodandanimalfeedingstuffsPreparationoftestsamples,initial
suspension and decimal dilution for microbiological examination Part 2: Specific rules for the
preparationofmeatandmeatproducts
5.***ISO68673,MicrobiologyoffoodandanimalfeedingstuffsPreparationoftestsamples,initial
suspension and decimal dilution for microbiological examination Part 3: Specific rules for the
preparationoffishandfishproducts.
6.***ISO68674,MicrobiologyoffoodandanimalfeedingstuffsPreparationoftestsamples,initial
suspension and decimal dilution for microbiological examination Part 4: Specific rules for the
preparationofproductsotherthanmilkandmilkproducts,meatandmeatproducts,andfishand
fisheryproducts.
7.***ISO6579,MicrobiologyoffoodandanimalfeedingstuffsHorizontalmethodforthedetection
ofSalmonellaspp.,2002.
8.***MicrobiologicalAnalyserTrac4200DeviceHandbook,Ed.SYLAB,2001.
9.***TheHiMediaManualforMicrobiologyLaboratoryPractice,Ed.Himedia,Mumbai1998.
10.***OfertadepreREDOX,2010.
11.***www.efsa.europa.euaccesatn04.03.2010.

1206


THEDETERMINATIONOFCOLISTINRESIDUESFROM
PORKMEATANDORGANSUSINGMICROBIOLOGICAL
ANDHPLCANALYSIS

V.TRIFAN1,MariaCRIVINEANU2,A.iOANCEA1,
C.LUPESCU3,V.NICORESCU2
1
CridaPharmS.R.L.Bucharest,Romania,trifanavasile@yahoo.com,
2FacultyofVeterinaryMedicineBucharest,Romania,
3ANSVSA,Bucharest,Romania

Abstract
The consequences of the drug residues presence in animal organism and in animal origin food
products represent a controversial subject and also an actual one in the conditions of a large
diversityofthesubstancesusedinveterinarytherapy.
The aimofthisstudywas todetermine colistinresidues in pork meatand organs after an oral
treatment in pigs with a commercial product based on colistin. There were collected samples
both from control and experimental lots. The determination of colistin residues were
comparatively effectuated, using microbiological and HPLC analysis. HPLC determinations
completed the microbiological study, in order to confirm the colistin levels in the days when
microbiologicalanalysisdidnotrevealthepresence ofcolistin.UsingHPLCmethod, theresults
weremoreaccurateandprecise.
The results obtained using the two methods allowed to establish the withdrawal period for
colistininporkmeatandorgans.

Keywords:colistin,pork,microbiologicalanalysis,HPLC.

Theconsequencesofdrugsresiduespresenceinanimalorganismandinanimalorigin
foodproductsrepresentacontroversialandactualsubjectintheconditionsofalargediversity
ofthesubstancesusedinveterinarytherapy(2).
Internationalorganizationsandgovernmentsareinvolvedinadaptingthenecessary
methodsforreducingandeliminatingthepotentialriskovertheconsumerhealthrepresented
bythepresenceoftheseresidues(5).
Colistin (colimycin, polymixin E) is an antibiotic produced by cultures of Bacillus
polymixa var. colistinus; it belongs to polymyxin therapeutic class. Colistin is used for the
prevention and treatment of diseases caused by sensitive bacteria in rabbits, pigs, poultry,
cattle, sheep and goats (6). After the oral administration, the absorption is low (1). The
eliminationismadebyrenalway,realizinghighurinaryconcentrations(4).Fortheoralwayis
usedcolistinsulphate,especiallyingastroenteritisproducedbysensiblegerms(3).
Theaimofthisstudywastoestablishthewithdrawalperiodforporkmeatandorgans
afteroraltreatmentwithcolistin.

MATERIALSANDMETHODS

The experiment was made on 3 monthaged pigs. It was administered commercial


productColicrid50%,buvablesolutioninadoseof50mg/kg/day,for5days.
After this period, 2 pigs were slaughtered in the first, second, third and fourth day
after the treatment interruption and there were collected muscle, liver, kidney and fat

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Lucrritiinificevol53seriaMedicinVeterinar
samples.
In order to detect colistin residues, all samples were analyzed using the
microbiologicalmethodbasedonthemeasurementoftheinhibitionareaproducedbycolistin
residuesinacultureofBordetellabronchiseptica.
Thedeterminationofthecolistinresiduesvalueintheanalyzedsamples(gresidue/
kgsample)wasrealizedbycomparingtheinhibitionareawithstandardcurvevalues.
Inthesametime,therewerecollectedsamplesinordertodetectcolistinresiduesby
highperformanceliquidchromatography(HPLC).
The HPLC study was intended to complete the microbiological analysis in order to
detect the values of colistin residues in the days in which the values obtained by
microbiological analysis were under the limit of detection, respectively in the 4th day after
treatmentinterruption.

RESULTSANDDISCUSSIONS

Colistinresiduesconcentrationsinmuscle,liver,kidneyandporkfatarepresentedin
tables14.
Having in view the interpretations of the obtained results, it was taken into
considerationthefactthatmaximumadmittedlimitsofcolistinresiduesaccordingtoCouncil
Regulation(CEE)Nr.2377/90,are:150g/kginporkmuscle,150g/kginliver,200g/kgin
kidney,150g/kginskinandfat.

Table1
Thelevelsofcolistinresiduesinporkmuscle(g/kg)

Slaughteredat
1dayafterthe
treatment

Slaughteredat
2daysafter
thetreatment

Slaughteredat
3daysafter
thetreatment

Pig1

Microbiol.
method
2200

Microbiol.
method
870

Microbiol.
method
313

Microbiol.
method
<LOD

23.20

Pig2

2020

754

253

<LOD

32.58

Slaughteredat4days
afterthetreatment
HPLC

Average
2110
812
283
<LOD
27.89
LOD=Limitofdetection

Fromtable1itcanbenoticedthatthehighestdecreaseoftheresiduesconcentration
wasproducedbetweendays12afterthetreatmentwasstopped.Itcouldalsobenoticedthat
therearentbigdifferencesbetweentheresiduesconcentrationsinthe2pigsmuscle.Inthe
4th day, although microbiological method didnt detect residues, by HPLC there were
determined quantities of colistin residues, but under European MRLs (Maximum Residues
Limits).

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UniversitateadetiineAgricoleiMedicinVeterinarIai

Table2
Thelevelsofcolistinresiduesinporkliver(g/kg)

Slaughteredat
1dayafterthe
treatment

Slaughteredat
2daysafter
thetreatment

Slaughteredat
3daysafter
thetreatment

Pig1

Microbiol.
method
604

Microbiol.
method
321

Microbiol.
method
170

Microbiol.
method
<LOD

22.39

Pig2

572

313

<LOD

<LOD

19.11

317

170

<LOD

20.75

Average
588
LOD=Limitofdetection

Slaughteredat4days
afterthetreatment
HPLC

Inthe2 tableitisshownthefactthat,inliver,theresiduesdecreasesignificantlyin
the first three days after the treatment and there arent major differences between the
concentrationdecreasingdynamicofthetwopigs.Inthe4thday,althoughbymicrobiological
methodtherewerenotdetectedresidues,byHPLCthereweredeterminedlowquantitiesof
colistinresidues,alsounderEuropeanMRLs(MaximumResiduesLimits).

Table3
Thelevelsofcolistinresiduesinporkkidney(g/kg)

nd

Slaughteredat
1dayafterthe
treatment

Slaughteredat
2daysafter
thetreatment

Slaughteredat
3daysafter
thetreatment

Pig1

Microbiol.
method
2145

Microbiol.
method
798

Microbiol.
method
335

Microbiol.
method
<LOD

44.24

Pig2

2233

854

<LOD

<LOD

47.04

826

335

<LOD

45.64

Average
2139
LOD=Limitofdetection

Slaughteredat4days
afterthetreatment
HPLC

In table 3 it can be noticed the dynamic of residues concentration decreasing in


kidneyinthetwopigs,thisbeingalmostthesamewiththeoneintheliver,startingobviously
fromdifferentlevels. Inthe4thday,althoughmicrobiologicalmethoddidntdetectresidues,
byHPLCthereweredeterminedquantitiesofcolistinresidues,higherthaninmuscleandliver,
butunderEuropeanMRLs(MaximumResiduesLimits).Thisfactisduetotherenalelimination
ofcolistin.

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Lucrritiinificevol53seriaMedicinVeterinar
Table4
Thelevelsofcolistinresiduesinporkfat(g/kg)

Slaughteredat
1dayafterthe
treatment

Slaughteredat
2daysafter
thetreatment

Slaughteredat
3daysafter
thetreatment

Pig1

Microbiol.
method
133

Microbiol.
method
<LOD

Microbiol.
method
<LOD

Microbiol.
method
<LOD

<LOD

Pig2

145

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Average
139
LOD=Limitofdetection

Slaughteredat4days
afterthetreatment
HPLC

Intable4itcanbenoticedthattheresidueslevelinfatismuchlowerinthefirstday
incomparisonwithotherorgans,butstartingthesecondday,itdecreasesunderthequantity
detectablebymicrobiologicalmethod.HPLCanalysisperformedinthe4thdaydidnotreveal
quantifiablevaluesofcolistinresidues.

Becauseitisknownthatcolistinabsorptionislowafteroraladministration,itcanbe
noticedthatinsometissues(liver,fat)thelevelsofthecolistinresiduesarelow,eveninthe
firstdays;inthefat,colistinresiduesareunderMRLsfromthefirstdayafterthetreatments
interruption.

CONCLUSIONS

1. Consecutive the determinations using microbiological method, the values of colistin


residues in pork meat and organs after the administration of Colicrid 50% product
decrease under maximum residues limits starting the 4th day after treatments
interruption.
2. HPLC method detects colistin residues in pork meat and organs more precise and
accuratethanmicrobiologicalmethod.
3. HPLCstudycompletesmicrobiologicalanalysis,providingvaluabledatainthedaysin
which the values obtained by microbiological analysis were under the limit of
detection.
4. Withdrawal period for pork meat and organs after the treatment with Colicrid 50%
productwasestablishedat4days.

REFERENCES

1.
2.
3.
4.
5.
6.

Cristea,AureliaNicoletaTreatyofPharmacology.Ed.Medicala,Bucharest,2005.
Cristina, R.T. Introduction in pharmacology and veterinary therapy. Ed. Solness, Timisoara,
2006.
FulgaI.Pharmacology.Ed.Medicala,Bucharest,2004.
Tang S.S., Gong L.J., He J.K., Jin X., Xiao X.L. Residue depletion of colistin in swine after
intramuscularadministration.J.S.Afr.Vet.Assoc.,80(1):4144,2009.
***EMEA/MRL/815/02FINALJanuary2002.
***EMEA/MRL/016/95FINALColistinSummaryReport.

1210

RESEARCHCONCERNINGTHEEXTRACTIONMETHODS
FORPATHOGENICYERSINIAENTEROCOLITICA
FROMPORKMEAT

La.TUDOR1,I.OGOE,1I.L.ILIE1,
ANCAMARIAGALI1,ANETALAURATUDOR2
1
FacultyofVeterinaryMedicineBucharest
2
FacultyofVeterinaryMedicineSpiruHaret

Abstract
Atthemoment,therearealotofmethodsfortheDNAextractionfrommeat,andmostofthem
do not succeed in removing the inhibitors for the PCR reaction. In this study, we compared 2
methods of DNA extraction in order to determine which one removes better and in a higher
quantity the PCR specific inhibitors. The amplification of the specific yadA gene from Y.
enterocoliticapure cultures can be carried outwith both of the protocols that we investigated
during this study. Both protocols were able to highly detect a lower limit of 0,4 fg/l of DNA
extractedfromY.enterocoliticapureculture.Themainconclusionwasthatbothprotocolscan
beusedforthedetectionofY.enterocoliticainmeat,wherethebackgroundmicrofloraisusually
veryrich.

Keywords:DNAextraction,PCRreaction,meatsamples.

Causingagreatvarietyofintestinalandextraintestinalsymptoms,Y.enterocolitica
isaverywellknownhumanpathogen(1,5,7,8,15).Thismostfrequentserotypesthatare
associatedwithhumanpathologyare:4/O:3,2/O:9,1B/O:8and2/O:5,27(6,9,10,11).From
all these 4/O:3 is the most spread and its frequently isolated from living pigs. Healthy pigs
weredemonstratedtobethemainreservoirofthisdiseaseallaroundtheworld(2,3),butthe
pork meat was not considered to be a vector of this bacterial species, especially for this
serotype (4, 12, 13). Of course, not all the serotypes are pathogenic, and the pathogenic
strains for this species contain a virulence plasmid which was called pYV, with great
importanceintheclinicalsciences.Amongthevirulencefactorswhichthisplasmidencodes,
therecanbementioned:Yersiniaputerproteins(Yops)andtheadhesineA(YadA).Thelatter
promotestheadhesiontotheintestineepithelialcellsandamongthebacteriathemselves,at
the same time producing autoagglutination and interference with the bactericidal action of
serum(14).InmostcasestherearealotofdifficultiesconcerningthecapacityofPCRreaction
todetectmicroorganisms,dependinguponthepurityofthetemplate,whichisusedastarget
andthenumberoftargetmolecules.Insometypesofsamples,meatforexample,alotofPCR
inhibitorsmaybepresent,indifferentformsandcompositions.
TheobjectiveofthisworkwastocomparetwodifferenttechniquesusedforDNA
extractioninordertodeterminewhichoneremovedahigherquantityofPCRinhibitors.

MATERIALSANDMETHODS

In this study, we used as inoculum, two strains of Y. enterocolitica, one from the
universitycollectionandtheotherisolateddirectlyfrompigs,raisedonindustrialconditionsin
ourcountry.ThesestrainswerekeptinLuriabroth,supplementedwith20%glycerolat20oC.A
colonyofY.e.obtainedonMacConkeyagarwasinoculatedin100mlofTSBandincubatedfor

1211

Lucrritiinificevol53seriaMedicinVeterinar
18hat25Ctoobtainaconcentrationof1x109CFU/ml.Onemilliliterofthisconcentration
wasusedtoperformtheDNAextractionwhentheextractionwasdonedirectlyfromthepure
cultureortoinoculatethesamples.
We used two types of meat samples: pork salami and minced meat. From each
sample, consisting of one of the two mentioned types, we obtained a 25 g aliquot and we
inoculateditwith1mlofthepreparedinoculum,andafterwardsweplaceditina225mlof
TSB.Theinoculatedsamplewenttoastomacher,whereitwaskeptfor90sat2000rpm.All
thesampleswereincubatedat25oC,andtheDNAwasafterwardsextracted.

RESULTSANDDISCUSSIONS

The DNAextractionprotocolsmaintainedtheDNAintact, andtheamplificationof


YadAgenewassuccessfulinbothcases.Bothofthesampletypescouldbeusedsuccessfully
for the Y.e. detection, even when the concentration of this bacteria was not higher than 1
CFU/ml. As well, both protocols detected up to the lowest concentration for the examined
samples, that of 0,4 fg/l. Since the concentration was in some cases very low, the PCR
reactionincreasedthesensitivityoftheassay,duetoitsspecificparameters.
Inthenextstage,weselectedtwomethodsofDNAextraction,forcomparison.The
firstoneincludedthenextsteps:100loftheenrichedsamplewerecentrifugedat16,000g
for10minutesat4oC,thepelletwasresuspendedin50lofPCRbuffersolution,with0,2mg
proteinase K/ each ml. After incubation at 37 C for 1 h, the suspension was boiled for 10
minutesandcentrifugedagainat16,000gfor5minutes,at4oC.Thesupernatantwasusedto
performthePCR.
Thesecondprotocolconsistedof:100mloftheenrichedsamplewaswashedwith
100lofammoniahydroxide,150lofethanol,200lofpetroletherand10lofSDS(15%).
Thesamplewascentrifugedat14,000gfor10minat4C,andthepelletwasresuspendedina
solutioncontaining100lof5Murea,90lofabsoluteethanol,150lofpetrolether,25l
ofSDS(10%)and10lof3Msodiumacetate.Asecondcentrifugationfor5minat12,000gat
4Cwasperformed,andthepelletwasresuspendedwith300lofTEbuffer(TrisEDTA)pH
7.5,15lofSDS(10%)and5lofDNasefreeRNase(10mg/ml).Thetubeswereincubatedat
37Cfor30minbeforetheadditionof15lofProteinaseK(20mg/ml).Thisprocessedsample
was incubated at 37 C for 30 min. Finally, 100 l of 5M sodium perchlorate and 300 l of
phenol chloroform isoamyl alcohol were added for DNA extraction. The sample tubes were
subjectedtocentrifugationat10,000gfor7minutes,andtheaqueousphasewascollected.
The nucleic acids were precipitated with alcohol and the DNA was dissolved in 15 l of
deionizedwater.
For the DNA integrity test, 2 l of each solution, containing the extracted DNA
obtainedbythetechniquesdescribedabovewereintroducedandtestedin0,9%agarosegel,
afterelectrophoresisat85Vfor30min.Thegelswerestainedwithethidiumbromideandthe
DNAbandswerevisualizedusingatransluminatorwithUVlightsource.
ThePCRreactionincludedaquantityof5loftemplatedforthefirstPCRstep,and
2 l for the second step. The reaction mixture contained: 1 U Taq DNApolymerase, buffer
solution,100MforeachdNTP,and0,1Mforeachprimer.Theampliconsizewas747bp
andtheproductsize529bp.Theseproductsweredeterminedbyusingelectrophoresisin1%
agarosegel.
ThePCRsensitivitywasdeterminedbyusingserialdilutionsfrom1x104CFU/mlof
Y.e., with virulence plasmid on TSB. From each concentration, 1 ml was inoculated on 25 g
samples.EachsamplewasintroducedinTSB(225ml)andhomogenizedinastomacherfor90

1212

UniversitateadetiineAgricoleiMedicinVeterinarIai
s.Afterincubationat25oC,1mlofeachconcentrationwastakenandtheDNAextractionand
PCRreactionwerecarriedout.
ThedetectionofY.e.strainsinthemeatsamplessimplifiedenormously,yadAwas
identifiedin18samples(25,7%)fromporksalamiand52(74,3%)frommincedmeat.

CONCLUSIONS

1.
ThepresenceofY.enterocoliticainmeatandmeatproductsisconsidereddebatable.
Culturetechniquesareveryprecise,buttimeconsuming,whilemolecularbiologytechniques
arerapid,butthePCRinhibitorscanleadtowrongresults.InthisworktwomethodsofDNA
extractionwerestudied,bothofthemappliedsuccessfullyconsideringtheDNAextractionand
the removal of PCR inhibitors. The two methods that we chose for comparison were used
beforebydifferentresearchersandmostofthemwerealsoabletoremoveanyPCRinhibiting
agents,consideringthesamplesthatwechosefortheassay:porksalamiandmincedmeat.
2.
Whenadirectdetectionofthesearchedpathogeniscarriedout,falsepositiveresults
canbeobservedduetothepresenceofdeadcellsinthesamplesstudied.Thiscouldleadto
an erroneous conclusion in relation to the potential risk for the consumer. Therefore, an
enrichment step was included in this work in order to promote the growth of the target
organism and dilute any dead bacterium or exogenous DNA present in the sample. The
differencesobservedbetweentheresultsobtainedby
3.
PCRandbytheculturemethodsmaybeduetothelowersensitivityoftheselective
culture media. It is also important to emphasize that the PCR is a rapid method for the
detectionofvirulentstrains.Thepossibleexistenceofasmallnumberofdamagedorstressed
cells could be the cause of long incubation times needed so that these cells may reach the
detectionlimitoftheculturemethods.
4.
Theresultsofthisworkallowustoconcludethatthestudiedprotocolswereableto
eliminatesatisfactorilythePCRinhibitorspresentinthestudiedfoods.

Acknowledgments.ThisstudyhasbeenfinancedandisaofID153grantCNCSIS,
thecontractIDEI289/2007.

REFERENCES

1. Aepfelbacher,M.,Zumbihl,R.,Ruckdeschel,K.,Christoph,A.J.,Barz,C.&Heesemann,J.(1999).The
tranquilizing injection of Yersinia proteins: a pathogens strategy to resist host defense. Biological
Chemistry,380,795802.
2.Bhaduri,S.,&Cottrell,B.(1998).AsimplifiedsamplepreparationmethodfromvariousfoodsforPCR
detection of pathogenic Yersinia enterocolitica: a possible model for other food pathogens.
MolecularandCellularProbe,12,7983.
3.Bhaduri,S.,Wesley,I.,&Bush,E.J.(2005).PrevalenceofpathogenicYersiniaenterocoliticastrainsin
pigsintheUnitedStates.AppliedandEnvironmentalMicrobiology,71,71177121.
4. Bottone, E. J. (1999). Yersinia enterocolitica: overview and epidemiologic correlates. Microbes
Infection,1,323333.
5.Boyapalle,S.,Wesley,I.V.,Hurd,H.S.,&Reddy,P.G.(2001).Comparisonofculture,multiplex,and
5_nuclease polymerase chain reaction assays for the rapid detection of Yersinia enterocolitica in
swineandporkproducts.JournalofFoodProtection,64,13521361.
6. FredrickssonAhoma, M., Hielm, S., & Korkeala, H. (1999). High prevalence of yadApositive Yersinia
enterocoliticainpigtonguesandmincedmeatatretaillevelinFinland.JournalofFoodProtection,
62,123127.

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Lucrritiinificevol53seriaMedicinVeterinar
7.FredrickssonAhoma,M.,&Korkeala,H.(2003).LowoccurrenceopathogenicYersiniaenterocoliticain
clinical,foodandenvironmentalsamples:amethodologicalproblem.ClinicalMicrobiologyReviews,
16,220229.
8. Ibrahim, A., Liesack, W., GriYths, M. W., & RobinsBrowne, R. M. (1997). Development of a highly
specific assay for rapid identification of pathogenic strains of Yersinia enterocolitica based on PCR
amplificationoftheYersiniaheatstableenterotoxinagene(yst).JournalofClinicalMicrobiology,35,
16361638.
9. Kapperud, G. (1991). Yersinia enterocolitica in food hygiene. International Journal of Food
Microbiology,12,5366.
10. Lamberts, S. T., & DanielssonTham, M.L. (2005). Identification and characterization of pathogenic
Yersinia enterocolitica isolated by PCR and pulseWeld gel electrophoresis. Applied and
EnvironmentalMicrobiology,71,36743681.
11. Logue, C. M., Sheridan, J. J., Wauters, G., Mc Dowell, D. A., & Blair, I. S. (1996). Yersinia spp. and
numbers,withparticularreferencetoYersiniaenterocoliticabio/serotypes,occurringonIrishmeat
andmeatsproducts,andtheinfluenceofalkalitreatmentontheirisolation.InternationalJournalof
FoodMicrobiology,33,257274.
12. Nesbakken, T. (1985). Comparison of sampling and isolation procedures for recovery of Yersinia
enterocoliticaserotypeO:3fromoralcavityofslaughterpigs.ActsVeterinaryScandinavian,26,127
135.
13.Powell,H.A.,Gooding,C.M.,Garret,S.D.,Lund,B.M.,&McKee,R.A.(1994).Proteinaseinhibition
of the detection of Listeria monocytogenes in milk using the polymerase chain reaction. Letters of
AppliedMicrobiology,18,5961.
14.Vishnubhatla,A.,Oberst,R.D.,Fung,D.Y.C.,Wonglumsom,W.,Hays,M.P.,&Nagaraja,T.G.(2001).
Evaluation of a 5nuclease (TaqMan) assay for the detection of virulent strains of Yersinia
enterocoliticainrawmeatandtofusamples.JournalofFoodProtection,64,355360.
15.Waage,A.S.,Vardund,T.,Lund,V.,&Kapperud,G.(1999).Detectionoflownumberofpathogenic
Yersinia enterocolitica in environmental water and sewage sample by nested polymerase chain
reaction.JournalofAppliedMicrobiology,87,727821.

1214

RESEARCHCONCERNINGTHEIDENTIFICATIONOFSEROTYPEO:9
OFYERSINIAENTEROCOLITICAFROMMEAT

L.TUDOR1,I.OGOE1,I.L.ILIE1,
ANETALAURATUDOR2,ANCAMARIAGALI1
1FacultyofVeterinaryMedicineBucharest
2
FacultyofVeterinaryMedicineSpiruHaret

Abstract
Based on a 181bp fragment originating from a cloned per gene, a real time PCR was used in
order to detectYersinia enterocolitica O:9 (Ye O:9). The validation comprised 50 Ye O:9, 25 Ye
nonO:9and40othercloselyrelatedstrains,whichcontainedhomologiesofthesamegene.The
PCRassaywasusedforspecificYeO:9.Thelimitsofdetectionwerefrom1to20genomecopies
ofDNAandtheamplificationindicatedalinearregression.
Yersiniaenterocoliticaspeciescomprisesaseriesofhumanpathogenserotypes,O:9beingoneof
them (2, 3, 9, 12). It presents an identical Oantigen structure to the ones from Brucella spp.,
consisting of a homopolymer of Nformylperosamine, encoded by per gene (1, 5, 7). Until this
moment, there have been developed different types of PCR tests in order to detect the two
generamentionedabove,fromvarioustypesofsamples(4,10,11).Withasuccessfulapplication
in this field of work, identification methods like the slide agglutination and biochemical tests
wereused.Incomparisontothesekindsoftests,PCRassayisamuchmorespecificandreliable
methodtouseinthisdirectionofresearch(6,8).

MATERIALANDMETHODS

Fifty strains of Ye O:9, 25 Ye nonO:9 strains and 40 other closely related strains
wereincludedinaselectivitytest.Thestrainswerecollectedfrommeatsamples,comingfrom
specieslikeswine,cattleandpoultry,fromalloverthesouthernpartofRomania.Inthisstudy
there has been used the MasterMix 2X (a diagnosis kit with premixed substances) and the
optimalprimers,andtheconcentrationofthesampleswas200nM,respectively80nM.The
reading was performed on the Light Cycler thermocycler 2.0 (Roche). A 2 l quantity was
added over 23 l of primary mix. The optimal temperature for 54oC normalization was
determined using the gradient characteristic included in the thermocycler software. The
specificity of the test considering the pure cultures. The specificity studies were performed
using32bacterialstrains.Yersiniaspp.pureculturesweremaintainedinBHIbrothat30oCand
forotherbacterialspeciesat37oC,for1618hours.ThebacterialDNAwasextractedfrom1l
quantity, from an enrichment medium left overnight, which was transferred in a 100 l
quantityofsteriledistilledwaterandsuccessivelyboiledfor10minutes,forthebacteriallysis
to take place. A 2l quantity from this bacterial suspension, subjected to the thermal
treatmentthroughboilingwasusedasapatternforthePCRtechnique.
The assay reagent was mixed with a uracilNglycosydase enzyme and
deoxythimidine(replacingdeoxyuridine).Thefinalreactioncomprisedthefollowing:
YeO:9:
250nmolforwardprimer5VTGTGCTGAAGCTTTTGGATCT3V;
230nmolofreverseprimer5VGAGGCCGATACACCTTGATT3V;
80nmolprobe(5VFAMTGGACGACATGTAGGTACCTTTGGTGATAMRA3V).
IACreaction:

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Lucrritiinificevol53seriaMedicinVeterinar

75nmolofforwardprimer5VCTGCTTAACACAAGTTGAGTAG3V;

60nmolofreverseprimer5VTTCCTTAGGTACCGTCAGAA3V;

45nmolofprobe5VVICRTTCATGAGGACACCTGAGTTGATAMRA3V.
All these quantities were added to 0,5 mM of each deoxynucleoside triphosphate
mixture(dNTPs),thereactionbuffer,0,8UofDNAzymeIIrecombinantpolymerase,7,5mM
MgCl2,2,5%v/vglycerol,and0.7mg/mlswineserumalbumin.
ThePCRreactionswereperformedinaLightCycler2.0(RocheAppliedBiosystems),
usingthenextprofile:initialdenaturationat96oC,10min,followedby40cyclesof96oC,20s;
60oC,30sand74oCC,30s,withthefluorescencemeasuringaftertheannealingstep.
TheDNAfrom3YeO:9wasseriallydilutedinbuffersolution,with0.15mMEDTA
overarangeof10logscorrespondingto150genomecopiesforeachreaction.

RESULTSANDDISCUSSIONS

TheDNAwasisolatedusingacontinuousheatingforcelldisruption.So,thestrains
were taken from refrigerated stocks and introduced in Yersinia Selective Medium and blood
agarplates.Fromeachofthem,acolonywassuspendedin100mldoubledistilledwater,and
placedat80oCfor20minutes,beforebeingcentrifugedfor10minutesat15,000xg.
The sequences of the per gene of the microorganisms were used to design the
primersforamplificationofdifferentpartsofthegene,whiletheampliconsweresubjectedto
sequencinginbothdirection.Afterwards,wesequencedaPCRampliconofpergene(541bp)
which contained a fragment of interest in seven Ye O:9 strains already isolated, in order to
assesthedegreeofgeneticvariationwithinthepergenefragmentwhichwasamplified.Inthe
endofthisstage,thesequencesweobtainedwere100%identical.
The next step comprised the hydrolysis of the samples, using TaqMan R, labeling
with 6 carboxyfluorescein (FAM) and suppression with 6 carboxytetramethylrhodamine
(TAMRA).Subsequently,theinternalamplificationcontrol(IAC)includedasecondsetofPCR
primers,asecondTaqManprobeandanartificial124bpamplicon.Thisstepwasperformedin
ordertominimizethecompetitionandalsotheinfluenceonthelimitsofdetection.
TheRTPCRtest,developedinordertodetectthepresenceofY.enterocoliticaail
positivestrains,isaspecificoneforallthestrainswithpathogenicpotential.Y.enterocolitica
strains (approx. 104 bacteria for a single PCR test), that belong to the pathogenic serotype
have showed a positive reaction at Ct values comprised between 25 and 26, meanwhile the
nonpathogenic strains of Y. enterocolitica, Y. pseudotuberculosis and other Yersinia species,
concludedinobtainingofnegativeresults.Thespecificityoftheprimersusedinthisstudywas
testedbyNakajimaandcol.(14).Theseobtainedsimilarresultsforthe14pathogenicstrains
ofY.enterocolitica,13nonpathogenicstrainsofY.enterocolitica,31forY.pseudotuberculosis,
11forY.frederiksenii,17forY.intermediaandother12bacterialstrainsweretestedthrough
conventionalPCRtechnique.ThelowestdetectionlimitofthisPCRtestinordertoidentifyY.
enterocoliticaailgeneinpurecultureswasof103104CFU/ml,becausetheDNAwasextracted
froma100lquantityofpurecultureanda2lquantitywasusedasapattern.However,the
detectionlimitofthePCRreactionwasof110CFUforaPCRtest.Thesedataarecorrelated
withthelateststudies,performedonthesamesubject.
After the enrichment step through incubation for 1618 hours at 25oC, the PCR
methodwasabletodetect110CFU/mlofliquidextractfrombiologicalproductsample.The
former enrichment step, before PCR technique application is necessary usually in order to
raise the sensitivity when studying the naturally contaminated samples. Moreover, the
enrichment steplowerstheriskof obtainingfalse positiveresults,incaseof PCR technique,

1216

UniversitateadetiineAgricoleiMedicinVeterinarIai
duetothepresenceofdeadcells.ThedetectionratesforailpositiveY.enterocoliticastrains,
especiallyconsideringthebiologicalproductsortheexteriorsources,naturallycontaminated,
were especially low by using bothwork methods. A reason for the high number of negative
resultscanbethelownumberofpathogenicstrainspresentinthesesamples.The2lpattern
usedinthisstudyhadlowvalues.Analternativeforraisingthesensitivitycouldbethewhen
using a 5 l pattern, which can be afterwards added to a 45 l quantity of primary mix.
Another reason for obtaining negative results can be the high number of microorganisms
whichcomposethemicrobialfloraofthemedium,consideringthesampleswhicharestudied.
The nonselective enrichment step used in this study allows the development of all bacteria
andthisfactcandetermineadecreaseinthesensitivityofthistest,ifthemicrobialfloraofthe
medium is present in a high concentration. It had been demonstrated that a high
concentration of external DNA can represent the cause of a negative effect on the
amplification efficiency. This fact could exist due to the different interactions between the
bacterial DNA and the PCR reagents. In order to overcome this problem, it can be used a
selective enrichment step, that doesnt allow the inhibition of the necessary factors for PCR
technique.
An explanation for obtaining low detection rates in case of using PCR technique
couldbethepresenceoftheenzymeswithabactericideandbacteriostaticeffect,aswellas
other inhibiting substances naturally present in biological products, fact which leads to
obtainingsomefalsenegativeresults.TheDNAextractionprocedureusedincaseofthisstudy
wasaquickandeasyone,butnotnecessarilythemostefficientfordiscardingtheinhibiting
agentsfromthebiologicalproducts.Apositiveinternalcontrolhastobeelaboratedasastep
ofthistest,inordertomonitorthepossiblefalsenegativeresults.

CONCLUSIONS

1. AlltheYeO:9strainsincludedinthestudyproducedFAMcyclethresholdvalues(Ct)
in the range of 9.5 to 32.5 with 97 % of these being between 10.5 and 15.5. Five strains
showed no signal for IAC, due to inhibition of FAM signal. A second PCR reaction of these
strainsconfirmedtheresults.
2. All the Ye nonO:9 presented values of FAM Ct above 55, and the Ct values for IAC
werenotinhibited,consideringthenegativeresults.ThePCRassaydetected5to15genomic
copiesofYeO:9DNA.TheRvalueswereabove1,16inallstages,withaoptimumcorrelation
betweenFAMCtvalueandDNAcontent.
3. The IAC is mandatory for standardization of RTPCR assay, especially for Ye O:9
reactions.Thisisthewaytodifferentiatetruenegativeresultsfromfalsenegativeones.
4. InwhatconcernstheapplianceofPCRreactiontothisspeciesandserotype,itisone
ofthemanytrialsandvariantsconsideringthissubject,andinthefuture,wehopethatthis
techniquewillimprove,inordertoofferusastandardmethodforthissectorofmicrobiology
testing.

Acknowledgments.ThisstudyhasbeenfinancedandisaofID153grantCNCSIS,
thecontractIDEI289/2007.

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Lucrritiinificevol53seriaMedicinVeterinar
REFERENCES

1.Anonymous,2002.Microbiologyoffoodandanimalfeedingstuffspolymerasechainreaction(PCR)
for the detection of foodborne pathogens general method specific requirements, Draft
InternationalStandardISO/DIS22174.Berlin,Germany.
2.Bogdanovich,T.,Skurnik,M.,Lqbeck,P.S.,Ahrens,P.,Hoorfar,J.,2004.Avalidated5VnucleasePCR
assayforrapididentificationofgenusBrucella.J.Clin.Microbiol.42,22612263.
3.Bottone,E.J.,1997.Yersiniaenterocolitica:thecharismacontinues.Clin.Microbiol.Rev.10,257276.
4.FredrikssonAhomaa,M.,Korkeala,H.,2003.LowoccurrenceofpathogenicYersiniaenterocoliticain
clinical,foodandenvironmentalsamples:amethodologicalproblem.Clin.Microbiol.Rev.16,220
229.
5. Godfroid, J., Saegerman, C., Wellemans, V., Walravens, K., Letesson, J.J., Tibor, A., Mc Millan, A.,
Spencer, S., Sanna, M., Bakker, D., Pouillot, R., GarinBastuji, B., 2002. How to substantiate
eradication of bovine brucellosis when aspecific serological reactions occur in the course of
brucellosistesting.Vet.Microbiol.90,461477.
6. Hoorfar, J., Malorny, B., Abdulmawjood, A., Cook, N.,Wagner, M., Fach, P., 2004. Practical
considerationsindesignofinternalamplificationcontrolfordiagnosticPCRassays.J.Clin.Microbiol.
42,18631868.
7. Jensen, A.N., Hoorfar, J., 2003. Optimal purification and sensitive quantification of DNA from fecal
samples.J.RapidMethodsAutom.Microbiol.10,231244.
8. Lantz, P.G., HahnHegerdal, B., Radstrofem, P., 1994. Sample preparation methods in PCRbased
detectionoffoodpathogens.TrendsFoodSci.Technol.5,384389.
9. Lobeck, P.S., Wolffs, P., On, S.L.W., Ahrens, P., Radstrofem, P., Hoorfar, J., 2003. Toward an
internationalstandardforPCRbaseddetectionoffoodbornethermotolerantcampylobacters:assay
developmentandanalyticalvalidation.Appl.Environ.Microbiol.69,56645669.
10. Malorny, B., Hoorfar, J., Hugas, M., Heuvelink, A., Fach, P., Ellerbroek, L., Bunge, C., Dorn, C.,
Helmuth, R., 2003. Interlaboratory diagnostic accuracy of a Salmonella specific PCRbased method.
Int.J.FoodMicrobiol.2796,19.
11. Malorny, B., Hoorfar, J., Bunge, C., Helmuth, R., 2003. Multicenter validation of the analytical
accuracy of Salmonella PCR: towards an international standard. Appl. Environ. Microbiol. 69, 290
296.
12.Moreno,E.,Moriyon,I.,2001.ThegenusBrucella.In:Dworkin,M.,etal.,(Eds.),TheProkaryotes:An
EvolvingElectronicResourcefortheMicrobiologicalCommunity.SpringerVerlag,NewYork.

1218

DIROFILARIAIMMITISINFECTIONANEWCHALLENGEFOR
VETERINARYPRACTITIONERSINIASICOUNTY

D.ACATRINEI,L.MIRON,SIMONADIMITRIU,
ANAMUSTEA,LARISAPARASCA,RAMONAORIC
FacultyofVeterinaryMedicineIai

Abstract
The first case report of canine heartworm disease in Iai was diagnosed in march 2009. Since
than,27newcaseswerediagnosedindogsownedbyIaicitizensand41invagrantdogsfrom
sheltersandpublickennelsbymicroscopicexaminationofbloodsmearsandspecificrapidtests
(SNAP Heartworm RT and Dirochek). The increase of the frequency of new cases, the zoonotic
characterofthediseaseaswellasthepresenceofmicrofilariainclinicallyhealthydogscanbea
serioushazardtobothhumanandanimalhealth.
Keywords:Dirofilariaimmitis,heartworm,dog

Dirofilaria immitis (Leidy, 1856) is a dixenic spirurid nematode from the Family
Onchocercidae.Theadultstageisusuallyhostedbythedog,although,therearealsoreported
infestationofthefelids(FeliscatusLiuetal.,2005,PantheraparduspantherMazzariolet
al.,2010,Pantheratigristiger,Pantheraleolion,Pantheraoncajar,Unciauncialsnow
leopard Murata et al., 2003), canids other than Canis familiaris ( Canis familiaris dingo
dingodogStarretal.,1988,CanislupuswolfDulceanuetal.,1994,Canislatranscoyote
Kazacosetal.,1979,CanisaureusgoldenjackalKesdangsakonwutetal.,2006,Vulpessp.
fox Kazacos et al., 1979, Nyctereutes procynoides raccoon dog Sato et al., 2009),
Mustelidae (Mustela putoriuus furo weasel), Hominidae (Homo sapiens sapiens human
Dulceanuetal.,1994,Kimetal.,2001,Genchietal.,2005).

Thelarvaestages1to3arehostedbythemosquitoesoftheFamilyCulicidaeandthe
ticksofIxodidaewhichingestthemtogetherwiththebloodtakenfromthedefinitive hosts.
TheL3 larvaeareinoculatedinthedefinitivehostduringanewfeeding.Theprepatentperiod
isofapproximately6months;meanwhilethelarvaemigratethroughtheblood,thenstopin
the pulmonary artery and become adult. The increase of the parasitic density in the
pulmonary artery leads to the retrograde migration of the parasites in the right atrium, the
cave vein and the portal vein. The symptoms of the canine dirofilariosis are generally
represented by cardiac and respiratory insufficiency, the clinical expression being directly
relatedtothehostbodyweight.Sometimes,thesedisordersarecomplicatedwithhepaticand
renalinsufficiencyduetothemicrofilariaandtheirmetabolitesandalsototheendosimbionts.

Ifinthedogcanbefoundmassiveinfestations(Acatrineietal.,2008),inthecat,only
twoadultwormscancausetheheartdilatationandseriousclinicalsigns(Liuetal.,2005).

The disease has a zoonotic character, the infestations lacking the adult stages, but
with the presence of the larvae in the blood vessels, the pulmonary tissue (Dulceanu et al.,
1994)orliver(Kimetal.,2001).Themostfrequentlocalizationisthepulmonaryoneinthe
shape of eosynophilic granulomas limited by connective tissue, radioopaque, that resemble
thepulmonaryneoplasms.Thehistologicaldamagescausedbythecardiovascularyparasitism
offilariaconsistofMPZAaccumulation,aneurisms,thehyalinationofthemusclefibersfrom
the arterial walls and the collagenation and the erosion of the endothelium due to the
mechanicalactionoftheluminalparasitesandthesubendothelialmigrationofthemyoblasts
(Acatrinei, 2008). In the lungs, the lesions are more diverse, being caused by the congestive

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Lucrritiinificevol53seriaMedicinVeterinar
cardiac insufficiency, by the intrapulmonary localization of the microfilaria and by the
reactivityoftheorganismtotheaggressionoftheparasitesandtheirendosimbionts(Genchi
M. et al, 2005). In the intrapulmonary bronchi was observed an intraluminal hemorrhagic
infiltrate caused by the parasitic migrations, the proliferation of the ciliary pseudolayered
epitheliumandaperibronchialhyperplasiaoftheconnectivetissue.Thesubpleuralpulmonary
emphysema and the fibrosis of some surface areas of the pleura were also present. In the
pulmonarytissuetherewascongestionandsubacutechronicinterstitialpneumonia,followed
bytheinterstitialvessels(Acatrineietal.,2005).

Thelongevityofadultfilariaisofapproximately5yearsinthecardiovascularsystem
ofthedog(figureno.1)andthelongevityofmicrofilariaisofapproximately2years.Inother
hosts, the longevity is shorter (Liu, 2001). So, a dog who gets infested today and remains
untreated,willbeasourceofinfestationfortheintermediaryhosts,forthenext5years.

Fig.1:MicrofilariaofD.immitis,blood
smear.ModifiedKnotttechnique.

Consideringthe2casesofcardiovasculardirofilariosisdiagnosedinnativedogsfrom
IaiCountyinMarch2009,ourgoalistoestablishwhethertheseareonlyisolatedcasesorwe
canacknowledgeanewpathologicalhazardfortheanimalandpublichealth.

Ourobjectivesare:
1.FurtherparasitologicalinvestigationsforthediagnosisoftheD.immitisinfestations
in dogs with cardiorespiratory distress presented for examination in the clinics of
FMVIai.
2.Thetestingofstraydogsfromthesheltersadministeredbythelocaladministration
andtheAnimalProtectionAssociations,becausethesedogsarethemostexposedto
theattackoftheintermediaryhostsandcanrepresentthereferalpopulationforthis
disease.

MATERIALANDMETHODS

Biologicalmaterial:
- Stray dogs from the shelters administered by the local administration and the
AnimalProtectionAssociations.
- DogswithcardiacdistresspresentedforexaminationattheclinicsofFMVIai.
The sampling of the dogs from the shelters was made according to the following
criteria:

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UniversitateadetiineAgricoleiMedicinVeterinarIai
age: only the adult dogs, preferably older than 2 years because of the long
prepatenceofparasites
- healthstatus:onlydogswithcardiacdisorders
- representativity : from each shelter, 10% of the dogs new sampled, along 6
months.
Thetestsused:
- SNAPHeartwormRTIDEOXLaboratories,USA
- DirocheckSynbioticsCorporationUSA
The positive diagnostic identifies a cuticle antigen expressed by the living
females, by the ones that died following specific treatment and by the live males,
antigenthatbindstotheimobilisedantibodyonthetestsupport.
Thepositiveresponseisrepresentedbyacoloredarealessormoreintensethanthe
positive standard. The tests alow the identification of the infestation before the
microfilariaproductionandalsotheidentificationofthelowunisexinfestationswhich
donotproducemicrofilaria.

Protocol:
1. The sampling of the resident canine population, the identification, the clinical
examination and the individual registration of cases. From the testing lot, the dogs
were selected following an effort test and the separation of the ones with cardio
respiratorydistressclinicallyexpressed.
2.Theharvestingandidentificationofthebloodsamplesusinggelclotactivatortubes
(forDirocheck)andEDTAvacutainers(forbloodsmearsandfortheSNAPheartworm
RT).
3. The examination of the blood smears using whole blood or hemolysed blood
enrichedbytheKnottmodifiedmethod.
4.Thenegativesampleshavebeensubmittedtoserologicalrapidtestsaccordingto
theproducersrecommendations.
5.Thecentralizationandtheinterpretationoftheresults.

RESULTSANDDISCUSSIONS

Theresultsfromthemicroscopicexaminationofthebloodsmearsandtheserological
testingarepresentedintable1.

42, 85% of the samples harvested from dogs with owners were positive. The
percentage is higher than the one described by the screenings of the traditional endemic
areas,situationexplainedbythefactthatwetestedonlydogssuspectedofdirofilariosis.Our
studyisnotascreening,butisaproofthatdirofilariosisispresentindogsfromIaiandsoon
willbearealhazardforthepublichealthandaprofessionalchalengefortheveterinarians.

The existence of 16 serologically positive samples (5,28% of the tested samples or


21,64% of the positive samples) that were initially negative at the microscopic examination,
indicate infestations with parasites in the prepatenic stage, unisex infestations or strong
reactivityofthehostwhichinhibitedthereproductionofparasites.Wecalledthe3situations
ocult dirofilariosis. The hidden dirofilariosis justifies the annual or twice a year serological
screeningusingtherapidteststhattakeonly815minutesforaresultwithhighsensibility(98,
7%)and100%specificity.

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Table1:PrevalenceofdirofilariosisatdogsfromIai
Positive
No.ofanalysed
Negative
samples
Direct Test
Shelter1
120
14
9
97
Shelter2
120
11
7
102
Clinic
63
27
0
36
Location

Total

303

52

16

235

%positivesamplesfromthe
analysedsamples
23.33
15,00
42.85
22.44

CONCLUSIONS

1.Therewere68positivecasesforDirofilaria.immitisamongstraydogsanddogswith
owners from Iai County, fact that proves that the disease is evolving and spreading among
dogs.

2.Intheinvestigatedlotsthereare5,28%infestationswithDirofilaria.immitis.

3. The annual or biannual testing for the identification of hidden infestations with
Dirofilaria.immitisisamustinordertoprotecttheanimalandpublichealth.

REFERENCES

1.

Acatrinei,D.,Paca,S.A.,Miron,L.D.,MihalachiDimitriuSimona,2008Investigaii
morfologiceninfestaiacuDirofilariaimmitislacine.Lucr.t.USAMVIai,vol51(10),Ed.Ion
IonescudelaBrad,Iai,ISSN14577406,200206
2. BritoA.C.,FontesG.,ElianaMMdaRocha,DeisyAMRocha,LdaRegis,1999,developmentof
Dirofilariaimmitis(Leidy)inAedesaegypti(L.)andCulexquinquefasciatus(Say)fromMacei,
Alagoas,Brazil,MemInstOswaldoCruz,RiodeJaneiro,Vol.94(4):575576
3. ByeonK.H.,,BongJinKimB.J.,,SunMiKimS.Mi,YUHakSun,JeongHaeJin,OCKMeeSun,
2007, A serological survey of Dirofilaria immitis infection in pet dogs of Busan, Korea, and
effectsofchemoprophylaxisKoreanJournalofParasitology,Vol.45,No.1:2732
4. DulceanuN.,CristinaTerinte,1994,Parazitologieveterinar,Vol.1,2,Ed.Moldova,Iai
5. Genchi M., Kramer, L., Simn, F., Tamarozzi, F.,., Bazzochi, C., 2005 Is Wolbachia
complicatingthepathologicaleffectsofDirofilariaimmitisinfections?.VeterinaryParasitology
133,133136
6. KazacosKR,EdbergEO.,1979,DirofilariaimmitisinfectioninfoxesandcoyotesinIndiana,JAm
VetMedAssoc.1;175(9):909910
7. Kesdangsakonwut S., S. Chungpivat, S. Lacharoje, R. Siriwattanarat, Y. Utrara, S.
Chotiapisitkul, 2006,Dirofilaria immitis infection in captive Asiatic jackal (Canis aureus),
ProceedingsofAZWMPChulalongkornUni.Fac.ofVet.Sc.,Bangkok,Thailand,p.19
8. Kreeger T.J., DelGiudice G.D., Mech L.D., 1997, Effects of fasting and refeding on body
compositionofcaptivegraywolves(Canislupus),Can.J.Zool.,75,15491552
9. Kim KyoungHo, YunMi Lee, SeungTae Oh, Cheol Jeong, TeaHo Han, Sung Mo Lee,
InvestigationofDirofilariaimmitisinfectionindogsofIncheonarea,Korean,2009,J.Vet.,Serv.,
32(4),385389
10. Liu J. , K.H. Song , S.E. Lee , J.Y. Lee , J.I. Lee , M. Hayasaki , M.J. You , D.H. Kim, 2005,
Serological and molecular survey of Dirofilaria immitis infection in stray cats in Gyunggi
province,SouthKorea,VeterinaryParasitology130,125129
11. MazzariolS.,CassiniR.,VoltanL.,AresuL.,FrangipanediRegalbonoA.,Heatworm(Dirofilaria
immitis)infectioninaleopard(Pantheraparduspardus)housedinazoologicalparkinnorth
easternItaly,Mazzarioletal.Parasites&Vectors2010,
http://www.parasitesandvectors.com/content/3/1/25

1222

UniversitateadetiineAgricoleiMedicinVeterinarIai
12.

13.
14.
15.

16.
17.
18.
19.
20.

MinKyungKim,ChulHwanKim,BeomWooYeom,SeongHwanPark,SangYongChoi*,Jong
SangChoi,2002,TheFirstHumanCaseofHepaticDirofilariasis,JKoreanMedSci,17:686690,
ISSN10118934
MurataK.,JanaiT.,AgatasumaT.,UniS.,2003,Dirofilariaimmitis,InfectionofasnowLeopard
inaJapanesezoowithMitchondrialDNAAnalysis,J.Vet.Med.Sci.,65(3),945947
Oge H., Oge S., Yildirim A., Kircali F., Kara M., 2005, Immunoblotting Analysis of Somatic
ComponentsofDirofilariaImmitis,Parasite,12,179182
Paca, S.A. Acatrinei, D., Miron, L., MihalachiDimitriu Simona, 2008, Both cardiovasculary
andsubcutaneousformsofdirofilariosisindog:acasereport,Lucr.t.USAMVIai,vol51(10),
Ed.IonIonescudelaBrad,Iai.ISSN14577406,123127
Peterson R.O., Thomas N.J., Thurter J.M., Vucetich J.A., Waite T.A., 1998, Population
limitationandthe wolwesofIsleRoyale,JournalofMammalogy,vol.79,No.3,828841
Rhee J.K., Yang S.S., Kim H.C., 1998, Periodicity exhibited by Dirofilaria immitis microfilariae
identifiedindogsofKorea,TheKoreanJournalofParasitology,Vol.36,No.4,235239
Starr T.W., Malley R.C., 1988, Dirofilaria immitis in the Dingo (Canis familiaris dingo) in a
TropicalregionoftheNorthernTeritory,Australia,JournalofWildlifeDiseases,24(1),164165
Vezzani D. and Anbal E. Carbajo, 2006, Spatial and temporal transmission risk of Dirofilaria
immitisinArgentinaInternational,JournalforParasitology,Volume36,Issue14,14631472
Experimental Infection of Dogs, Cats, Ferrets, and Rodents with Dirofilaria immitis, Brugia
malayi, B. pahangi, or Dipetalonema reconditum, Filariasis research reagent resource center:
ugastandardoperatingprocedure.

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STUDIESCONCERNINGTHEHUMORALIMMUNERESPONSEAT
GOATSAFTERVACCINATION
AGAINSTGANGRENOUSMASTITIS

A.Tudose,TurcuD.,T.Perianu2,MarianaOporanu1,P.Grigorescu1,D.Condur1,T.Petru11
FacultyofVeterinaryMedicine,SpiruHaretUniversity
2
FacultyofVeterinaryMedicine,IasiUniversity
drturcudan@yahoo.com

Abstract
The humoral immune response at goats was studied using the following dynamics of some
effectors(immunologicalproteinserumfractionsIgG,totalprotein,lysozyme)2groupsofgoats
were studied:group Athat didnt get the vaccine was used as the counted group and group B
that was inoculated. The second dose of vaccine was given 21 days after the first dose. The
experimentlasted65days,duringwhich4bloodsamplesweretaken.Thedatawasstatistically
processedusingtheStudentFishermethod.Usingthecomputeranalysisoftheelectrophoresis,
aslightincreasewasdeterminedinaveragevaluesof1globulin,2globulin,
globulinandespeciallyofglobuliningroupBincomparisonwithgroupAaftertheseconddose
ofvaccine.Afterthesecondsample,itcomeoutthattheaveragevaluesofIgGandofthetotal
proteinincreasedbyahighlysignificantlyvalue(p<0,001)ingroupBincomparisonwithgroupA.
After the third and the fourth sample the lysozyme concentration presented an important
increase,statisticallyspealing(p<0,001),ingroupBincomparisonwithgroupA.

Keywords:proteicserumfractions,IgG,lysozyme

INTRODUCTION

The animal immunological profile consists of the all the data that characterizes the
cellular and humoral effectors of immunity. The changes that can occur in one or more
segmentsoftheimmunemechanismestablishthebodysabilitytorecognizeandtoneutralize
the harmful factors and to give a suitable answer when prophylactic biological products are
inoculated. The main methods that are used for the immunological profile definition can be
groupedaccordingtomultiplecriteria,fromwhichthemostimportantistheonewhichrefers
totheresearchedeffectors.Thustheeffectorsinvolvedintheimmuneresponsearedividedin
cellular effectors (lymphocyte) and humoral effectors (immunoglobulins, complement,
lysozyme, cytokine). Gjessing and all (1978) showed that goats have serumwounds, present
some changes in the serum protein fractions concentration and also in the lipids one. The
values of the total protein and the electrophoretic model in goats where also studied
dependingonageandsex(Castro,1997).ThealbuminandtheA/Gratiopresentedimportant
differences depending on sex. The value of the total protein in goats of different ages was
significantlyvaried(p<0,005).Quilesandall(2009)studiedthecontentintotalproteinsfrom
the goats lactoserum during the lactation. The protein fractions increased significantly
(p<0,01) during thus period. The proteins of acute phase represents another important
diagnosis indicator in animals' disorders. Gonzales and all (2008) studied the changes
producedintheproteinsconcentrationsoftheacutephaseaftertheyweresubduedtosome
internalandexternalfactors,thatistheconcentrationinhaptoglobinandCreactiveprotein.
Mauser(1986)andVassiliadou(2009)determinedtheprevalenceandthecausesofsubclinical

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Lucrritiinificevol53seriaMedicinVeterinar
mastitisin170samplesofgoatmilkandfoundoutthat80%ofStaphylococcusarenegative
coagulase,16%arepositivecoagulaseand2%arehaemolytics.ThereforeafterStaphylococcus
infection,theparameterofthecellularandhumoralimmuneresponseundergochanges.
In this paper the research's results are presented regarding the humoral immune
responseatgoatsaftergangrenousmastitisvaccination.

MATERIALANDMETHODS

Animals.Theexperimentwasdoneon2groupsofgoats:groupA(n=10)thatdidn't
get the vaccine was used as the counted group and group B got the vaccine against
gangrenousmastitis.Theseconddoseofvaccinewasgiven21daysafterthefirstdose.Blood
samplesweretakenfromtheseanimals,inday0(sampleI),day21(sampleII),day47(sample
III)andday64(sampleIV).
The1%agarosegelelectrophoresiswasdoneinTrisbarbitalswab,pH=8,6,usingthe
horizontalelectrophoresismachine,Line1.1.Themigrationtimewas30minutesat100Vand
37mA.Aftertheslidedriedout,theywerecolouredwith1%10BAmidoblacksolution.The
electrophoregramswereobtainedthroughcomputerintegrationandrelative(%)andabsolute
(g/dl)valuesofserumproteinfractionswereshowed.
Simpleradialimmunodiffusiontest(IDSRMancini)forIgGdosagewasdoneusinga
plate 5 cm in diameter. For making this test possible, we used a reagents set prepared in
FacultyofVeterinayMedicineSpiruHaretlabswhichcontains:rabbitserumgoatantiIgGand
goatserumasacontrolserumwithaknownconcentrationofIgG.TheIgG(g/dl)concentration
valuesfromthesampleswerereadonastandardcurvedependingontheprecipitationring
diameters(mm).
Thelysozymemeasurementusingthemethodofplatelysisdeterminationwasdone
using plates 9 cm diameter in which we moulded 2% agar gel, prepared in phosphate swab
pH=6,2,inwhichtheMicrococcuslysodeicticusculturewascomprised.Theserumswereputin
gelpitting.Thereactionreadingwasdoneafter24hoursbymeasurementoflysisdiameters
(mm)andthevaluesoflysozymeconcentration(g/ml)werereadonastandardcurve.Asa
controllysozymeweused100g/mlsolutionofpurifiedlysozyme(Merck).
The biuret method, for the protein concentration measurement we used the
colorimetry(Spekol)atwavelengthof545nm.
The statistic analysis was done using the average calculation (x) the standard
deviation(cv%)andthesignificancecoefficienttestswerecalculatedusingtheStudentFisher
method(ttest).

RESULTSANDDISCUSSION

The computer integration of electrophoresis showed 5 different protein fractions,


sortedoutdependingontheirelectriccharge:albumin,1globulin,2globulin,globulinand
globulins. The relative (%) and absolute (g/dl) average values of protein fractions in goats
fromgroupAandgroupBaresynthesizedintable1and2.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
Table 1. The relative (%) and absolute (g/dl) average values (xds) and the variability
coefficient(cv%)oftheproteicfractionsingoatsfromgroupA.

(xds)
Fractions
Samples
I
II
III
IV
Albumina
%
77,620,59
73,150,64
58,740,77
78,070,61
g/dl
5,320,28
5,720,03
5,620,27
5,601,25
cv(%)
5,61
4,49
4,80
4,58
%
1globulina
4,940,44
4,750,40
4,740,45
1,930,08
g/dl
0,340,02
0,140,02
0,420,05
0,370,06
cv(%)
11,90
16,21
15,88
14,28
%
2globulina
8,190,17
4,390,16
6,640,95
6,000,87
g/dl
0,690,05
0,340,05
0,470,06
0,430,09
cv(%)
7,24
14,70
12,5
11,62
%
globulina
1,430,40
0,770,03
1,260,21
4,680,44
g/dl
0,090,02
0,300,01
0,120,04
0,060,01
cv(%)
33,33
16,66
22,22
22,94
%
globuline
26,691,81
12,020,24
12,751,37
14,241,31
g/dl
1,700,06
1,730,04
1,790,07
1,860,28
cv(%)
2,65
4,30
9,85
7,18

Table 2. The relative (%) and absolute (g/dl) average values (xds) and the variability
coefficient(cv%)oftheproteinfractionsingoatsfromgroupB.

(xds)
Fractions
Samples
I
II
III
IV
Albumina
%
66,384,37
73,960,51
72,471,42
70,234,49
g/dl
5,390,15
5,350,30
5,200,18
5,010,51
cv(%)
2,78
5,24
3,11
8,76
%
1globulina
7,743,99
5,210,27
4,901,43
5,341,73
g/dl
0,630,34
0,400,07
0,370,03
0,400,10
cv(%)
23,96
17,5
18,1
25,00
%
2globulina
9,340,17
7,301,05
6,310,60
5,911,71
g/dl
0,760,04
0,560,02
0,480,05
0,440,10
cv(%)
5,26
3,57
10,46
22,72
%
globulina
2,310,36
2,140,25
1,600,53
1,960,65
g/dl
0,120,01
0,150,05
0,190,36
0,160,02
cv(%)
30,47
12,5
18,3
33,33
%
globuline
16,400,59
13,371,36
13,712,56
14,203,37
g/dl
1,720,09
1,820,09
1,820,21
2,260,22
cv(%)
7,75
10,3
23,33
27,5

Theelectrophoreticanalysisofalbuminconcentrationafterwetookthefirstsample
of blood from goats in group A and B showed close values (group A: 5,32g/dl and group B:
5,39g/dl)(figure1).

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Lucrritiinificevol53seriaMedicinVeterinar
6
5.32 5.39
5

g/dl

Lot A

Lot B

1.7

1
0.42

0.63

1.72

0.69 0.76
0.12 0.19

0
Albumin

1-globulin

2 -globulin

-globulin

-globulins

Serum protein fractions

Figure1.Theabsolute(g/dl)averagevaluesofserousproteinfractionsingoatsfromgroupA
andB(sampleI)

The variation rate of the relative (%) average values (xds) of 1globulin was
between 4,940,44% and 7,743,99% meaning 0,42g/dl in group A and 0,63g/dl in group B.
The 2globulin concentration showed average values (g/dl) 0f 0,69 g/dl for group A (8,19%
0,1) and of 0,76g/dl for group B (9,34 0,17). The globulin level recorded
homogenousconcentrationsbetweenthe2groups(groupA0,12g/dl0,04meaning1,43%
0,40)andgroupB0,19g/dl0,36meaning2,310,36).
IngroupAtheglobulinsconcentrationwas1,70g/dl0,06(26,691,81)andingroup
B1,720,09(26,400,59).
Infigure2weshowedtheabsoluteaveragevaluesoftheproteinfractionsfromthe
serumatgoatsfromgroupAnotvaccinatedandfromgroupB21daysafterthefirstdoseof
vaccine(sampleII).

5.72
5.35

4
g/dl

Lot A
Lot B

1.73

1
0.37

0.63

0.34

1.82

0.56
0.06

0.12

0
Albumin

1-globulin

2 -globulin

-globulin

-globulins

Serum protein fractions

Figure2.Theabsolute(g/dl)averagevaluesofserousproteinfractionsingoatsfromgroupA
andB(sampleII)
A slight decrease of albumin concentration can be see in group A 5,72g/dl
(78,070,61) in comparison with group B 5,35g/dl (73,060,51). The 1globulin values from
thesecondsampleremainedconstantincomparisonwiththefirstsampleingroupAandB.

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UniversitateadetiineAgricoleiMedicinVeterinarIai
The globulins level was higher in group B 1,82g/dl (13,37%1,36) than in group A
1,73g/dl0,04(12%0,24)table1and2.
From the data analysis in table 1 and 2 and figure 3 a decrease of albumin
concentration can be seen after the third sample was taken in group B 5,20g/dl0,18
(72,42%1,42) in comparison with group A 5,62 g/dl0,27 (77,620,59). Slightly increased
values of 1globulin, 2globulin and globulin can be seen, also in group B in comparison
withgroupA.Theglobulinsconcentrationhadincreasedvalues1,79g/dl0,07ingroupAand
1,82g/dl0,21ingroupBinthesecondsample(figure3).

5.62
5.2

g/dl

4
Lot A

Lot B
1.79 1.82

1
0.34

0.48

0.47 0.48
0.09 0.12

0
Albumin

1-globulin

2 -globulin

-globulin

-globulins

Serum protein fractions

Figure3.Theabsolute(g/dl)averagevaluesofserousproteinfractionsingoatsfromgroupA
andB(sampleIII)
In figure 4 there are shown the absolute (g/dl) average values of serous protein
fractions in the group studied in the fourth sample. In the group B the decreased level of
albumin (5,01g/dl1,25) if we analyse the data a slight increase of the average values in 1
globulin, 2globulin, globulin and especially in globulins can be seen in group B
(2,26g/dl0,22 meaning 14,20%3,37) in comparison with group A (1,86g/dl0,28 meaning
14,24%1,31). The variability coefficient (cv%) was calculated in the analysed group. This
experimentprovesthatthetestsresultsareconstant,ensuringsamedataacquirementwhich
isinaccordancewithhomogeneousvaluepopulations(table1and2).

5.6
5.01

g/dl

4
Lot A

Lot B
2.26
1.86

1
0.14

0.4

0.43

0.44
0.1

0.15

0
Albumin

1-globulin

2 -globulin

-globulin

-globulins

Serum protein fractions

Figure4.Theabsolute(g/dl)averagevaluesofserousproteinfractionsingoatsfrom
groupAandB(sampleIV)

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Lucrritiinificevol53seriaMedicinVeterinar
Intable3andfigure5,5,7theresultsarepresentedindynamicsincomparisonwith
eachotherconcerningtheaveragevaluesofIgG(g/dl)andlysozyme(g/ml)ingroupAandB,
andthestatisticmeaningofthedifferencebetweenthem.

Table3.Thecomparativeresultsconcerningtheaveragevalues(xds)ofIgG,totalproteinand
lysozymingroupAandBandthestatisticmeaningofthedifferencebetweenthe2groups
Para
meter

IgG
(g/dl)
Total
protein
(g/dl)

Samples
I

II

III

IV

LotA
LotB
LotA
LotB
LotA
LotB
1,690,04 1,710,05 1,690,05 1,770,13 1,700,22 1,890,25
p<0,5*
p<0,05**
p<0,001***
7,700,25 7,730,35 7,730,13 7,890,10 7,730,23 8,490,27
p<0,5*
p<0,05**
p<0,001***

Lyso
5,60,95 5,60,90
zym
p<0,5*
(g/ml)

5,60,79

7,81,25

5,70,26

p<0,05**

LotA
LotB
1,711,72 1,890,08
p<0,025***
7,740,24 8,360,36
p<0,001***

10,860,05 5,70,38

p<0,001***

8,60,05

p<0,001***

*=unsignifiantdifference**=distinctsignifiantlydifference***signifiantlyhighdifference

The IgG concentration in group A was 1,690,04 and in group B 1,21g/dl0,05, the
difference between the 2 groups was unsignificant (p<0,5) in sample I. In sample II the IgG
concentration in group B (1,77g/dl0,05) increased significantly (p<0,05) in comparison with
group A (1,69g/dl0,05). The IgG concentration in group B increased significantly high
(p<0,001)incomparisonwithgroupA(1,700,22)inthethirdsample.Inthefourthsamplea
significantly high difference was seen between group B (1,89g/dl0,08) and group A
(1,71g/dl1,72)(figure5).

p<0,025

2.5

p<0,001
p<0,5

2.26

p<0,05

1.89
1.71

1.69

1.77

1.69

1.71

1.7

1.5
g/dl

Lot A
Lot B
1

0.5

0
I

II

III

IV

Samples

Figure5.TheIgG(g/dl)valuesdynamicsgoatsfromgroupAandBforthose4samples

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UniversitateadetiineAgricoleiMedicinVeterinarIai
ThetotalproteinconcentrationingroupB(3,49g/dl0,27)increasedsignificantlyhigh
(p<0,001) in comparison with group A (7,79g/dl 0,13) in the third sample, the difference
between group B (8,36g/dl1,36) and group A (7,74g/dl0,23) stays significantly high
(p<0,001)figure6.

8.6

8.49

p<0,001
8.36

8.4

8.2

p<0,5

p<0,05

g/dl

Lot A

7.84
7.8

7.73

7.7

7.73

Lot B

7.74

7.73

7.6

7.4

7.2
I

II

III

IV

Samples

Figure 6. The total protein concentration (g/dl) for goats from group A and B for those 4
samples

p<0,001
p<0,001
12
10.86

10.86

g/dl

10

p<0,05

p<0,05

5.6

5.6

5.6

5.8

5.7

Lot A

5.7

Lot B

0
I

II

III

IV

Samples

Figure7.Thelysozymeconcentration(g/ml)forgoatsfromgroupAandB

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Lucrritiinificevol53seriaMedicinVeterinar
In the third and fourth sample the lysozyme concentration showed an important
increasestatisticallyspeaking(p<0,001)ingroupB(sampleIIIandIV)(10,86g/mlingroupB
incomparisonwith5,7g/mlingroupA).
The results herby presented prove that the vaccination against gangrenous mastitis
has determined a meaningful increase of some humoral immunologic parameter at a
significanthighstatisticallevelincomparisonwiththecriticalvalues.

CONCLUSIONS

The study of the dynamics of serous protein fractions at the goats from the
inoculatedgroup(B)againstthegangrenousmastitisandtheuninoculatedgroup(A)showed
theincreaseofglobulinsconcentrationandthedecreaseofalbuminlevel.Thevaccination
induced humoral immune high significant statistic response by the increase of IgG
concentration, total protein and lysozym. Studied immunologic parameter revealed a
significant increase after the second dose of vaccine. The study of the humoral immune
effectors can reveal correct evaluation criteria of the efficiency of inoculation against
gangrenousmastitis.

REFERENCES
1

4.
5.
6.

Castro A., D.S. Dhindsa, A.S. Hoversland, J. Metcalfe Serum proteins and protein
elctrophoreticpatterninnormalpygmygoats.AmericanjournalofveterinaryResearch,1997,
38,(5),665667.
Gjessing E.C., S Ludewig, A. Chautin Fractionation, electrophoresis and chemical studies of
proteinsinseraofcontrolandinjuredgoats.JournalofBiologyandChemistry,1978,170,551
565.
GonzalesF.H.D.,F.Tecles,SilviaMartinezSubiela,A.Tvarijonavicinte,LauraSoler,J.J.Ceron
AcutephaseproteinresponseingoatsJournalofVeterinaryDiagnosticinvestigation,2008,
20,5,580584.
QuilesA.,C.Gonzalo,Y.Barcina, F.Fuetes, M.Hevia Proteinquality of Spanish Murciana
Granadinagoatmilkduringlactation.SmallRuminantResearch,2009,14,1,6772
ManserP.A.Prevalence,causesandlaboratorydiagnosisofsubclinicalmastitisinthegoat
TheVeterinaryRecord,1986,118,20,552554.
VassiliadouK.D.MastitisrelatedpathogensingoatmilkSmallRuminantResearch,2009,4,
2,203212.

1231


THEEVOLUTIONOFRABIESINTHENEIGHBORINGCOUNTRIESOF
ROMANIAINTHEPERIOD20052008

IuliaAdelinaTURIAC ,F.MAZDRAG ,C.VASIU .


1
NationalSanitaryVeterinaryandFoodSafetyAuthority
2
FacultyofVeterinaryMedicine,ClujNapoca
1

TheevolutionofrabieswasfollowedintheneighboringcountriesofRomania(Ukraine,Moldova
Republic,Bulgaria,SerbiaandMontenegroandHungary)duringtheperiodoftime20052008.
For this period of time there were recorded an total number of 10201 cases of rabies,
distributionwhosesufferingonlyminordifferencesfromyeartoyear.
Ukrainerepresentthecountrywiththelargestnumberofcases,9229,withahigherproportion
ofcasesindomesticanimals(55.4%fromthetotalnumberofcases),thecatbeingplacedinfirst
place with 2167 cases (23.48% of the total number). In wild animals, most cases have been
recordedinfoxes(respectively3668cases,whichrepresent39.74%ofthetotalnumberofcases
recorded).
Thehighestincidencewasfoundinyear2007(35,02%)andthelowestin2006(23.76%).
Hungary and Bulgaria had the lowest number of recorded cases, which are due to the
implementationofasustainedprogramtoeradicaterabiesbyoralvaccinationoffoxes.

Keywords:rabies,neighboringcountries,distribution,cases,fox

In the late nineteenth century, the rabies virus reservoir was represented by dog
(Canis canis) in most European countries and the main wildlife reservoir the wolf (Canis
lupus).In these areas, rabies control programs in which parenteral mass vaccination
campaignsandreductionofthepopulationofstraydogshavebeenshowntobeeffective.
Duringthesecondhalfofthetwentiethcentury,rabiesamongthecaninepopulation
hasdisappearedgraduallyinmostEuropeancountriesandineasternPolandoccurredrabies
in foxes which was spread rapidly in all directions with an 3060 km annual prevalence
rate.Except the British Isles (UK, Ireland) and Scandinavia, most European countries were
infected.
Thefox(Vulpesvulpes)isthemainspeciesinvolvedintheepidemiologyofrabiesin
western and central Europe and is the origin of rabies transmission to other species of wild
and domestic animals, including, pets, cattle, sheep and goats.Raccoon dog (Nyctereutes
procyonoides)isanimportantfactorinrabiesepidemiologyandepizootologyineasternand
northernEurope.
Despite the substantial progress in the twentieth century that have been made in
reducingtheburdenofcostsistocombatrabies,particularlyinCentralandEasternEurope,
thediseaseremainsendemicinmanycountries.

MATERIALSANDMETHOD

AnalysisontheevolutionofrabiesinanimalsintheneighboringcountriesofRomania
was based on data supplied by them to "Rabies Information System of the WHO Centre for
RabiesSurveillanceandCollaborationResearch"datapublishedonthewebsiteoftheRabies
BulletinofEurope.
Collected data were statistically processed by comparing the number of cases
reportedbyeachcountry,forthemaindomesticandwildspeciesofanimals,everyyear,using

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Lucrritiinificevol53seriaMedicinVeterinar
graphsandchartstohighlightthecasesfrequenceofrabiesinanimalspecies,theirshareand
temporalevolution.
Because the number of cases of rabies in wild animals other than foxes are small,
thesecasesweregroupedunder"otherwildlife"thusingraphicrepresentationsarepresented
threemaincategoriesnamely:"Fox","Otherwildanimalsand"Domesticanimals."

RESULTSANDDISCUSSION

The temporal evolution and share of rabies cases during the period of time 2005
2008inUkrainearerepresentedingraphandchart1.
In2005inUkrainewerediagnosedpositivea