319

CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
9
Mycobacteriosis and Nocardiosis
S. Chinabut
Aquatic Animal Health Research Institute, Department of Fisheries, Kasetsart
University Campus, Jatujak 10900, Bangkok, Thailand.
MYCOBACTERIOSIS
History
The discovery of the tubercle bacillus by Robert Koch in 1882 stands as the most
important contribution to the study of tuberculosis in any host. Before the end of
the eighteenth century, piscine mycobacteria had been described by a group of
French scientists (Bataillon et al., 1897). They reported that Mycobacterium
piscium (a name which is now obsolete; Van Duijn, 1981) was a pathogen of
diseased carp, Cyprinus carpio. Since that time, much research on mycobacterial
diseases in fish has been carried out.
Von Betegh first reported mycobacteriosis in marine fish in 1910.
Mycobacterium marinum was isolated and described in 1926 by Aronson as
the cause of tuberculosis-like lesions in the liver, spleen and kidney of
tropical coral fish kept in the Philadelphia aquarium. This organism was
initially thought to infect only marine fish, but it has since been isolated from
freshwater species and human beings (Barrow and Hewitt, 1971; Van Duijn,
1981). Mycobacterium fortuitum was another acid-fast bacillus repeatedly
found from diseased neon tetra, Parocheirodon innesi, in 1953, although the
taxonomic identification of the species was later described by Ross and
Brancato (1959).
Many acid-fast bacteria have been recorded as the aetiological agents
causing mycobacteriosis in fish. However, some of them were synonymous
with either M. marinum, M. fortuitum or Mycobacterium chelonei and
some species names were not considered valid by the International Working
Group on Mycobacterial Taxonomy. These included M. piscium, Myco-
bacterium salmoniphilum and Mycobacterium anabanti (Thoen and
Schliesser, 1984).
320
S. Chinabut
Species of fish affected and geographical distribution
Mycobacterium is ubiquitous in the water and sediment, and so mycobacteriosis
in fish populations continues to be documented worldwide, and no country can
claim that this long-recognized bacterial fish disease has been eradicated as a
cause of illness and death. Since many fish live in close contact with soil and
water during their entire lives, these environments should be considered
potential sources of infection. Some 167 species of both freshwater and salt-
water fish have been reported as hosts for this disease, and it has been described
from a wide variety of aquarium fish (Aronson, 1926; Besse, 1949; Nigrelli and
Vogel, 1968; Giavenni et al., 1980) and food fish (Alexander, 1913; Johnstone,
1913; Sutherland, 1922; Wood and Ordal, 1958; Hublou et al., 1959; Parisot and
Wood, 1960; Lawhavinit et al., 1988; Chinabut et al., 1990; Hatai et al., 1993).
Mycobacteriosis was reported in the freshwater prawn. Macrobrachium rosen-
bergii (Brook et al., 1986) and is widespread in all penaeids (Lightner, 1996).
As mycobacteriosis is a subacute to chronic disease, it seems likely that fish
maintained in aquaria will show a higher incidence of this disease than cultured
or wild species, because aquarium fish are often kept for long periods of time
compared with fish raised for commercial purposes.
Wild stocks of fish have been reported to suffer from mycobacteriosis,
including cod, Gadus morhua (Alexander, 1913); halibut, Hippoglossus
hippoglossus (Sutherland, 1922); striped bass, Morone saxatilis (Aronson,
1926); North-East Atlantic mackerel, Scomber scombrus (MacKenzie, 1988);
and yellow perch, Perca flavescens (Daoust et al., 1989). In intensive fish-
culture systems, mycobacteriosis was first documented in the salmon industry of
the Pacific North-West (Parisot, 1958; Wood and Ordal, 1958). Mycobacterial
disease has also been recorded from pejerrey, Odonthestes bonariensis
(Lawhavinit et al., 1988; Hatai et al., 1993) and snakehead fish, Channa striatus
(Chinabut et al., 1990), which are both cultured in ponds.
The incidence of mycobacteriosis in aquarium fish has been reported to vary
from 10 to 22% (Wolke and Stroud, 1978; Santacana et al., 1982). The
prevalence of infected fish in natural populations varies from 10 to 100%
(Abernethy and Lund, 1978; Sakanari et al., 1983; Hedrick et al., 1987,
Lawhavinit et al., 1988; MacKenzie, 1988). There appears to be no bias towards
the sex of the fish in the prevalence of mycobacteriosis, but the severity of the
infection is apparently related to age (Abernethy and Lund, 1978; MacKenzie,
1988).
Disease outbreaks in cultured fish appear to be related to management
factors, such as, the quality and quantity of nutrient and water supplied and the
stocking density. Poor management results in abnormal stress and a reduction in
the normal resistance of the host.
Clinical signs
Piscine mycobacteriosis is a slowly developing chronic disease, which may take
2 or more years for the number of organisms to grow to readily detectable
321 Mycobacteriosis and Nocardiosis
numbers (Ashburner, 1977). Most species of fish may manifest few or no
external signs of disease. However, in advanced stages, emaciation, cachexia,
exophthalmia, lordosis, haemorrhagic and dermal ulcerative lesions or loss of
scales may be observed. Other signs of infection can be seen in the gills, which
are paler than normal and show thickened areas on some filaments. Small lesions
may be observed around the mouth and vent. Changes in cutaneous
pigmentation include a fading of normal colour in aquarium fish (Snieszko,
1978) and a bright coloration in salmonids (Ross, 1970). Affected fish generally
exhibit lethargic behaviour, floating impassively on the surface of the water,
with concurrent loss of appetite.
Gross pathology
The gross signs of infection include an enlargement and softening of spleen,
kidney and liver. Sometimes a number of greyish-white nodules are seen
peppered throughout these organs. In severe cases, almost all the visceral organs
are swollen and fused by whitish membranes around the mesenteries, and fluid
accumulates in the peritoneal cavity.
Histopathology
Mycobacteriosis lesions in fish are normally localized in the skin (Fig. 9.1) and
internal organs (Fig. 9.2.) and consist of nodular structures with characteristic
Fig. 9.1. Subcutaneous granuloma in Siamese fighting fish infected with Mycobacterium. 130
(haematoxylin and eosin (H & E) stain).
322
S. Chinabut
Fig. 9.2. (and Opposite) Tubercle granuloma in snakehead fish infected with Mycobacterium: (a)
gills 130, (b) muscles 250 and (c) brain 500 (H & E stain).
(a)
(b)
323 Mycobacteriosis and Nocardiosis
focal granulomas. These are composed of centrally located epithelioid cells and
macrophages. The size of the granulomas vary from 80 to 500 m (Sakanari et
al., 1983). The condition of mycobacteriosis can be divided into subacute and
chronic forms (Wolke and Stroud, 1978). In the subacute form, there is a diffuse
distribution of reticuloendothelial cells and macrophages (Fig. 9.3), with large
caseous necrotic areas. Acid-fast bacilli are found scattered among the
reticuloendothelial cells and within the cytoplasm of phagocytic macrophages.
The chronic proliferative form is characterized by the production of both hard
and soft granulomas. Soft granulomas have four distinguishable layers. In the
centre is an area of caseous necrosis, with or without nuclear debris, surrounded
by a layer of spindle-shaped epithelioid cells. The third layer contains highly
eosinophilic, flattened, epithelioid cells. The outermost layer is composed of
fine fibrous connective tissue encircling to form a thin capsule. Hard granulomas
are composed of epithelioid cells encapsulated by fibrous connective tissue. The
layer of epithelioid cells closest to the zone of caseation and fibrous capsule may
not be seen in all granulomas. Noga et al. (1989) have indicated that cells
participating in the chronic inflammatory response to mycobacteria may not be
derived from mononuclear phagocytes. As the true classification of these cells is
now uncertain, they proposed the name chronic inflammatory foci (CIF), instead
of tubercle granuloma.
Calcification in the caseous necrotic centre occurs in more chronic
infections (Majeed et al., 1981; Van Duijn, 1981; Anderson et al., 1987).
Melanization and vacuolation may be found surrounding the cutaneous
granulomas (Noga et al., 1990). Such granulomas are all of the soft tubercle
type, occurring in the stratum spongiosum of the dermis (Anderson et al., 1987).
Melanomacrophage centres were found close to the granulomas in the spleen
(c)
324
S. Chinabut
and kidney of infected fish. The presence of giant cells is not typical in piscine
mycobacteriosis (Wolke and Stroud, 1978; Anderson et al., 1987). However,
they may be found at the early stage of the granulomatous formation (Timur and
Roberts, 1977).
At the early stages in the development of the disease, the spleen, kidney and
liver are the primary target organs for infection. In severe cases, a number of
granulomas spread to almost all the visceral organs and some of them fuse to
form a large granuloma enclosed by loose connective tissue (Giavenni et al.,
1980). Acid-fast bacilli have been seen occasionally in the caseous necrotic
centre and in the cytoplasm of the surrounding epithelioid cells and
macrophages. These bacilli may also be found free within the swim-bladder
lumen or within the cytoplasm of sloughed epithelial cells (Anderson et al.,
1987).
Characterization and taxonomy
Mycobacteriosis in fish can be caused by several distinct species of
Mycobacterium. This genus is a member of the order Actinomycetales and
family Mycobacteriaceae. Fish mycobacteria can grow in several types of media
(Table 9.1). Identification can be made using a number of criteria, including
acid-fastness, growth rate, pigment production, colonial morphology and
homogeneity of a suspension of the organism in a liquid medium. However, the
definitive identification of each species is based on specific biochemical test
reactions (Table 9.2), the deoxyribonucleic acid (DNA) level and the guanine-
plus-cytosine content. Mycobacterium spp. are mostly resistant to conventional
Fig. 9.3. Macrophage with lipofuscin in the cytoplasm accumulating at the edge of the
granuloma to form a melanomacrophage centre in an infected fish. 500 (H & E stain).
325 Mycobacteriosis and Nocardiosis
Table 9.1. Media used to culture Mycobacteria.
Media References
BactoLowenstein medium (BLM) Anderson et al., 1987
Shamsudin et al., 1990
Dubos agar Abernethy and Lund, 1978
Dorsets egg agar MacKenzie, 1988
Dubos blood agar Giavenni et al., 1980
Harolds egg medium Wolke and Stroud, 1978
International Union against
Tuberculosis Medium (IUTM) medium Giavenni et al., 1980
LowensteinJ ensen medium MacKenzie, 1988
MacConkey agar Shively et al., 1981
Arakawa and Fryer, 1984
Middlebrook 7H10 agar Giavenni et al., 1980
Mycobacteria 7H11 agar MacKenzie, 1988
Ogawa egg medium Shamsudin et al., 1990
Petrignanis medium Hedrick et al., 1987
Petroff egg agar Giavenni et al., 1980
Potassium-tellurite agar Arakawa and Fryer, 1984
Table 9.2. Characteristics of Mycobacterium marinum and Mycobacterium
fortuitum (from Tsukamura, 1966).
Character M. marinum M. fortuitum
Colony morphology Rough Smooth
Pigmentation +
Photochromogeneity
Growth rate Rapid Rapid
Catalase + +
NaNO
3
reduction + +
Third-day arylsulphatase +
Second-week arylsulphatase + +
Salicylate degradation +
PAS degradation +
Ammonium hydroxide (NH
3
OH) resistance +
Picric acid tolerance (0.1%) +
Picric acid tolerance (0.2%) +
Growth at 28C + +
Growth at 37C + +
Growth at 45C
Growth at 52C
Acetamidase +
Benzamidase +
Urease + +
Isonicotinamidase +
Nicotinamidase
Pyrazinamidase +
Allantoinase + +
Succinamidase
continued overleaf
326
S. Chinabut
Utilization of organic acids as sole carbon source
Acetate +
Citrate +
Succinate +
Maltate +
Pyruvate +
Benzoate
Malonate
Fumarate +
Acid from:
Glucose +
Mannose +
Galactose
Arabinose
Xylose
Rhamnose
Trehalose +
Inositol
Mannitol
Sorbitol
Utilization of carbohydrates as sole carbon source
Glycerol + +
Glucose +
Fructose +
Sucrose
Mannose +
Galactose
Arabinose
Xylose
Rhamnose
Trehalose +
Raffinose
Inositol
Mannitol +
Sorbitol +
Ethanol +
Propanol +
Propylene glycol
1,3-Butylene glycol +
2,3-Butylene glycol +
Utilization of nitrogen compounds as sole nitrogen source
L-Glutamate + +
L-Serine +
L-Methionine +
Acetamide +
Benzamide
Urea + +
Pyrazinamide + +
Isonicotinamide +
Table 9.2. Continued
Character M. marinum M. fortuitum
327 Mycobacteriosis and Nocardiosis
Nicotinamide +
Succinamide + +
Nitrate + +
Nitrite +
Utilization of nitrogen compounds as sole nitrogen and carbon source
L-Glutamate +
L-Serine
Glucosamine +
Acetamide +
Benzamide
Nicotinamide
Monoethanolamine +
Trimethylene diamine +
NaNO
3
, sodium nitrate; PAS, periodic acid-Schiff.
Table 9.2. Continued
Character M. marinum M. fortuitum
antibiotics. This resistance can be used as a tool for taxonomic purposes.
Seroagglutination tests are particularly valuable in identifying strains of
mycobacteria (Sato, 1962). It is, however, recommended that the cultures be
submitted to a reference laboratory for final identification.
Morphologically, mycobacteria are pleomorphic, acid-fast, non-motile,
non-sporulated, Gram-positive, non-branching rods, 0.20.6 m in diameter and
1.53.0 m long. They are slender in shape and can be beaded or barred. They
are occasionally seen as filaments up to 10 m in length (Van Duijn, 1981).
Mycobacterium found in diseased fish can be divided into two groups, one
showing a slow growth rate and another that can grow rapidly and requires less
than 5 days for culture.
An important staining characteristic of Mycobacterium is its resistance to
acid decolorizing agents. Several staining procedures are available for
demonstrating acid-fast bacilli, but the ZiehlNeelsen or Kinyoun techniques
with basic fuchsin are most widely used (Thoen and Karlson, 1985).
From a review of the literature, there are six species of Mycobacterium
known to cause mycobacterial disease in fish. Mycobacterium marinum was
isolated from coral fish (Aronson, 1926), M. fortuitum was named and described
in 1938 by Cruz and found infecting diseased neon tetra (Ross and Brancato,
1959), M. chelonei was isolated from salmonid fish (Arakawa and Fryer, 1984)
and Mycobacterium neoaurum from chinook salmon (Backman et al., 1990).
Mycobacterium simiae and Mycobacterium scrofulaceum were first reported
from black acara (Cichlasoma bimaculatum) and Pacific staghorn sculpin
(Leptocottus armatus), respectively (Lansdell et al., 1993). However, the species
of Mycobacterium most commonly isolated from fish are M. marinum and M.
fortuitum.
328
S. Chinabut
Diagnostic methods
Diagnosis of mycobacteriosis depends on clinical and histological signs and
identification of the bacterial pathogen. Mycobacteriosis in fish is normally
localized in the skin and internal organs and appears as nodular structures with a
typical granulomatous pattern. Smears from scrapings of the cut surface of
spleen and kidney tissues should be made and stained with the Kinyoun
modification of the ZiehlNeelsen stain. The smears should be air-dried. The
stained slides are examined with an ordinary light microscope for the presence of
acid-fast bacilli, which appear as coccoidal or bacillary rods, 13 m in length.
Fluorescing dyes, such as auramine or rhodamine, are recommended with either
blue or ultraviolet and a microscope equipped with special filters. Acid-fast
bacilli are seen as short, yellow-fluorescing rods. An immunocytochemical
method using the avidinbiotin complex (ABC) was recommended by Gomez et
al. (1993) to demonstrate the small number of mycobacteria in the section of
affected tissues.
A specific diagnosis of mycobacterial infection requires the isolation and
identification of the organisms from skin lesions, spleen or kidney. The pro-
cedures for isolating mycobacteria are the same as those used for other bacteria.
Specimens must be treated to kill contaminants, as indicated in Fig. 9.4.
Enzyme-linked immunosorbent assays (ELISAs) have been developed for
detecting mycobacterial antigens in exotic animals (Thoen et al., 1980) but not
fish.
Transmission of the disease
Fish may be infected by ingesting feed and water contaminated with faecal
material, urine or exudates from diseased animals that contain mycobacteria
(Ross and Johnson, 1962). The sources and modes of transmission of myco-
bacterial infections in fish may be related to the infection of invertebrates, such
as arthropods (Beerwerth et al., 1979), freshwater snails (Michelson, 1961) or
freshwater prawns (Brook et al., 1986). The entry of mycobacteria through skin
and gill lesions caused by injury or parasitic infection should also be considered.
After the organisms enter the body they may cause skin lesions or spread to other
organs through the circulatory or lymphatic system.
Some scientists have tried to prove that Mycobacterium tuberculosis
excreted from human patients may transform to cause disease in cold-blooded
animals. However, it is now clear that such transformations do not occur (Thoen
and Schliesser, 1984).
The observation of mycobacteria in the piscine ova and tubercle granulomas
in the ovary wall suggests that transovarian transmission is a definite possibility.
Johnstone (1913) reported that the ova of infected fish were small and markedly
undeveloped and contained numerous acid-fast bacteria. A report from an
Australian fish hatchery has provided the evidence that Mycobacterium can be
introduced by eggs and transmitted to the F1 generation (Ashburner, 1977).
However, this observation does not confirm that ovarian transmission takes
329 Mycobacteriosis and Nocardiosis
Fig. 9.4. Isolation procedure for mycobacteria (Thoen and Schliesser, 1984). NaOH, sodium
hydroxide.
place, as the egg surface may be contaminated by peritoneal fluid containing
mycobacteria (Conroy, 1964a). Recently, Chinabut et al. (1994) confirmed the
transmission of mycobacteria in Siamese fighting fish, Betta splendens, via
transovarian passage. Acid-fast bacteria were found in the ova of diseased
female Siamese fighting fish, using the fluorochrome technique.
330
S. Chinabut
Control and treatment
The implementation of preventive measures in controlling chronic myco-
bacteriosis is particularly relevant, due to difficulties in treatment. Anderson et
al. (1987) consider that chemotherapy is often unsuccessful. The importance of
good management practices should be recognized. Fish should be obtained from
farms known to be free of disease. Imported fish should require a period of
quarantine. Trash fish or dead fish carcasses used as a source of protein in the
feed should be heated at 76C for 30 min (Thoen and Schliesser, 1984) to kill
any pathogenic mycobacteria. Formalin or phenolic compounds should be used
as disinfectants in farms at which mycobacteriosis infections have been
recorded. Dead fish should be destroyed by burning or burying in quicklime.
Chloramine B or T at a concentration of 10 mg l
1
for 24 h is recommended for
bath treatment (Van Duijn, 1981).
Therapeutic measures, such as adding tetracycline to the water at a
concentration of 30 mg l
1
, have been efficacious in treating acute stages of the
disease (Van Duijn, 1981; Thoen and Schliesser, 1984). A combination of
antimicrobials, including streptomycin, ethambutol, cycloserine, cotrimoxazole,
rifampicin and tetracyclines, has been used and thought to be effective. Among
these drugs, cotrimoxazole or rifampicin with ethambutol have been used most
frequently (Pattyn, 1984). Isoniazid and rifampicin have been recommended for
treating mycobacteriosis in valuable exotic marine fish (Dulin, 1979).
Pathogenesis and immunity
Intramuscular injections of a suspension of Mycobacterium sp. isolated from
diseased pejerry, O. bonariensis, at concentrations of 10 and 1 mg l
1
demonstrated 100% mortality at 14 and 30 days after the injection, respectively.
The bacterium could be easily reisolated from the kidney of dead fish. In all
injected fish, many typical focal granulomas were found in the site of injection
and in the examined internal organs (Hatai et al., 1988). Another study was
carried out on rainbow trout, which was injected intraperitoneally with M.
chelonei suspension. Total mortality ranged from 20 to 52% at 12C and
increased to 98% by the tenth day after injection at 18C. Mycobacterium
isolates were recovered on Ogawa egg medium and observed using Ziehl
Neelsen stain. Moribund fish often had swollen abdomens and haemorrhaging
lesions around the vents and along the lateral surface (Arakawa and Fryer,
1984).
Vaccines against fish mycobacteriosis are not available at present. However,
Austin and Austin (1987) suggested that there is a possibility of developing a
Mycobacterium vaccine, because fish show a delayed hypersensitivity reaction
after immunization with M. salmoniphilum mixed with Freunds adjuvant
(Bartos and Sommer, 1981).
331 Mycobacteriosis and Nocardiosis
Public health
An important aspect of fish mycobacteriosis is that some of the causative
mycobacteria also cause skin disease in humans. Human infection with M.
marinum and M. fortuitum has been widely reported. Mycobacterium marinum
infections in humans have been known since 1951 and have been described in
some temperate and tropical countries, such as Sweden, the Netherlands,
Belgium, the UK (Pattyn, 1984), Canada (Brown et al., 1977), the USA
(Mollohan and Romer, 1961) and Thailand (Bovornkitti et al., 1991). These
outbreaks were associated with cutaneous abrasions and exposure to swimming-
pool water contaminated with M. marinum. Allergic dermatopathies have also
been reported on the skin of aquarists handling water in which affected fish have
been reared (Barrow and Hewitt, 1971; Giavenni et al., 1980; Huminer et al.,
1986; Kullavanijaya et al., 1993).
The rapidly growing M. fortuitum has been cultured from patients with
pulmonary disease and local abscesses (Cruz, 1938). Mycobacterium chelonei
has been isolated from heterograph heart-valve transplants, and lesions have also
been found in synovial fluid and muscle (Blacklock and Dawson, 1979; Thoen
and Schliesser, 1984).
Topics for further study
Fish mycobacteriosis has been studied for more than a century but the successful
vaccination of fish against mycobacteria is yet to be achieved, so emphasis
should be placed on research into the development of a mycobacteriosis vaccine.
The development of rapid diagnosis techniques such as ELISA for detecting
mycobacteriosis will be very useful for fish quarantine systems in the near
future.
NOCARDIOSIS
Nocardiosis is a disease of both salt-water and freshwater fish caused by
actinomycetes of the genus Nocardia. Cases of piscine Nocardia are not as
widely reported as mycobacteriosis in fish. Some nocardial infections in fish
may be misinterpreted as mycobacterial disease, as they both result in similar
clinical signs and gross pathology. Nocardia asteroides, described by Eppinger
in 1891, is considered the type species of the genus (Gordon and Mihm, 1962).
Nocardia farcinica had been formerly regarded as the type species, but this was
changed due to the absence of an authentic strain in any culture collection and
the paucity of references to N. farcinica in the literature. The first case of fish
nocardiosis caused by N. asteroides was reported in neon tetra, Hyphessobrycon
innesi (Valdez and Conroy, 1962).
332
S. Chinabut
Species of fish affected and geographical distribution
Information on fish nocardiosis is very limited in comparison with myco-
bacteriosis in fish. However, this does not mean that nocardial infection is less of
a problem in fish than mycobacteriosis. Nocardia asteroides has been reported to
be the cause of granulomatous disease in neon tetras kept in aquaria in Argentina
(Conroy, 1964b). Snieszko et al. (1964) reported nocardial infection in hatchery-
reared fingerling rainbow trout, Salmo gairdneri, in Lee Town, West Virginia,
USA, and subsequently Campbell and MacKelvie (1968) isolated Nocardia in
brook trout, Salvelinus fontinalis, from Canada. Nocardiosis was also observed
in cultured yellowtails, Seriola quinqueradiata and Seriola purpurascens, in
Japan (Kubota et al., 1968). An outbreak of nocardiosis was recorded in
Formosa snakehead fish (Channidae) from Taiwan (Hsu et al., 1987). Nocardia
seriolae was later isolated from cultured yellowtail (S. quinqueradiata) and
Japanese flounder (Paralichthys olivaceus) (Kudo et al., 1988). Recently,
nocardioform actinomycetic organisms were isolated from many species of
freshwater fish affected with epizootic ulcerative syndrome (EUS) in India
(Chakrabarty and Dastidar, 1991).
Pathology
Nocardiosis is a systemic chronic granulomatous disease of fish caused by
several species of the bacterium Nocardia. Severe emaciation, inactivity and
skin discoloration are the clinical signs of this disease. In advanced stages,
cachexia, ascites, dermal ulceration, focal necrotic areas within skeletal muscle
and pale areas in the swollen kidney, spleen, heart and liver may be observed
(Kubota et al., 1968). The appearance of small nodules on the gills of infected
yellowtails has been reported (Kusuda et al., 1974).
Histopathological examinations of infected organs have revealed character-
istic granulomas, each with a centre of necrotic material, a peripheral cellular
zone of numerous histiocytes, lymphocytes and a few multinucleated giant cells,
all of which are partially circumscribed by a fibrous capsule. Engelhardt (1987)
noted that colonies of weakly acid-fast bacteria were present within and at the
periphery of several granulomas. Hsu et al. (1987) observed numerous
disseminated abscesses, encapsulated by granulation tissue, in the pleura and
viscera of infected Formosa snakehead fish.
Characterization and taxonomy
Nocardioform bacteria are tentatively identified by their rapid growth rate,
which ranges from 3 to 7 days, and the characteristic small, heaped and
wrinkled, moist to chalky colonies. The morphology of Nocardia varies from a
branched filamentous cell to a fragmented, irregularly shaped, pleomorphic or
coccobacillary cell, depending upon the phase of growth (Beaman et al., 1975).
The cell walls of Nocardia contain branched, hydroxylated fatty acids, called
333 Mycobacteriosis and Nocardiosis
mycolic acids or nocardiomycolic acids (Lechevalier et al., 1971). The partial
acid-fast staining property of this organism can distinguish it from other genera
of actinomycete bacteria. It is considered difficult to differentiate between
Nocardia and rapidly growing mycobacteria by their physiological and
morphological characteristics alone (Uesaka, 1956; Gordon, 1966; Tsukamura et
al., 1981). Some characteristics of N. asteroides and Nocardia kampachi are
shown in Table 9.3.
The use of certain antibiotics, at suitable concentrations, has provided
valuable data for the classification and identification of nocardioform bacteria.
Sensitivity tests were found to be particularly useful for this purpose
(Goodfellow and Orchard, 1974). The comparative immunodiffusion method
was used to classify Nocardia by Ridell (1981). Direct fluorescence antibody
techniques have also provided reasonable results for the identification of N.
kampachi (Kusuda and Kawahara, 1987).
The various species of Nocardia can be differentiated by the temperature
ranges at which they grow. Nocardia asteroides grows at 37C and can survive at
50C for more than 4 h but N. kampachi does not grow at 35C (Kusuda and
Taki, 1973; Kusuda, 1975).
Diagnostic methods
As already mentioned, nocardiosis may be misdiagnosed as mycobacteriosis,
because of the similarity between the clinical signs and gross pathology
associated with the two diseases. Positive differentiation can only be made by
isolation and identification of the causative agent. In addition, histological
sections of the infected organs should be examined. Nocardioform bacteria can
grow on similar kinds of media to mycobacteria. After 24 h of growth on nutrient
agar, typical colonies of Nocardia are seen. These are raised, folded, granular or
powdery, 14 mm in diameter, yellow or tan, and with aerial mycelium around
the edges (Engelhardt, 1987). Nocardial granulomas without the epithelioid cells
in the earliest stages of development are easily confused with piscine
mycobacteriosis. Furthermore, while acid-fast organisms are present in the
Nocardia lesion, they only show a positive reaction for Nocardia with Fite
Faraco acid-fast stain. Morphologically, Nocardia appear filamentous, branched
and beaded and are 550 m long, while mycobacteria are usually 13 m in
length (Wolke and Stroud, 1978).
Transmission of the disease
The routes by which infection takes place are not known. Most cases of piscine
nocardiosis involve the oral cavity, but, experimentally, oral exposure has not
been established as a primary route of infection.
334
S. Chinabut
Table 9.3. Characteristics of Nocardia asteroides (from Goodfellow, 1971)
and Nocardia kampachi (from Kusuda et al., 1974).
Character N. asteroides N. kampachi
Presence of Gram-positive rods + +
and coccci
Presence of weakly acid-fast + +
staining reaction
Production of aerial hyphae + +
Motility
Growth at 10C v
Production of
Catalase v +
H
2
S +
Indole .
Oxidase
Nitrate reduction + +
Degradation of
Casein
Gelatin
Hypoxanthine (weak) +
Starch . +
Tyrosine (weak) +
Urea (weak) +
Xanthine
Production of acid from
Fructose + +
Glucose + +
Glycerol + +
Utilization of
Adonitol
Arabinose
Cellobiose
Dextrin v
Dulcitol v
Fructose + +
Glucose + +
Glycerol + v
Glycogen
Inositol
Inulin
Lactose
Maltose v
Mannose + v
Rhamnose v
Salicin
Sodium acetate + +
Sodium benzoate
Sodium citrate +
Sodium lactate + +
Sodium malate + +
335 Mycobacteriosis and Nocardiosis
Control and treatment
No chemotherapeutant has been proved to be effective. The development of
nocardiosis in fish probably results from environmental stress and so the best
method of disease control is by providing good husbandry in the culture system
(Kusuda and Nakagawa, 1978). In aquarium fish, stressors, such as over-
crowding, elevated water temperature (above optimum) or inadequate nutrients
are predisposing causes of this disease (Conroy, 1964b; Ghittino, 1972).
Pathogenicity and immunity
No studies have yet been undertaken on the pathogenicity of nocardioform
bacteria. Kusuda et al. (1989) demonstrated that yellowtails immunized with
attenuated live N. kampachi showed an increase in the number of active
lymphocytes and granulocytes within 72 h.
Topics for further study
The taxonomy of bacteria in the genus Nocardia is not as well organized as in
other groups (Austin and Austin, 1987), so there is a need for bacteriological
taxonomists to modify the classification of nocardioform bacteria and help to
eliminate any confusion.
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