A survey and review of hepatobiliary lesions in Australian macropods

J. Comp.

Path.

1992 Vol.

A Survey

107, 147-167

and Review of Hepatobiliary Australian Macropods P. J. Canfield

and

Lesions

in

W. J. Hartley*

Department of Veterinary Pathology, University of Sydney, JvewSouth Wales, 2006 Australia and * Taronga zoo, Veterinary Quarantine Centre, P-0. Box 20, Mosman, .New South Wales, 2088 Australia

Summary

This report presents information on the range of diseasesand lesions that occurred in sectionsof livers of macropods held in the Non-Domestic Animal Registry at Taronga Zoo. Of 142 affected livers, 52 were due to parasites, 24 to bacteria, nine to Macropod Herpesvirus, four to fungal agents and ten to tumours. In addition, 17 livers had acute degenerative or necrotic lesions,22 were affected by fibrosis or other chronic lesionsand four had miscellaneous degenerative lesions. Common parasitic diseasesincluded toxoplasmosis, coccidial cholangitis, cestode cholangitis (Progamotaenia sp.), fascioliasis and capillarial hepatitis. Bacterial diseaseswere varied but lesionsdue to anaerobic bacteria or Mycobacteria spp. were most common. Many of the acute degenerative lesionswere associatedwith cardio-respiratory disease.Chronic lesions were often of unknown origin, although it was speculated that parasitic and bacterial organisms contributed to the aetiology. There were four primary and six metastatic turnout-s.

Introduction It is accepted that in man and in animals the liver is one of the more frequently damaged organs in the body (O’Brien and Gottlieb, 1979; MacLachlin and Cullen, 1988). This is mainly a result of its central role in metabolism, occupying a strategic location between the portal and systemic circulations, which continually exposes it to potentially injurious toxins and infectious agents (MacLachlin and Cullen, 1988). A s a consequence of its anatomical connections and key functions, diseases of the liver may not only develop as a result of primary insults but also secondarily to dysfunction and disease in other tissues (Kelly, 1985). The tremendous reserve capacity of the liver is in some way a counterbalance for the high frequency of liver disease and ensures that only the more severe lesions will give rise to clinical signs of liver dysfunction. Consequently, hepatic lesions are far more common than hepatic dysfunction. Although liver diseases have been mainly recorded in domestic animals, some have been reported for Australian marsupials. Many of these are of interest because of their potential disease significance for man or domestic animals, such as the prevalence of Fasciola hepatica in macropods, possums and wombats (Spratt and Presidente, 1981) and the occurrence of 0021-9975/92/060147

+ 21 $08.00/O

0

1992 Academic

Press Limited

148

P. J. Canfield

and W. J. Hartley

Salmonellaspp. in orphaned

kangaroo and wallaby joeys (Speare and Thomas, 1988), but some have been recorded because of a developing interest in conservation and the control of disease in captive populations of Australian marsupials, especially macropods (Speare 1988; Speare, Donovan, Thomas and Speare, 1989). This article presents information on the range of diseases and lesions which occurred as either important or incidental findings within the liver of macropods held in a registry for Australian native fauna and exotic species (NonDomestic Animal Registry) at Taronga Zoo. This Registry draws its material from the Taronga and Western Plains Zoos, from submitted wildlife (live and cadavers) and from histological sections submitted by various institutions. Materials

and Methods

Histological sectionsof liver lesionsheld in the Registry were examined by the authors. Sixty two caseswere from macropods held at Taronga and Western Plains zoos, and at sanctuariesand captive coloniesin Sydney and surrounding districts. Eighty caseswere

histological

sections from macropods submitted

by other Australian

zoos, wildlife

reserves, government veterinary laboratories, research institutes, universities and private individuals. Information on individual cases,where available, was obtained

from necropsy reports and anamnesis. All cases derived from local sources were from captive macropods. From available records, most of the submitted caseswere alsofrom captive macropods. Histological sections of paraffin wax-embedded tissueswere routinely stained with haematoxylin and eosin (HE). Additional stains included periodic acid-Schiff (PAS) reagent for carbohydrates, Gomori’s methenamine silver for fungi, Giemsa, Masson’s trichrome, Van Gieson’s method for collagen, the Warthin Starry method for spirochaetes, Congo red for amyloid and Brown-Rrenn Gram stain for bacteria. Results

The designation of specific diagnoses for some cases in the study was prohibited by a lack of adequate ante-mortem and post-mortem information and the absence of wet formalin-fixed tissue and paraffin blocks. These cases have not been excluded but included in broader categories. At the time of compilation of this publication (1991), the Registry contained 142 liver lesions in a total of 602 disease cases held. These had been catalogued from 1972 to 199 1. Liver lesions were examined from 42 macropods held at Taronga and Western Plains Zoos, 11 held at university compounds in Sydney and nine presented to Taronga Zoo by individuals. Nineteen histological sections of liver lesions in macropods were obtained from Victorian government laboratories and universities, 15 from Queensland government laboratories and universities, 13 from a Tasmanian government laboratory, nine from New South Wales government laboratories, six from South Australian government laboratories, six from Western Australian government laboratories and universities, five from New Zealand government laboratories, three from Northern territory government laboratories, two from an Australian Capital Territory government laboratory and two from London Zoo. These animals were primarily derived from wildlife reserves, zoos and individuals who were caring for sick or injured macropods. The lesions definitely occurred in 25 species of macropods

Hepatobiliary

Lesions

149

in Macropods

(in 16 from Taronga Zoo sources and in 21 from other sources). The more common species included 15 Macropus giganteus, 14 Macropus eugenii, 12 Bettongia penicillata, 11 Macropus parma, 11 Macropus rufogriseus, 11 Macropus rufus and seven Macropus robustus. Lesions were placed in categories according to aetiology or basic pathological change (Table 1). The impact of the liver disease on the well-being of the macropod was not taken into consideration when determining categories. Cases placed in parasitological or microbiological categories included those with definite identification of the aetiological agent or those that showed the presence of the agent within the histological section but without exact identification. Some of the cases placed in the general category of hepatic fibrosis/amyloidosis and other chronic lesions of undetermined cause could have been due to parasitic or microbiological causes. Detailed information on individual cases is presented in Table 2. The source of the case is presented as the Australian state or other country if not derived from local sources. Parasitic Lesions

Of the 52 macropods with definite parasitic lesions, 16 had lesions related to Toxoplasmagondii, eight had coccidial cholangitis, seven had capillarial hepatitis (including one case with concomitant Toxoplasma gondii), six had cestode (Progamotaenia spp.) cholangitis, four had cholangiohepatitis due to Fasciola hepatica, three had portal vasculitis due to Durikainema macropi, two had filarial vasculitis and cholangitis and one had amoebic hepatitis. In addition, six macropods had hepatic granulomas related to the presence of unidentified parasitic fragments or eggs. Hepatic lesions associated with toxoplasmosis have been described in detail in a previous paper (Canfield, Hartley and Dubey, 1990). The liver usually showed no gross changes, but occasionally there was swelling with patchy congestion. In most cases, focal areas of coagulative necrosis and neutrophilic debris were present in lobules while lymphocytes, plasma cells and some macrophages infiltrated the portal regions (portal hepatitis). Toxoplasma1 Categories

Category and total number cares in brackets

of liver

of

Parasitic lesions (52) Bacterial lesions (24) Viral lesions (9) Fungal lesions (4) Acute degenerative or necrotic lesions (17) Miscellaneous degenerative lesions (4) Fibrosis/amyloidosis and other chronic lesions of undetermined Tumours (10)

Table lesions

1 in

142 macropods

Number ofcases from Taronga zoo S!xmx 24 5 3 13

28 19 6 4 4

2

2

12

10

2

8

c:ause (22)

150

P. J. Canfield Details

Parasitic lesions M. parma M. parma M. parma M. rufogriseus M. rufogriseus B. penicillata M. parma M. fuliginosus M. rufogriseus S. brachyuris 1. billardierii M. eugenii M. giganteus W. bicolor M. eugenii

Table 2 macropods

of individual

Species

and W. J. Hartley

Diagnosis

Adult Adult Adult Young adult 8 months

F F F M M

TZ TZ TZ

Adult

F

TZ

9 months

M

Adult

F M M

NSW NZ VIC TAS VIC

Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis; capillarial cholangiohepatitis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Generalized toxoplasmosis Biliary stasis and fatty change secondary to toxoplasma1 pancreatitis Coccidial cholangitis Coccidial cholangitis Coccidial cholangitis Coccidial cholangitis Coccidial cholangitis Coccidial cholangitis Coccidial cholangitis Coccidial cholangitis Capillarial hepatitis Capillarial hepatitis Capillarial hepatitis and adhesions to liver Capillarial hepatitis Capillarial hepatitis Capillarial hepatitis Cestode cholangitis (Progamotaenia sp.) and portal hepatitis Cestode cholangitis (Progamotaenia sp.) Cestode cholangitis (Progamotaenia sp.) Cestode cholangitis (Progamotaenia sp.) Cestode cholangitis (Progamotaenia sp. ! Cestode cholangitis (Progamotaenia sp.); nodular regeneration and fibrosis Fasciola hepatica cholangiohepatitis Fasciola hepatica cholangiohepatitis Fasciola hepatica cholangiohepatitis Fasciola hepatica cholangiohepatitis Durikainema macropi portal vasculitis Durikainema macropi portal vasculitis Durikainema macropi portal vasculitis; hepatitis Filarioid eosinophilic vasculitis and cholangitis Filarioid eosinophilic vasculitis and cholangitis Amoebic hepatitis Nematode granuloma (nematode [filarial?] fragment present) Nematode granuloma (nematode fragment present); marked hepatic leukopoiesis Granulomas (parasitic fragment seen)

5 years

MCQ TZ

MCQ

QLD QL”

M. eugenii

TZ

Species unknown

MCQ

2 years

M

10 months

M

1 year -

F

Adult Aged 7 years

M M F

M. M. W. 7. M. M. M. M.

giganteus giganteus bicolor billardierii agilis robustus rufw agilis

NSW WC VIC VIC SA ACT TZ 7’2 TZ TZ TZ

MCQ

NSW

Species unknown M. rufus M. giganteus L. conspicullatus L. conspicullatus M. rufus

lesions

Source

SA UK

parma eugenii eugenii eugenii eugenii eugenii rufus rufescens penicillata agilis penicillata fmna

liver

St?.%

Species unknown

M. M. M. M. M. M. M. A. B. M. B. M.

with

Adult

F

NSW NSW

M

QLD QL”

F

F M

SA

VIC VIC NSW TAS

QLD QLD QW QLD

-

M. agilis

Adult

M

M. robustus M. parryi

-

B. penicillata

12 months

F

TZ

A. rufescens

Adult

F

TZ

QLD QLD

TZ

Hepatobiliary

Lesions Table

151

in Macropods

2 (continued)

Species

Age

sex

source

Diagnosis

M.

rufus

18 months

M

TZ

M.

eugenii

Adult

F

MCQ

Adult

M

VIC

Protozoa1 or nematode granulomas; portal hepatitis Granulomas (possible calcified parasite material) Granulomas (capillarial or fasciolarial eggs present)

M

TAS SA NZ NZ VIC

M. giganteus

Bacterial lesions M. rufogriseus A. rufescens

Species unknown Species unknown M. eugenii

2 years

M. giganteus M. parryi M. fuliginosus

Aged

TZ

Abscessation

F

Adult

M

QU’

B. gaimardi M. rufogriseus P. longipes

1 year 2 years Adult

M F M

WA TAS TAS WC

P. longipes

Adult

M

VIC

M. rufogriseus M. giganteus M. Parryi

3 years Adult

M F

TAS TAS TZ

M. fuliginosus

Joey

Ml. agilis

Adult

sp.

M

WA

Adult

WPZ TZ TZ

Dendrolagus

adult

Focal listerial abscesses Focal listerial abscesses Focal nocardial abscesses Focal nocardial abscesses Necrotic abscesses (Bacterioides sp. and Fusobacterium necrophorum) and chronic cholangitis Hepatic/gastric abscesses (necrobacillosis) Necrotic abscesses (F. necrophorum) Abscesses (Pasteur& multocida plus anaerobes) and portal hepatitis Melioidosis Mycobacterial pyogranulomatous hepatitis Mycobacterial pyogranulomatous hepatitis Mycobacterial pyogranulomatous hepatitis Pyogranulomatous hepatitis (acid-fast organisms) Pyogranulomatous hepatitis (acid-fast organisms) Abscesses (club colonies) Focal necrotic hepatitis (Proteus morgani) Focal hepatic necrosis and cholangitis (Salmonellla sp.) Focal necrotic hepatitis (Salmonella

M. eugenii

&?hiphimurium)

F

(mixed

organisms

but mainly

E. coli 7. the&s

-

B. penicillata M. eugenii

2 years

M

M. M.

Joey 18 months

M

eugenii robustus

Viral lesions 7. brunii M. parma P. penicillata B. B. S. T. P. 7.

penicillata penicillata brachyuris brunii co&ma brmii

Fungal lesions S. brachyurus M. giganteus

NSW VIC WC NSW NSW

Hepatic abscesses (Bacteria obvious but no growth) Old hepatic abscesses (not cultured) Vasculitis with bacterial thrombi in portal triads (not cultured) Capsular abscessation (not cultured) Old abscesses (not cultured)

TZ

Adult

M F F

Adult 5 years 10 years

M F F M

Juvenile

F

WA WA VIC VIC VIC SA

Macropod Macropod Macropod cholangitis Macropod Macropod Macropod Macropod Macropod Macropod

WA NSW

Generalized cryptococcosis Mycotic hepatitis (Candida

Sub-adult

Joey

MCQ MCQ

Herpesvirus Herpesvirus Herpesvirus

hepatitis hepatitis hepatitis

Herpesvirus Herpesvirus Herpesvirus Herpesvirus Herpesvirus Herpesvirus

hepatitis* hepatitis* hepatitis? hepatitis hepatitis hepatitis

sp.)

and mild

P. J. Canfield

152

and W. J. Hartley

Table Species

sex

2 (continued) Source

Diagnosis

Joey

-

8 months

F

NSW VIC

Mycotic Mycotic

M. parma M. parma M. robustus

Adult Sub-Adult Juvenile

F M F

TZ TZ WPZ

M. eugenii

-

ACT

W. M. M. P. A.

-

Centrilobular necrosis Centrilobular necrosis and calcification Focal necrosis and congestion, with sinusoidal fibrosis Centrilobular and mid-zonal vacuolar change and portal hepatitis Centrilobular congestion and portal hepatitis Extensive centrilobular necrosis (toxic)? Massive necrosis and haemorrhage (toxic?) Massive necrosis and myonecrosis (toxic?) Acute centrilobular necrosis (secondary to pneumonia?) Massive necrosis/infarcts seconday to bacterial pneumonia Acute centrilobular necrosis and congestion due to non-suppurative myocarditis Centrilobular atrophy, fibrosis and congestion due to chronic fibrinous endocarditis Centrilobular congestion and necrosis secondary to myocarditis Centrilobular congestion due to non-suppurative myocarditis. Portal hepatitis Centrilobular congestion, necrosis and fibrosis due to chronic pulmonary disease Centrilobular necrosis related to haemolytic anaemia Massive haemorrhage (warfarin poisoning)

M. giganteus M. giganteus

Acute degenerative necrotic lesions

M.

(Candida (Candida

sp.) sp.)

or

bicolor rob&us mfis xanthopus rufescem

Adult

F

NSW WPZ WPZ WPZ NSW

Adult

F

TZ

-

rufogriseus

Macropus

hepatitis hepatitis

-

sp.

WPZ

D. goodfellow

15 years

M. giganteus

-

M. pana

-

Species unknown

Joey

VIC

M. rufogriseus

-

NZ

Macropus sp. Miscellaneous degenerative lesions

-

-

VIC

M. mfus

8 months 12 months Adult Juvenile

F F F

NZ NSW SA NSW

Neurovisceral storage disease: Neurovisceral storage disease: Focal hepatic lipidosis (xanthomas in skin). Focal hepatitis and necrosis with accumulations of lakes of PAS positive acellular material; acute enteropathy

Adult Adult Adult Adult

E F

TZ TZ WPZ NSW TZ

P. xanthopus P. ranthopus

-

-

TZ

M. fuliginosus

18 months

M

TZ

B. penicillata 7. billardierii

Adult

F M

WA TAS

Focal fibrosis Focal fibrosis and portal hepatitis Focal fibrosis and possible telangiectasis Focal telangiectasis Dissecting lobular fibrosis secondary to duodenal carcinoma Centrilobular fibrosis or amyloidosis Focal amyloidosis, biliary hyperplasia and hepatocyte hypertrophy Biliary hyperplasia/hypertrophy secondary to enteropathy (Campylobacter sp.) Focal fibrosis Sinusoidal fibrosis or amyloidosis and focal neutrophilic hepatitis

M. giganteus

Potorus sp. M. giganteus

Hepaticjbrosis/amyloidosis arrd other chronic lesions undetermined cause B. penicillata B. penicillata M. rufus M. rufus B. penicillata

F

TZ

WPZ -

MCQ

of

-

F F

MCQ

Hepatobiliary

Lesions Table

Species

Adult

B. penicillata Bettongia

sp.

SOUW

Diagnosis

F

TZ

Focal fibrosis or amyloidosis (parasitic tracts?) Focal granulomatous hepatitis with calcification Focal granulomatous hepatitis and cholangitis Focal granulomatous hepatitis and portal hepatitis Focal chronic hepatitis with calcification (parasitic tracts?) Chronic cholangitis Chronic cholangitis and megalocytosis Chronic cholangitis and acute hepatitis Chronic cholangitis and vasculopathy Chronic cholangitis (parasitic?) Portal hepatitis Portal hepatitis or leukopoiesis

TZ M

L. hirsutus B. penicillata M.

nlfus

Adult

h4. mfogriseus M. rufiu

Species unknown Petrogale sp. M. panyi A. rafesteas Tumours M. antilopinus M. mfogriseus M. agilis M. robustur M. rufogriseus M. panyi 7. billardierii M. parma M. giganteus M. rob&us

QLD NT

Adult

2 years Adult

M

NSW NSW TAS NT NSW

QLD WPZ NSW NT TAS

Sub-adult

153

2 (continued)

sex

-

M. giganteus

in Macropods

QLD TAS TAS WPZ TAS UK WPZ

QLD

Biliary adenocarcinoma Cystic proliferation of bile ducts5 Focal hepatobiliaty proliferation Hepatic haemangioma§ Lymphoid neoplasia$ Lymphoblastic leukaemiag Lymphoid leukaemia Metastatic mammary carcinoma Metastatic haemangiosarcoma Metastatic neoplasia

-=

not known, F = Female, M = male, TZ = Taronga Zoo, MCQ= Macquarie University compound, NSW, NZ = New Zealand, WPZ = Western Plains Zoo, TAS = Mt Pleasant Laboratories, Tasmania, VIC=Victoria, QLD = Queensland, Australia, SA= South Australia, NT=Northern Territory, WA = Western NSW = New South Wales, UK = United Kingdom zoo, ACT = Australian Capital Territory. * Dickson, Hopkinson, Coackley, Spence and Fairfax ( 1980). t Finnie ( 1980). $ Rothwell, Harper, Hartley, Gumbrell and Meischke (1990). $ Canfield et al. (1990).

organisms were definitely seen in two cases in association with the areas of focal inflammatory necrosis (other cases had detectable toxoplasma1 organisms only in other organs or tissues). In two cases, there were focal aggregates of lymphocytes and macrophages within lobules, but no recent areas of inflammatory necrosis. One case within this category did not have lesions directly attributable to proliferation of toxoplasma1 organisms, but had biliary stasis and hepatocyte vacuolar change secondary to toxoplasma1 pancreatitis. Hepatic coccidiosis was seen as white nodules 2 to 5 mm in diameter and pale areas surrounded by reddened borders diffusely scattered through the liver. The lesions were often in association with intestinal coccidiosis and six of the eight affected macropods were M. eugenii. Microscopically, multilocular coccidial schizonts (megaloschizonts) were present in hypertrophied cells. These were surrounded by haemorrhage and necrosis in the early stages (Fig. 1), but

154

PJ.

Canfield

and

W. J. Hartley

with development and signs of rupture. Lymphocytes and plasma cells had penetrated the scarring areas (Fig. 2). Later, healing lesions consisted of focal areas of dense collagen, Bile ducts showed some hyperplasia and hypertrophy with neutrophilic contents. Lymphocytes and plasma cells infiltrated the portal regions and there was mild biliary fibrosis. Some bile ducts appeared ruptured and showed fibrosis and accumulations of heavily brown pigmented macrophages. Capillarial hepatitis primarily occurred in macropods held at Taronga Zoo. Grossly, livers showed diffusely scattered white nodules up to 5 mm in diameter. In two cases, fibrinous adhesions occurred between the surface of the liver and other abdominal structures. Microscopically, aggregates of capillarial eggs, and sometimes adults, were present in or adjacent to hepatic lobules. Early egg accumulates caused little cellular response (Fig. 3), but then became progressively surrounded by connective tissue and mononuclear inflammatory cells (lymphocytes, plasma cells and macrophages). Later nodules showed calcification, giant cell infiltration and extensive fibrosis. Some chronic nodules were devoid of eggs. Portal regions displayed some fibrosis and were heavily infiltrated with lymphocytes, plasma cells and brown pigmented macrophages. Cholangitis due to Progamotaenia spp. has been well recorded (Presidente and Beveridge, 1978; Beveridge, 1986). These macropods were presented for cystic dilatation of the larger bile ducts. Microscopically, the reaction to the presence of the parasites was minimal and restricted to mild fibrosis and infiltration with lymphocytes, and hypertrophy of biliary epithelium. Bile duct proliferation in the larger portal triads was present in one macropod, while prominent portal hepatitis was present in the smaller portal triads of another. One macropod (M. rufus), from the South Australian Central Veterinary Laboratory, also had extensive nodular regeneration and fibrosis apparently unrelated to the presence of Progamotaenia spp. Cholangiohepatitis due to Fasciola hepatica has been well recorded in various macropod species in south-eastern Australia (Beveridge, 1978; McManus, 1979; Spratt and Presidente, 1981). In the four cases presented to the registry, the most severe lesions occurred in the liver of a Thylogale billardierii. The liver had numerous haemorrhagic and necrotic tracts throughout the liver parenchyma in addition to bile duct hyperplasia, fibrosis and mononuclear and eosinophilic inflammatory infiltrates associated with the presence of flukes. The other three animals had fibrosed livers which showed grossly thickened, distorted large bile ducts. Microscopically, flukes or their eggs were associated with biliary hyperplasia and inflammation. Old fibrosed tracts were present and, in one animal, a granulomatous response occurred around apparently extra-biliary eggs. Three cases of hepatic vasculitis due to Durikainema macropi were presented from Queensland. The pathology of the eosinophilic phlebitis of the terminal radicules of the portal venules has been adequately described by previous authors (Speare and Spratt, 198’2). In addition, two cases of eosinophilic portal vasculitis and cholangitis due to suspected filarioid parasites were presented by the James Cook University of North Queensland. The lesions consisted of eosinophilic infiltrates of portal triads with some fibrosis, giant cell formation and eosinophilic abscessation.

Hepatobiliary

Fig.

1.

Lesions

in Macropods

155

Three developing coccidial infected cells (arrows) are surrounded by haemorrhage (H) and adjacent to hypertrophied, hyperplastic bile ducts. HE X 150. Fig. 2. Large hypertrophied host cell, with nucleus and filled with coccidial schizonts (arrow), is adjacent to hypertrophied, hyperplastic biliary tracts and surrounded by fibrous tissue and mononuclear inflammatory ceils. HE X 150. Fig. 3. Typical capillarial eggs lie free among hepatocytes and early fibrous tissue. HE x 250. Fig. 4. Amoebae (arrows) are circulating in the hepatic sinusoids without apparent host reaction. PAS x 250.

156

P. J. Canfield

and W. J. Hartley

One case of amoebic hepatitis was received from a Queensland source. The macropod had a severe amoebic gastritis which had spread to the liver. Amoebic forms were associated with randomly scattered focal areas of necrosis and neutrophilic infiltrates, and were present circulating within sinusoids and portal vessels, but without host reaction (Fig. 4). Six other cases had scattered hepatic nodules which were either true granulomas (epithelioid and giant cells), associated with protozoa1 or metazoal fragments, or fibrosed areas. Since four of the cases were from Taronga Zoo, capillarial or coccidial infections were likely possibilities. Bacterial Lesions

Hepatic abscessation and necrotic hepatitis were associated with a wide variety of organisms. In some cases, the organisms involved were pure, such as in the cases of Listeria monocytogenes or Salmonella spp., but many cases had mixed organisms. The number of organisms isolated may have been even greater if anaerobic culture had been attempted in all cases. The type of hepatic lesion produced varied with the organisms involved and the duration of infection. Lobular lesions of focal necrosis and neutrophilic exudate were present in Listeria and Salmonellainfections. Larger necrotic or purulent lesions, with or without encapsulation, commonly occurred in anaerobic or mixed bacterial infections. Portal hepatitis, and sometimes purulent cholangitis, was not uncommon in association with hepatic abscesses, especially those that were fibrosing. Caseating pyogranulomatous lesions were present in infections with fiocardia spp. and Mycobacteria spp. Four cases of confirmed or suspected necrobacillosis were recorded. Three of these cases were from Taronga and Western Plains Zoos. Hepatic lesions were usually a component of widespread or alimentary tract abscessation. Hepatic abscesses tended to be massive and consisted of large central areas of necrosis with organisms and neutrophils situated mainly on the perimeter. Older lesions tended to become encapsulated. Cholangitis, characterized by fibrosis and mild biliary hyperplasia was detected in one case. Five cases of pyogranulomatous hepatitis due to acid-fast organisms were recorded. Only one of these was cultured successfully (Mycobacterium avium in a B. gaimardi from Tasmania). These were derived from wildlife sanctuaries or zoos in Victoria (2)) Tasmania (2) and Western Australia (1). Lesions were widespread in visceral organs (spleen, gut, liver and lung) for those cases with documented information. Early lesions in the liver were characterized by focal aggregates of macrophages and occasional neutrophils. Later lesions displayed large central areas of caseation and, sometimes, calcification, surrounded by rims of neutrophils, lymphoid cells, epithelioid cells, giant cells and fibrovascular tissue. Acid-fast bacilli were plentiful in the caseating centres and epithelioid cells of the lesions of the case considered to be caused by M. avium, but were variable in number in the other cases. Viral Lesions

Liver lesions were detected in nine cases of confirmed

or suspected generalized

Hepatobiliary

Lesions

in Macropods

157

Macropod Herpesvirus (MHV) infection. These animals were from six zoos or captive colonies and included three Thylogale brunii and three Petrogalepenicillata. The Setonix bra&Uris has been previously reported by Finnie (1980). Grossly, livers were usually congested and contained pale circular foci, commonly of 1 to 5 mm in diameter. Microscopically, focal areas of coagulative necrosis and scattered neutrophils were often associated with eosinophilic to slightly basophilic intranuclear inclusions in hepatocytes on the margins (Fig. 5). Portal hepatitis was present in two cases. Fungal Lesions

Three young Macropus giganteus, being artificially reared in northern New South Wales (2) and Victoria (l), had numerous pale foci in the liver in addition to necrotic gastroenteritis. Microscopically, the acute lesions consisted of circular areas of necrosis and haemorrhage without an inflammatory with fungal elements that disresponse. The areas were densely populated played hyphae, pseudohyphae and blastospores (Fig. 6). These fungi were considered to be Candida spp. on the basis of morphology. In one animal, fungal elements also occurred in the lungs. Acute Degenerativeor .Necrotic Lesions

Three cases of centrilobular necrosis or massive necrosis could have been due to unidentified toxic insult. A further nine cases had defined causes such as cardiac or pulmonary lesions in seven cases, haemolytic anaemia in one case and warfarin poisoning in one case. No causes were determined for another five cases. MiscellaneousDegenerativeLesions

In this group, two suspected cases of congenital neurovisceral storage disease are included which have been reported previously by Rothwell et al. (1990). One was presented to a New Zealand government laboratory from a wildlife park, while another was from a joey being reared in southern New South Wales. Both animals had vacuolation of neurones and various parenchymal cells due to the accumulation of PAS-positive material. In the liver, irregularly scattered aggregates of ballooned foamy hepatocytes were present. In addition to these two, another macropod had periportal aggregates of either vacuolated hepatocytes or macrophages. This animal was an adult and had multiple xanthomas of the skin. FibrosislAmyloidosis and Other Chronic Lesionsof UndeterminedCause

Included in this category are focal chronic as a result of parasitic or, possibly, bacterial from focal to difIirse dissecting lobular deposits were often assumed to be amyloid case in five stained with Congo red and

lesions which could have occurred infection. Fibrotic patterns varied (Fig. 7). Focal dense eosinophilic on HE staining. However, only one when viewed with polarized light

P. J. Canfield

158

Fig.

5.

Macropod necrosis.

HE

Herpesvirus x 450.

inclusions

(arrows)

Fig.

6.

Liver affected by Cundida sp., showing response. PAS X 250.

Fig.

7.

Hepatacytes Scattered

Fig.

8.

appear groups

Undifferentiated band of spindle

yeast

and W. J. Hartley

are present forms,

hyphae

to be in clusters due to the diffuse of mononuclear inflammatory cells

metastatic neoplasm shaped cells adjacent

in hepatocyte and

deposition are present.

nuclei

pseudohyphae, of fine bands Van Gieson’s

adjacent and

to an area lack

of

of cellular

ofcollagen x 250.

(arrow).

present in liver. Neoplastic cells show pleomorphism to a sheet of round to square cells. HE X 450.

with

a

Hepatobiliary

Lesions

in Macropods

159

proved to be positive for amyloid. Paraffin wax-embedded blocks from other suspected cases of amyloidosis were not available for staining. Chronic cholangitis was characterized by biliary hyperplasia, fibrosis and peribiliary infiltrates of lymphocytes, plasma cells and eosinophils. Biliary stasis was apparent in two cases. Once again, there was the possibility of some of the lesions being parasitic or bacterial in origin. One case of chronic cholangitis and megalocytosis, in a M. rufogriseusfrom Tasmania, could have been due to the ingestion of pyrrolizidine alkaloids. Turnours

Of the ten tumours, four have been described in a previous article (Canfield, Hartley and Reddacliff, 1990). Three of the four primary tumours were benign and could have been hamartomas of the various tissues of the liver. Three tumours were lymphoid in origin, while three were metastatic. One metastatic tumour was derived from widespread haemangiosarcoma, while one was derived from a mammary carcinoma. The origin and histogenesis of the final case of metastatic neoplasia was not determined. It appeared undifferentiated with cell shapes varying from spindle to round (Fig. 8). Discussion

In the main, the information provided in this survey on the types of liver disease likely to be present in macropods applies to captive animals, but many of the diseases have been recorded in free living macropods (McManus, 1979; Obendorf and Munday, 1983; Munday, 1988; Speare et al., 1989). In this report only the more commonly detected or previously unreported diseases will be discussed. Liver lesions due to parasitic infections were by far the most commonly occurring diseases. This applies in particular to Toxoplasmagondii infections for which macropods, and some other marsupials, appear to have a high susceptibility (Dubey and Beattie, 1988; Canfield et al., 1990). Although Toxoplasma infection can have devastating effects on a wide range of tissues, it appears from our study that the effects on the liver are mainly limited to portal hepatitis and scattered focal areas of inflammatory necrosis, which would be contributing little to the demise of the animal. Coccidial cholangitis has been poorly described in the literature. Information was first provided by Presidente (1978) who commented on its occurrence in a mild form in free-ranging tammar wallabies (M. eugerzii) on Kangaroo island in South Australia and in a more severe form in juvenile tammar wallabies held in captivity. The captive wallabies also had intestinal coccidiosis due to Eimeria sp. Munday ( 1988) later suggested that overcrowding and other stressful conditions probably predispose young macropods to coccidiosis but did not suggest why tammar wallabies appear to be particularly susceptible to hepatic coccidiosis. Our survey supports the finding that M. eugenii is most susceptible to coccidial cholangitis. Apart from the M. parma affected in our survey, reports of occurrences in other species of macropods do not appear to exist. However, it is possible, from pathological descriptions, that a case of

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“sporozoal biliary hepatitis” reported by Griner (1983) in a M. eugeniiheld at San Diego Zoo could have been a case of coccidial cholangitis. Although wild rats appear to be the main hosts of Capillaria hepatica, a wide range of animals can be infected and develop hepatic lesions (Hsu, 1979). In addition, many of the reports of the parasite in hosts other than in rodents appear to emanate from zoos (Spratt and Singleton, 1987). Capillarial hepatitis has not been recorded previously in macropods, but experimental infection of brushtail possums (Trichosurus vulpecula) with the parasite has been noted (Spratt and Singleton, 1986). In our study it was presumed that the hepatic lesions were caused by Capillaria hepatica and that the macropods acquired infection via the ingestion of embryonated eggs present in soil. This mode of transmission, preceded by essential cannibalism, has been postulated for infection in mice (Spratt and Singleton, 1986). At Taronga Zoo, where most of the cases of capillarial hepatitis came from, wild rats, mice and rabbits are present in the grounds and commonly have capillarial granulomas in their livers. These animals, in addition to foxes which are also present, are likely to be involved in the spread of the nematode to macropods. The lesions produced in the macropods were primarily granulomatous in nature and often accompanied by portal hepatitis. These lesions are consistent with the progressive granulomas, commonly occurring adjacent to the portal triad regions, reported for other species (Hsu, 1979). Cholangitis due to Progamotaeniaspp. has been recorded for macropods and wombats (Beveridge, 1978). P. f estiva is the more commonly occurring species in both groups, with P. ejigia apparently restricted to M. fuliginosus and P. diaphana restricted to wombats (Spratt, Beveridge and Walter, 1990). Most information is available on the effects of P. festiva, and it appears that the effects of the parasite on the biliary tree vary with the species of macropods infected; for example, M. rufus and M. agilis are least affected (Presidente and Beveridge, 1978; Speare, Beveridge, Johnson and Corner, 1983). In macropods, disease is subclinical and is limited to mild biliary hyperplasia and hypertrophy, variable biliary fibrosis and mild mononuclear infiltrates (Presidente and Beveridge, 1978). Both the larger bile ducts and the gall bladder can be affected (Speare et al., 1983). In our survey, on the basis of species infected, it was presumed that most infections were due to P. festiva.. The lesions were minimal and appeared not to have caused clinical disease. Fasciola hepatica infection is more commonly seen in free-ranging macropods sharing pastures with infected sheep and cattle than in those in captivity (Spratt and Presidente, 1981). It is commonly seen in south-east Australia, where the climatic conditions are suitable for the survival of the intermediate host, the snail Lymnea tomentosa(Boray, 1969). The effects of infection are varied depending on the species of macropod infected (Beveridge, 1978). Although M. giganteus is the most common macropod species affected by the parasite (Spratt and Presidente, 1981), it tolerates moderate infections of the fluke usually without clinical signs of the disease (Beveridge, 1978). This is in contrast to T. billardierii, in which fluke burdens may be fatal (Beveridge, 1978). In our survey, two of the four infected macropods were derived from a wildlife park in Victoria, and infection could have been by a direct macropod-

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Lesions

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snail cycle as described by Spratt and Presidente (1981). Both were M. giganteus and in one, acute fascioliasis was considered to be the cause of death. Durikainema macropiinfection of portal veins has been reported in a variety of macropods in Queensland and other northern locations (Speare et al., 1983), and in South Australia (Speare and Spratt, 1982). The parasite can be identified in tissue sections and is not. reported as causing significant clinical disease (Speare and Spratt, 1982). I n our survey, in addition to three cases of D. macropi presented from Queensland, two cases of suspected filarioid vasculitis and cholangitis were reviewed, although the parasites were not identified. It is not possible to be sure whether D. macropiwas the cause since a variety of filarioids have been recorded in macropods (Beveridge and Presidente, 1982; Spratt et al., 1990). In addition to parasitic lesions caused by identified species, there were lesions caused by unidentified parasites. Other lesions included in the category of fibrosis/amyloidosis and other chronic lesions of undetermined cause could have been due to parasites. In all cases the lesions were limited and thought not to have led to clinical illness or to the demise of the animals. A case of amoebic hepatitis in M. robustushad previously been established through amoebic gastritis and ulceration leading to parasitaemia. Naturally occurring gastric amoebiasis was first recorded in macropods, in two wallaroos (M. robustus) held at Baton Rouge Zoo, Louisiana (Roberts, Williams and Pirie, 1973). The parasites were identified as Entamoebasp., but the source of the infection was not identified, although it was speculated that African ruminants may have been the source of infection. Griner ( 1983) also reported gastric amoebiasis and resultant peritonitis in six wallaroos held at San Diego Zoo during 1972 to 1973. Organisms were identified as Entamoebasp. by indirect immunofluorescent staining techniques. Although all these affected animals were wallaroos, Munday (1988) reported sub-clinical infections of Entamoeba sp. in M. giganteus and M. eugenii. The amoebae were confined to parasitic nodules containing Labiostrongylus spp. Entamoebahaemolyticacan parasitize and produce disease in a wide variety of animals, and in dogs can cause not only gastro-intestinal lesions, but also spread to the liver by extension or parasitaenematode parasitism can predispose the mia (Anderson, 1975). Concurrent dog to more severe amoebic infection (Anderson, 1975). In our study, hepatic lesions due to a wide range of identified and unidentified bacteria were common. In marsupials, many of the infections are of a sporadic nature and liver involvement is commonly part of a systemic process (Munday, 1988). However, outbreaks of infectious disease can occur in captive macropods exposed to stressful management conditions: for example, Wells and Montali (1985) recorded an outbreak of pasteurellosis in Potorus tridactylus stressed by overcrowding and aggression. Pasteurella multocida, a normal inhabitant of the upper respiratory tract, was isolated from lesions in five animals, with one animal having necrotic hepatitis associated with septicaemia. Although the range of bacteria affecting marsupials is similar to that affecting

commonly commonly

eutherian

mammals, there are some types of bacteria

that

cause infection in marsupials (Butler, 1986). In macropods, some recorded bacterial infections include necrobacillosis, mycobacterio-

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sis and salmonellosis (Speare et al., 1989). All these infections are capable of producing liver lesions of varying intensity, but there is some contention as to the exact identification of some of the organisms involved in the infections (Munday, 1988). Th is was the case in our study, with hepatic infections due to these particular groups of organisms being established via extension and the blood system. Necrobacillosis, most often due to Fusobacterium necrophorumalone or in association with other bacteria, commonly occurs in macropods in the form of “lumpy jaw”, but other sites such as stomach, liver, lung and hindlegs can be affected (Smith, 1988). Although captive macropods are most prone to the disease, “lumpy jaw” can develop in wild macropods (Speare et al., 1989). There is contention as to the pathogenesis and species of bacteria involved in “lumpy jaw” and associated conditions but it is accepted that obligate anaerobes, such as F. necrophorum,F. nucleatumand Bacteriodesspp., need to be present apart from other bacteria for characteristic lesions to occur (Oliphant, Parsons and Smith, 1984). Liver lesions are generally necrotic abscessesand it is thought that vascular spread is most likely to allow development (Munday, 1988; Griner, 1983). In our study, the suspected cases of necrobacillosis were all from captive colonies and liver lesions appeared to accompany, for the most part, gastro-intestinal or other abdominal organ lesions. Therefore, lesions could have become established by extension as well as by vascular spread. Mycobacterial infections in macropods have been well documented in captive animals, but not in free-living macropods (Speare et al., 1989). Although Mycobacterium tuberculosiswas reported in macropods in zoos at the turn of the century, most organisms detected in macropods since then belong to the atypical mycobacteria group (Munday, 1988). M. avium infections were recorded in macropods held at London Zoo during the 1930s (Hamerton, 1932). Since then, reports of unclassified acid-fast bacteria have been recorded in Setonix brachyuris (Kakulas, 1964) and in tree kangaroos held in San Diego Zoo (Griner, 1983). Other authors have been reported in M. eugenii, Macropus irma (Peet, Dickson and Nickels, 1982) and Bettongia penicillata (Richardson and Read, 1986). The granulomatous lesions affected a variety of organs, including the liver in two animals (Peet, Dickson and Nickels, 1982; Griner, 1983). A recent report stated that tree kangaroos appear to be particularly susceptible to various atypical mycobacteria, including M. avium and M. avium intracellulare, and that infection is acquired from the environment (Ott Jaslin, 1990). The acid-fast bacilli detected in liver lesions in our survey were not classified through non-culture or failed culture in four of the five cases. However, it is likely that the organisms were of the atypical mycobacterial group and that spread to the liver was via bacteraemia. Confirmed salmonellosis was not common in our collection of cases, but it is possible that other liver lesions were caused by this genus of organisms. A wide range of Salmonella spp. has been isolated from both free-living and captive macropods (Samuel, 198 1; Griner, 1983; Speare et al., 1989)) but it appears that clinical diseaseis restricted to captive adult macropods and joeys (Speare, 1988; Speare and Thomas, 1988). It is suggested, therefore, that isolation of Salmonellaspp. from macropods be interpreted with caution since there is a high

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Lesions

in Macropods

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carrier rate in normal, animals (Munday, 1988). In joeys, salmonellosis can be particularly severe (Butler, 1986), with clinical signs related to gastroenteritis and septicaemia (Speare et al., 1989). Lesions in the liver are thought to be the result of bacteraemia and vary from focal necrotic lesions to aggregates of neutrophils and histiocytes (Carlton and Hunt, 1978). Macropod Herpesvirus (MHV) causes fatal systemic disease characterized by widespread lesions, especially pneumonia, hepatitis and gastroenteritis (Callinan and Kefford, 1981; Wilks, Kefford and Callinan, 1981). The condition was first recorded in parma wallabies (Macropus parma) (Finnie, Littlejohns, and Acland, 1976), but since then it has been recorded in a wide variety of macropod species and the virus designated as various strains (Munday, 1988). Although neutralizing antibodies to MHV have been detected in a wide range of captive and free-living species of macropods, clinical disease has not been reported in free-living macropods (Speare et al., 1989). Consequently, stress in captivity is thought to play a role in the activation of latent virus and the expression of disease (Speare et al., 1989). In our survey, MHV infection was suspected on the basis of types of lesions and the presence of intranuclear basophilic to eosinophilic inclusions. The species were varied and all were from zoos or captive colonies. Mucocutaneous mycoses, reputedly due to Candidu albicans, particularly affect young captive macropods (Obendorf, 1980; Finnie, 1986; Munday, 1988). It is speculated that the organism becomes established through stress of rearing or debilitation due to intercurrent disease (Munday, 1988). The lesions are usually restricted to the buccal cavity, but if untreated can spread to cause a mycotic oesophagitis or gastritis (Finnie, 1986). For example, Griner (1983) reported oesophagitis in two M. agilis, and gastritis in a M. robustus due to an unspecified Candida sp. Systemic candidiasis has been reported in man and in a variety of mammals, especially in calves and piglets (Chandler, Kaplin and Ajello, 1980). Several saprophytic Cundidu spp., in addition to the endogenous species Candida albicuns, have been reported as causing systemic candidiasis in man, with kidneys and lungs appearing to be predilection sites (Chandler et al., 1980). In our survey, the three cases of hepatic and gastric fungal lesions were designated as being caused by Cundidu spp. on the basis of characteristic fungal morphology (pseudohyphae, hyphae and blastospores), but further delineation was not carried out. The hepatic lesions were devoid of an inflammatory response, but apparently this is not uncommon in man in early infections or in severely immunosuppressed individuals (Chandler et al., 1980). Obendorf (1980) also reported a lack of inflammatory response accompanying Cundida albicans invasion of deep muscle wall and psoas muscle in three joeys. These animals were all in poor nutritional condition and, coincidentally, all M. giganteus.

The acute degenerative lesions detected in macropods in our survey were, for the most part, related to cardiopulmonary compromise. Consequently, most lesions were of a centrilobular nature. Very little has been recorded about hepatoses in macropods, but it is accepted that suppurative and non-suppurative myocarditis, and cardiomyopathy due to capture or nutritional aberrations are common (Munday, 1988). In the case of non-suppurative myocardi-

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tis, the aetiology is poorly understood, but Toxoplasmagondii is accepted as one cause (Canfield et al., 1990). Acute toxic hepatic necrosis was suspected for some cases in our survey, but history did not incriminate specific agents. Munday (1988) suggests that plant poisons and pesticides should be considered, but apart from information on yew (Taxus baccata) (Barker, Calaby and Sharman, 1963), phalaris plants (Munday, 1988) and sodium fluoroacetate (Speare et al., 1989), little is available. Liver neoplasia in macropods is poorly recorded except for a hepatoma (Lombard and Witte, 1959)) an adenomatous proliferation of intrahepatic bile duct (Effron, Griner and Benirschke, 1977) and four cases of this study presented previously (Canfield et al., 1990). In other marsupial groups, primary tumours of the liver, especially hepatomas, have been recorded for possums (Hopkins, Dickson and Gaynor, 1984) and dasyurids (Griner, 1979), while lymphosarcoma involving the liver is well recorded for dasyurids (Attwood and Woolley, 1973) and koalas (Canfield, Brown, Kelly and Sutton, 1987). This survey failed to identify the cause of a variety of chronic liver lesions. This is to be expected since a variety of aetiological agents may produce, over a long term, similar morphological changes. The variety of fibrotic lesions was probably primarily related to past parasitic infection, but scarring could have developed secondary to toxic necrosis, chronic inflammation or metabolic disturbances (MacLachlin and Cullen, 1988). The density of the amorphous deposits in a group of the cases did raise the possibility of amyloid deposition, but this was confirmed in only one case. The amyloid was in the spaces of Disse and in small nodules, possibly associated with vessels. In domestic animals, most cases of amyloidosis occur secondarily to prolonged antigenic stimulation (MacLachlin and Cullen, 1988). It is obvious that this study does not give an accurate reflection of the incidence of specific liver diseases in macropods. Nor does it depict the commonness and range of liver disease. This is because, despite asking for both commonplace and unusual lesions, contributors tend to present what they regard as spectacular lesions. Ordinary lesions are often not stored or not preserved for histopathology. However, the study has provided information on the appearance of some specific liver lesions and has presented new information on specific aetiological agents. Acknowledgments We thank the numerous individuals, government and university institutions who so willingly gave to the Non-Domestic Animal Registry. We acknowledge the roles of the Elizabeth Macarthur Agricultural Institute and the Orange Regional Veterinary Laboratory, New South Wales Department of Agriculture and Fisheries in handling and reviewing cases from Taronga and Western Plains Zoos. Our special thanks go to D. L. Obendorf., I. Beveridge and R. Speare for advice and assistance. Special histological and photographic assistance was provided by Beverley Horsburgh, Karen Wadwell and Darren Head, of the Department of Veterinary Pathology, University of Sydney.

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in Macropods

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