A murine xenograft model for a transmissible cancer in Tasmanian devils.

The number of Tasmanian devils in the wild is rapidly declining owing to a transmissible cancer, devil facial tumor disease (DFTD). Although progress has been made to understand the spread of this disease, crucial research on the pathogenesis of DFTD has been limited because of the threatened status of the host species. Here, the authors describe the development of a NOD/SCID (nonobese diabetic / severe combined immunodeficiency) mouse model that reproduces DFTD and provides a much-needed model to undertake studies into this intriguing transmissible cancer. Histologically, the DFTD produced in NOD/SCID mice (xenografted DFTD) was indistinguishable from the DFTD identified in Tasmanian devils. At the protein level, all xenografted DFTD tumors expressed periaxin, a marker that confirmed the diagnosis of DFTD. The karyotype of DFTD in NOD/SCID mice reproduced similar chromosomal alterations as seen in diseased devils. Furthermore, each NOD/SCID mouse inoculated with cultured DFTD tumor cells developed tumors, whereas DFTD did not develop in any of the inoculated immune-competent BALB/c mice.

Serological responses against the pathogenic dimorphic fungus Mucor amphibiorum in populations of platypus (Ornithorhynchus anatinus) with and without ulcerative mycotic dermatitis.

Mucor amphibiorum, a dimorphic fungus, causes ulcerative dermatitis and systemic infections in the platypus Ornithorhynchus anatinus in some river systems in Tasmania but apparently not in other regions of Australia. As yet there are no suitable tests for population surveys, nor for detection of internal lesions in live animals. Consequently, immunoglobulins were purified from the serum of platypuses and anti-immunoglobulin antisera were prepared in rabbits in order to develop an enzyme-linked immunosorbent assay (ELISA) for anti-M. amphibiorum antibodies. Antigens from plate-grown cultures resulted in greater signal-to-noise ratios in indirect ELISA than those from broth-grown cultures. Platypuses with clinical ulcerative dermatitis had elevated anti-Mucor antibody levels compared to apparently unaffected individuals. Seroconversion was observed in one animal coincident with the development of cutaneous ulcers. The results suggested that platypuses in affected rivers were exposed to M. amphibiorum at a higher frequency than the occurrence of clinical disease. Some platypuses from New South Wales had elevated antibody levels but these increased significantly with age suggesting exposure to cross-reactive antigens, although exposure to M. amphibiorum cannot be excluded. Further studies are warranted to determine factors that result in progression from infection to disease, the occurrence of the fungus in areas where disease has not been observed and the specificity of antigen used in ELISA.

Surveillance and monitoring of wildlife diseases.

It is now recognised that those countries which conduct disease surveillance of their wild animal populations are more likely to detect the presence of infectious and zoonotic diseases and to swiftly adopt counter measures. The surveillance and monitoring of disease outbreaks in wildlife populations are particularly relevant in these days of rapid human and animal translocation, when the contact between wild and domestic animals is close and the threat of a bioterrorist attack is very real. The authors describe the problems inherent in wildlife disease surveillance and stress the importance of the establishment of national strategies for disease detection. The various sampling methods employed for monitoring outbreaks of disease and mortality in wildlife populations are discussed and their strengths and weaknesses described. A major advantage of an efficient disease monitoring programme for wildlife is the early detection of new and 'emerging' diseases, some of which may have serious zoonotic and economic implications. The authors conclude that wildlife disease monitoring programmes that are integrated within national animal health surveillance infrastructures should have the capacity to respond promptly to the detection of unusual wildlife mortality and to institute epizootiological research into new and emerging wildlife diseases.

Gross, histological and immunohistochemical features of mucormycosis in the platypus.

Nine male and five female adult free-living platypuses, obtained in a prospective capture-release study from northern Tasmania, exhibited gross features of cutaneous mycosis caused by Mucor amphibiorum. The lesions were present on the hind limbs (six cases), front limbs (four), tail (five), dorsal trunk (three) and ventral trunk (one). They varied in size, and ranged from raised red nodules or plaques, which sometimes exuded purulent material, to ulcerated lesions with central cavitation, red exuding centres and raised epidermal margins. Older lesions were covered either partly or fully by thickened and irregular epidermis. Histological examination of skin biopsies revealed discrete, poorly encapsulated granulomas, or more commonly a diffuse granulomatous or pyogranulomatous inflammation. Inflammatory cells consisted of neutrophils or eosinophils, sparse plasma cells and lymphocytes, many macrophages and occasional multinucleated giant cells. Fibrovascular tissue was diffusely and irregularly scattered in the granulomatous regions. Sphaerules characteristic of M. amphibiorum infection were observed in all lesions. The cutaneous distribution of the lesions and the natural history of the platypus indicated that entry of M. amphibiorum may have been via superficial skin wounds. T cells were the predominant infiltrating lymphoid cells in the diffuse lesions, indicating the importance of the cell-mediated response to infection.

Pathology of experimental toxoplasmosis in eastern barred bandicoots in Tasmania.

Wild-caught eastern barred bandicoots (Perameles gunnii) initially seronegative to Toxoplasma gondii, were inoculated orally with approximately 100 T. gondii oocysts. The bandicoots were maintained in indoor pens under laboratory conditions and observed daily. Serial blood samples were tested for agglutinating antibodies to T. gondii. Inoculated bandicoots died 15 and 17 days post infection. A rise in Direct Agglutination Test (DAT) titres was detected at the time of death (1:256, 1:64 respectively). Clinical observations, serological changes, gross findings at necropsy, and histopathological changes were consistent with acute toxoplasmosis. The findings indicate that eastern barred bandicoots are likely to die from primary T. gondii infection, often even before detectable antibodies are produced, reinforcing the significance of toxoplasmosis as a potential contributor to the reduction in numbers of wild populations of eastern barred bandicoots.

Earthworms as paratenic hosts of toxoplasmosis in eastern barred bandicoots in Tasmania.

An experimental feeding study was designed to assess the role of earthworms in the transmission of Toxoplasma gondii infection to eastern barred bandicoots (Perameles gunnii). Six animals with no agglutinating antibodies to T. gondii were fed artificially cultured earthworms that had been maintained in autoclaved nutrient-enriched soil. Two animals were given earthworms that had been maintained in soil contaminated with T. gondii oocysts (P89/VEG strain); two animals were fed on earthworms, which initially had been exposed to soil containing T. gondii oocysts then transferred through three changes of sterile soil; two control bandicoots were fed earthworms maintained in sterile soil. Both bandicoots fed earthworms maintained in T. gondii contaminated soil died 11 and 14 days after feeding. The necropsy findings were consistent with acute toxoplasmosis. Bandicoots fed earthworms exposed to oocysts but then transferred through changes of sterilized soil remained healthy as did control animals. All surviving animals remained seronegative over the 6 wk observation period after feeding. These findings confirm that earthworms, a major component of the natural diet of P. gunnii, can transmit T. gondii infection. It appears that oocysts present in the alimentary tracts of the worms, rather than infective stages of T. gondii in worm somatic tissues, are responsible for these infections.

Haematological, serum biochemical and serological features of platypuses with and without mycotic granulomatous dermatitis.

OBJECTIVE: To determine whether there are haematological, serum biochemical and serological differences between platypuses (Ornithorhynchus anatinus) with and without granulomatous dermatitis due to Mucor amphibiorum infection. An additional objective was to establish reference haematological and serum biochemical ranges for the species in Tasmania. DESIGN: A clinicopathological and serological study. ANIMALS: A total of 37 free-living adult platypuses captured from streams and dams in Northern Tasmania were used in the clinicopathological study. Twenty-seven were clinically normal and 10 had mycotic granulomatous dermatitis. A total of 22 platypuses (20 adult and 2 juvenile) were used for the serosurvey. Eighteen were captured from streams in Northern Tasmania, and four were submitted for necropsy. RESULTS: Platypuses with mycotic ulcerative dermatitis had significantly smaller packed red cell volumes, haemoglobin concentrations, lymphocyte counts, serum cholesterol and calcium concentrations, and higher serum globulin and potassium concentrations than clinically normal animals. The lymphopenia and hyperkalaemia were thought to be clinically significant. Numbers of Trypanosoma binneyi in blood smears were similar between the two groups. Diseased platypuses had higher concentrations of serum antibody against Mucor amphibiorum as determined by ELISA compared to clinically normal platypuses. CONCLUSION: Platypuses affected by mycotic granulomatous dermatitis showed haematological and serum biochemical changes when compared to clinically normal animals from the same Tasmanian sites. A serological survey may be a useful method for detecting the prevalence of exposure to Mucor amphibiorum and humoral immunity in platypus populations both in Tasmania and the mainland of Australia.

Comparison of the inflammatory responses of mice infected with American and Australian Trichinella pseudospiralis or Trichinella spiralis.

This study was designed to determine if the Tasmanian devil isolate of Trichinella pseudospiralis suppressed inflammation as does the original isolate. While adult worm numbers were similar in all groups, lower enteritis occurred in devil isolate-infected mice compared with mice infected with the original isolate of T. pseudospiralis or with Trichinella spiralis. Diaphragm muscle inflammation was greater in T. spiralis-infected than in mice infected with either isolate of T. pseudospiralis or concurrently infected with T. spiralis and either of the isolates of T. pseudospiralis. Granuloma inflammation was lower in mice infected with either isolate of T. pseudospiralis compared with uninfected or T. spiralis-infected mice. The devil isolate down-regulated inflammation more profoundly during the intestinal phase than the muscle phase compared to the original isolate, differences which may be related to the biology of their natural hosts.

Detection of agglutinating antibodies to Toxoplasma gondii in sera from free-ranging eastern barred bandicoots (Perameles gunnii).

Sera from 150 eastern barred bandicoots (Perameles gunnii) were collected from two study sites in southern Tasmania between 1992 and 1995. Samples were tested for antibodies to the protozoan parasite, Toxoplasma gondii, using formalin-treated tachyzoites as the antigen in direct (DAT) and modified agglutination tests (MAT). Cut-off titers were set based on confirmed cases of toxoplasmosis in this species. A total of 133 animals (89%) were classified as negative, seven (4.6%) had suspicious reactions, and 10 (6.7%) were diagnosed as positive. Five of the 10 positive animals were not retrapped after initial seroconversion; another three animals recorded high MAT titers on two consecutive bleedings, three months apart. Of the remaining two sero-positive bandicoots, one was found dead in a trap and generalized toxoplasmosis was diagnosed at necropsy, while the other animal had central nervous system disabilities consistent with toxoplasmosis but was accidently released and never recaptured. Based on these findings we propose that eastern barred bandicoots are likely to be highly susceptible to primary T. gondii infection.

Protozoan parasites and pathological findings in Hydromys chrysogaster (Muridae: Hydromyinae) from Queensland.

Tissues from 23 Australian water rats (Hydromys chrysogaster) collected from five localities in central and northern Queensland, Australia, between February 1992 and May 1993, were examined for protozoan parasites and additional pathological changes. We found Klossiella hydromyos in the kidneys, Toxoplasma gondii in the brain and skeletal muscles and Sarcocystis sp. in the somatic musculature. Other pathological findings, including interstitial nephritis, interstitial pneumonia and a tongue abscess, as well as helminth-induced lesions in the lungs, mesenteries, stomach wall and cecal wall were also noted.

Tuberculosis in a wild Australian fur seal (Arctocephalus pusillus doriferus) from Tasmania.

Tuberculosis was found in a wild, mature male Australian fur seal (Arctocephalus pusillus doriferus) at Hobart, Tasmania on 8 March 1992. We observed fibrogranulomatous and pyogranulomatous lesions in the lung, pleura, lymph nodes and spleen. The SDS/PAGE profile of this Tasmanian isolate was similar to other seal strains; however, differences were detected using pTBN12 and insertion sequence IS6110 probes.

Application of a composite faecal egg count procedure in diagnostic parasitology.

Egg counts from a simple composite faecal counting procedure using equal amounts of sample from ten sheep were compared statistically against the arithmetic means of the same ten samples prepared by a conventional egg counting method. Forty separate data sets were analysed in an untransformed bivariate plot and after natural logarithmic transformation. A sign test analysis indicated a high degree of similarity between the two data sets. A confidence interval for the composite count (n = 10) was calculated to give a result between five eggs more and 15 eggs less than the arithmetic mean count of the ten samples. When multiple faecal samples are to be examined, the composite method has significant advantages in time saving and increased throughput whilst still providing an accurate result. This technique has been used to monitor gastrointestinal helminthosis and for faecal egg count reduction testing to assess anthelmintic efficacy.

Mucor amphibiorum infection in platypus (Ornithorhynchus anatinus) from Tasmania.

Mucor amphibiorum, a fungus previously isolated from frogs and toads, is reported from free-living platypus, Ornithorhynchus anatinus, from rivers in northern Tasmania. This fungus is responsible for the severe ulcerative skin condition originally described by Munday and Peel (1983). Mucor amphibiorum was isolated from dermal lesions on four separate occasions. The gross and histopathological appearance of the fungal lesions were similar to the earlier description. In vivo this fungus develops as spherical forms containing a number of daughter spherules; no mycelia are seen in tissue sections. By contrast, the in vitro growth consists of aerial aseptate mycelia and sporangia, features typical of the genus Mucor. This is the first report of this organism causing a fatal disease in a mammal. Susceptibility to infection may be due to the platypus having a body temperature of 32 C while the maximum temperature for growth of M. amphibiorum is 36 C.

Allozymic and biological characters of Trichinella pseudospiralis isolates from free-ranging animals.

To evaluate biological and biochemical variability in nonencapsulated Trichinella isolates, biological and allozymic studies were conducted on isolates of Trichinella collected from a raptoral bird (Aquila rapax) and a fox (Vulpes corsac) in Kazakhstan and from a dasyurid marsupial (Dasyurus maculatus) on the island of Tasmania, Australia. Allozyme profiles of bird and marsupial isolates showed close similarity with the type isolate of Trichinella pseudospiralis. The avian and fox isolates successfully interbred with the type T. pseudospiralis isolate, but they failed to interbreed with 3 encapsulating species, Trichinella spiralis, Trichinella nativa, and Trichinella britovi. The reproductive index assessed in 4 inbred and 1 outbred strains of mice was lower for the avian isolate than for the marsupial and the type T. pseudospiralis isolates (P < 0.001).

The origin of a new focus of infection with Echinococcus granulosus in Tasmania.

Hydatid cysts were discovered in cattle on King Island, north of Tasmania, where Echinococcus granulosus was thought to have been eradicated. Using enzyme electrophoresis, isolates from King Island were compared genetically with isolates from Tasmania and the mainland of Australia. The genetic distinctness of the King Island isolates make it unlikely that they originated from a recent introduction from either Tasmania or mainland Australia. Alternative possibilities, that the infection resulted from a recent introduction from another source or from previously undetected persistence of E. granulosus on King Island, could not be distinguished from available data.