David L. Obendorf

The Immune Response of the Tasmanian Devil (Sarcophilus harrisii) and Devil Facial Tumour Disease

One of the most remarkable aspects of Devil Facial Tumour Disease (DFTD) is its infectious nature, and for successful transmission it must avoid detection by the devil’s immune system. For this to occur, the devil either is severely immunosuppressed or factors produced by the tumor contribute to its avoidance of immune detection. An analysis of the devil’s immune system revealed the presence of normal-looking lymphoid organs and lymphoid cells. At a functional level the lymphocytes proliferated in response to mitogen stimulation. Subcutaneous injection of a cellular antigen produced a strong antibody response, providing compelling evidence that the devil has a competent immune system. Tumor cell analysis demonstrated that the tumor expresses the genes of the major histocompatibility complex; however, there was a limited diversity. Therefore, the most likely explanation for devil-to-devil transmission of DFTD is that the tumor is not recognized by the devil as “non-self” because of the limited genetic diversity. With its consistent morphology and relatively stable genome, this tumor would provide a reasonable target for a vaccine approach, provided the immune system can be coaxed into recognizing the tumor as “non-self.”

CAUSES OF MORTALITY AND MORBIDITY OF WILD KOALAS, PHASCOLARCTOS CINEREUS (GOLDFUSS), IN VICTORIA, AUSTRALIA

Between 1975 and 1980, necropsy investigations were conducted on 44 wild koalas (24 males, 20 females) from several localities in Victoria, Australia. An additional 11 (5 males, 6 females) were presented for clinical appraisal and treatment. Traumatic injuries resulting from motor vehicle accidents and intra-specific conflict were the commonest reason for submission (19 of 55; 35%). Keratoconjunctivitis (8 of 55; 15%), ascending urinary tract infections (6 of 20 females; 30%), ascending genital tract inflammation (10 of 20 females; 50%) and sarcoptic mange (2 of 55; 4%) were recognized as specific diseases or disease syndromes. A peracute syndrome characterized by lassitude, depression, anorexia and coma was identified in moribund koalas submitted from the wild and also in hospitalized animals. The condition, termed koala stress syndrome, was thought to be initiated by intercurrent disease or trauma, long term hospitalization and frequent manipulation and treatments. Hematological observations in 54 apparently healthy wild koalas from five different populations and on 17 sick or injured animals are also presented. Certain blood parameters are discussed in relation to the health status of the populations or individuals. Areas for further research into koala diseases are also discussed.

Tumor-specific diagnostic marker for transmissible facial tumors of Tasmanian devils: immunohistochemistry studies

Devil facial tumor disease (DFTD) is a transmissible neoplasm that is threatening the survival of the Tasmanian devil. Genetic analyses have indicated that the disease is a peripheral nerve sheath neoplasm of Schwann cell origin. DFTD cells express genes characteristic of myelinating Schwann cells, and periaxin, a Schwann cell protein, has been proposed as a marker for the disease. Diagnosis of DFTD is currently based on histopathology, cytogenetics, and clinical appearance of the disease in affected animals. As devils are susceptible to a variety of neoplastic processes, a specific diagnostic test is required to differentiate DFTD from cancers of similar morphological appearance. This study presents a thorough examination of the expression of a set of Schwann cell and other neural crest markers in DFTD tumors and normal devil tissues. Samples from 20 primary DFTD tumors and 10 DFTD metastases were evaluated by immunohistochemistry for the expression of periaxin, S100 protein, peripheral myelin protein 22, nerve growth factor receptor, nestin, neuron specific enolase, chromogranin A, and myelin basic protein. Of these, periaxin was confirmed as the most sensitive and specific marker, labeling the majority of DFTD cells in 100% of primary DFTD tumors and DFTD metastases. In normal tissues, periaxin showed specificity for Schwann cells in peripheral nerve bundles. This marker was then evaluated in cultured devil Schwann cells, DFTD cell lines, and xenografted DFTD tumors. Periaxin expression was maintained in all these models, validating its utility as a diagnostic marker for the disease.

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 seropositive 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.

Sarcocystis AND RELATED ORGANISMS IN AUSTRALIAN WILDLIFE: II. SURVEY FINDINGS IN BIRDS, REPTILES, AMPHIBIANS AND FISH

Sarcocystis was found in 105 (44 species, 25 families) of 832 (12.6%) (129 species, 43 families) avian muscle samples. Muscle sarcocysts were most prevalent (16-29%) in certain carnivorous, omnivorous or insectivorous birds. Lower prevalences (6-13%) were recorded in other carnivorous, ground-feeding omnivorous and insectivorous birds. In waterfowl, prevalence of infection was only 3.5%. Generally, sarcocysts in bird muscle were thin-walled (<0.5 μm) with small zoites. However, those in little pied cormorants (Phalacrocorax melanoleucos), hoary-headed grebes (Podiceps poliocephalus) and a pelican (Pelecanus conspicillatus) examined in Victoria had thicker (0.5-1 μm) walls. Three of 53 (5.7%) muscle samples from reptiles (14 species, four families) had Sarcocystis. Greatest prevalence was found in goannas (Varanus spp.) where two of three specimens were positive. Ninety samples from nine amphibian species (two families) and seven from five species of fish (five families) were negative for muscle sarcocysts. Sporocysts and oocysts of Sarcocystis or Frenkelia were found in intestinal scrapings from eight of 18 (44.4%) barn owls (Tyto spp.), two of five (40.0%) spotted owls (Ninox novaeseelandiae), and two of seven (28.6%) brown falcons (Falco berigora). Mucosal scrapings from five of 12 (41.7%) elapid snakes (one Austrelaps superba and four Notechis ater) were positive for sporocysts and oocysts.

Sarcocystis AND RELATED ORGANISMS IN AUSTRALIAN WILDLIFE: I. SURVEY FINDINGS IN MAMMALS.

Muscle samples from 1497 Australian mammals, comprising 73 species (2 monotreme, 48 marsupial, 3 cetacean, 1 lagomorph, 10 rodent, 2 canid, 3 chiropteran and 4 cervid) were examined histologically for cysts of Sarcocystis. Cysts were recorded in 126 (8.4%) of the mammals examined. Type A cysts (cysts with thick walls and/or large zoites and/or internal trabeculation) were found in 77 (5.1%) of these mammals, representing 13 species (4 marsupial, 1 cetacean, 1 lagomorph and 7 rodent). Type B cysts (cysts with thin walls and small zoites) were found in 50 (3.3%) of the animals, representing 20 species (14 marsupial, 1 cetacean and 5 rodent). The greatest prevalence of type A cysts (21.1%) was found in rabbits, Oryctolagus cuniculus. Among the rodents examined, type A cysts were more frequently encountered (11.1%) than type B (2.2%). In four species, Rattus fuscipes, R. lutreolus, R. rattus and Mus musculus, both types of cyst were found. Both types of cyst also were recorded in the macropodid marsupials, but type B cysts were encountered more frequently (7.2%) than type A cysts (1.7%) and only in one species, Macropus rufus, were both forms detected. Type B cysts were found in macropods in Tasmania as well as on the mainland of Australia, whereas only the mainland marsupials harbored type A cysts. Scrapings of intestinal mucosa or feces were examined from 92 dasyurid marsupials, comprising 5 species, and from 55 feral cats (Felis domesticus). Sporulated sporocysts typical of Sarcocystis or Frenkelia were found in 1 Tasmanian devil, Sarcophilus harrisii, 1 tiger cat, Dasyurus maculatus, and 1 feral cat.

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.

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.

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.

A Histological and Immunohistochemical Analysis of Lymphoid Tissues of the Tasmanian Devil

Tasmanian devil lymphoid tissues (thymus, spleen, and lymph node) from seven animals, including pouch young, juvenile, and adult devils, were investigated using histological and immunohistochemical techniques. Antibodies against the conserved intracytoplasmic portion of CD3 and CD79b (T‐ and B‐cell markers, respectively) and MHC II were used to label immune cells. The thymus from the juvenile devils and the pouch young had CD3+ cells that were primarily located in the medulla of the organ. The spleen consisted of red and white pulp areas with characteristic lymphoid follicles with CD79b+ and MHC II+ cells and nonfollicular T‐cell‐dominated periarteriolar lymphoid sheaths. Peripheral lymph nodes presented three distinct regions, outer cortex and medulla (both with primarily CD79b+ and MHC II+ cells) and paracortex (mainly CD3+ cells). Tissue architecture and distribution of the immune cells were similar to that seen in eutherian mammals and other marsupials, indicating that the Tasmanian devil has all the structural elements necessary for effective adaptive immunity. Anat Rec, 292:611–620, 2009.