INVESTIGATION INTO INDIVIDUAL HEALTH AND EXPOSURE TO INFECTIOUS AGENTS OF PLATYPUSES (ORNITHORHYNCHUS ANATINUS) IN TWO RIVER CATCHMENTS IN NORTHWEST TASMANIA.

Changes in the health of individuals within wildlife populations can be a cause or effect of population declines in wildlife species. Aspects of individual platypus ( Ornithorhynchus anatinus ) health have been reported. However, holistic studies investigating potential synergistic effects of both pathogens and environmental factors are needed to expand understanding of platypus individual health. We collected baseline data on the health of platypuses in two Tasmanian river catchments (including evidence of the potentially fatal fungal disease mucormycosis) and on individual, demographic, and geographic patterns associated with health data results. We examined 130 wild platypuses from the Inglis River Catchment and 24 platypuses from the Seabrook Creek Catchment in northwest Tasmania between 29 August 2011 and 31 August 2013. More than 90% of captured platypuses were infected with ticks, Theileria spp., and trypanosomes. Evidence of exposure to other infections, including Salmonella spp., Leptospira spp., and intestinal parasites, was low (<10%). Three platypuses had single fungal granulomas in the webbing of a forefoot, but no evidence of mucormycosis was found in any of the study animals. Possible subclinical hepatopathies or cholangiohepatopathies were found in six platypuses. Exposure to infectious agents did not cluster geographically, demographically, or in individuals, and there was minimal evidence of morbidity resulting from infection. This study has provided important baseline data for monitoring the effects of threatening processes, including mucormycosis, on the health of infected populations.

Dermatophilus congolensis Infection in Platypus ( Ornithorhynchus anatinus ), Tasmania, Australia, 2015.

We report disease due to Dermatophilus congolensis infection in three of 13 (23%) platypuses ( Ornithorhynchus anatinus ) from a catchment in Tasmania, Australia. This pathogen has not previously been reported in platypuses. Two of the three infected platypuses had extensive scab formations, but no substantial hair loss was apparent.

Preliminary investigation into the prevalence of mucormycosis in the platypus (Ornithorhynchus anatinus) in three catchments in north-west Tasmania.

OBJECTIVE: To investigate the distribution and prevalence of mucormycosis in platypus (Ornithorhynchus anatinus) from the Inglis, Emu and Black-Detention catchment areas in north-west Tasmania. PROCEDURE: A field study was performed and resulted in the examination of 44 wild platypuses; in addition, one dead platypus and two live platypuses were examined after they were independently submitted to a local veterinary clinic. RESULTS: No cases of mucormycosis were conclusively diagnosed. One platypus with signs consistent with those previously described in cases of mucormycosis was captured in the Emu River catchment. However, laboratory tests did not provide a definitive diagnosis for the lesion. Two platypuses from the Inglis catchment area had signs very similar to those previously described in cases of mucormycosis, but laboratory tests found Corynebacterium ulcerans to be the likely cause of the cutaneous ulcers on one of these platypuses and an unidentified fungal agent to be the cause of a cutaneous nodule in the other. CONCLUSIONS: These findings do not prove that mucormycosis is absent from the populations studied. However, they may indicate that the prevalence of disease is low. The possibility that Mucor amphibiorum is present in a subclinical form in platypuses, or infecting another reservoir, is not excluded. The findings also suggest that caution should be exercised when diagnosing mucormycosis based on clinical findings alone and raise the possibility that some cases may have been incorrectly diagnosed.

Genotypic analysis of Mucor from the platypus in Australia.

Mucor amphibiorum is the only pathogen known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads (Bufo marinus) and green tree frogs (Litoria caerulea) from mainland Australia but has not been confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of 21 Mucor isolates representing isolates from the platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of most of the isolates. Representative isolates from infected platypuses formed a clade containing the reference isolates of M. amphibiorum from the Centraal Bureau voor Schimmelcultures repository. The M. amphibiorum isolates showed a close sequence identity with Mucor indicus and consisted of two haplotypes, differentiated by single nucleotide polymorphisms within the ITS1 and ITS2 regions. With the exception of isolate 96-4049, all isolates from platypuses were in one haplotype. Multilocus fingerprinting via the use of intersimple sequence repeats polymerase chain reaction identified 19 genotypes. Two major clusters were evident: 1) M. amphibiorum and Mucor racemosus; and 2) Mucor circinelloides, Mucor ramosissimus, and Mucor fragilis. Seven M. amphibiorum isolates from platypuses were present in two subclusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate subcluster, distinct from other Mucor spp. The combination of sequencing and multilocus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functional diversity will provide critical information for its management in Tasmanian river systems.

Serological survey for evidence of Leptospira interrogans in free-living platypuses (Ornithorhynchus anatinus).

OBJECTIVE: To survey free-living platypuses for evidence of Leptospira interrogans. METHODS: A serological study of Leptospira antibodies was carried out on 21 platypuses captured between May and October 2001 in the Wollondilly River 200 km south of Sydney, New South Wales. RESULTS: Positive reactions, all to the L Hardjo serovar, were seen in 14 (66%) of the captured animals, with adult males showing a higher prevalence of antibodies than adult females. Several individual platypuses showed a high titre of L Hardjo antibodies, and some animals demonstrated cross-reactions to the serovars L Medanensis and L Kremastos. CONCLUSION: The serological findings demonstrate that these animals are constantly exposed to infection with Leptospira in their environment, but it is not known if platypuses suffer from clinical leptospirosis or if they mount an immune response, but are unaffected by the bacteria. The prevalence of Leptospira infection among the platypus population could not be precisely estimated because of the unknown number of individuals inhabiting the Wollondilly River inside the survey property. Domestic livestock, mostly cattle, may be the major source of Leptospira infection. The effects of this disease on population dynamics and on reproduction in wild platypuses are not well understood. The role of other wildlife in the transmission and maintenance of Leptospira in the environment is unknown.

Possible differences in pathogenicity between cane toad-, frog- and platypus-derived isolates of Mucor amphibiorum, and a platypus-derived isolate of Mucor circinelloides.

Platypuses (Ornithorhynchus anatinus) in the north of the island state of Tasmania, Australia, suffer from a serious disease called ulcerative mycosis, which is responsible for high morbidity and, presumably, mortality rates in areas where it occurs. The disease is caused by the dimorphic fungus Mucor amphibiorum, which is also found in Queensland, New South Wales and Victoria. However, it does not cause disease in platypuses in those states. It has been previously reported that a closely related fungus, Mucor circinelloides, may also be capable of causing this disease. This paper describes pathogenicity trials involving cane toads (Bufo marinus) as the experimental model. The toads were infected with either Tasmanian, platypus-derived M. amphibiorum, West Australian, frog-derived M. amphibiorum, Queensland cane-toad-derived M. amphibiorum or Tasmanian platypus-derived M. circinelloides. The Tasmanian isolates of M. amphibiorum were more likely to cause a serious, long-term infection than were Queensland or West Australian isolates, and (+) mating types caused a more serious infection than the (-) mating type. The isolate of M. circinelloides was incapable of infecting the toads, lending further weight to the theory that it represents an environmental contaminant. The results suggest that an endemic strain of M. amphibiorum has mutated and become pathogenic to platypuses. Alternatively, a pathogenic strain of M. amphibiorum may have been introduced into Tasmania, where it is infecting a naïve population.

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.

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.

Isolation of Mucor circinelloides from a case of ulcerative mycosis of platypus (Ornithorhynchus anatinus), and a comparison of the response of Mucor circinelloides and Mucor amphibiorum to different culture temperatures.

The fungus Mucor circinelloides was isolated from a platypus (Ornithorhynchus anatinus) suffering from ulcerative mycosis. On horse blood agar at 20, 25 and 30 degrees C, the fungus formed sphaerule-like bodies, a morphology previously associated with Mucor amphibiorum, the species thought to be responsible for the disease in platypus. A biopsy taken from the ulcer was fixed, cut and stained. The sections were compared with sections taken from other platypuses suffering from ulcerative mycosis, and from which M. amphibiorum had been isolated. There were no discernible differences between the sphaerule-like bodies found in any of the sections. The presence of sphaerule-like bodies in tissues of ulcerated animals can, therefore, probably no longer be relied upon as a definitive method for the diagnosis of M. amphibiorum infection. It is possible that M. circinelloides is either a primary or a secondary pathogen of platypuses, and further work is required to resolve this point. The isolate of M. circinelloides grew at temperatures up to 38 degrees C, with an optimum temperature for growth of 30 degrees C. Of six isolates of M. amphibiorum derived from both platypus and amphibians, two grew well at 38 degrees C. The growth of one of these isolates at elevated temperatures may be explained by the hot climate of the area in Queensland in which it was found. All of the isolates tested had maximum temperatures for growth in excess of the body temperature of platypuses (32 degrees C).

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.