Andrew C Thompson

The genus Trypanosoma comprises numerous species of flagellated vector-borne protozoa that parasitise the blood and tissues of vertebrates. They are ubiquitous in terms of their geographical distribution and host range. In mammals, most species of Trypanosoma have been described from wildlife yet apart from their taxonomy, we know very little about the host parasite relationship, particularly those species in Australia. Most of what is known about their host parasite relationships, life history, and developmental biology has been obtained from studies on the two species that invade cells and infect humans - T. brucei and T. cruzi - species endemic in Africa and South America respectively, and which are major causes of disease and death in humans and domestic animals. T. brucei is the cause of sleeping sickness as a result of its association with the nervous system, whereas T. cruzi is the cause of Chagas disease that results in cardiac, neurological, and digestive disorders. Importantly, an Australian species - T. copemani G2 - has also been found to invade cells, thus demonstrating a pathogenic potential previously not associated with trypanosomes from Australia. Recently several new species of Trypanosoma in Australian marsupials (T. copemani (G1 & G2), T. vergrandis, and T. noyesi) have been characterised. These have varying affinities to T. cruzi, including surprisingly similar genetic relationships (e.g. close genetic link of T. noyesi and T. cruzi) and behavioural traits (e.g. cellular invasion by both T. copemani and T. cruzi). Such observations not only raise concerns about the impact of Australian trypanosomes on wildlife health and conservation but also in terms of biosecurity and human health given the potential for local transmission of imported cases of Chagas disease. Here we present correlative data across a range of length scales demonstrating the ongoing characterisation of several Trypanosoma spp. from Australian wildlife. In particular we have used live cell imaging to show host cell-pathogen interactions (Figure 1), scanning (SEM) (Figure 2) and transmission (TEM) (Figure 3) electron microscopy for structural analysis, and Slice and ViewTM focussed ion beam-scanning electron microscopy (FIB-SEM)(Figure 4) to begin to image key structural features (e.g. kinetoplast, flagellum) at high resolution in 3-dimensions. Together these data are i) providing a greater understanding of the pathogenic potential and host-parasite relationships of trypanosomes in Australian marsupials; ii) allowing for identification of biosecurity issues relating to potential local hosts and transmission of exotic species; and iii) generating information about the role of trypanosomes as a potential cause of disease in threatened and endangered Australian marsupials.

Toxoplasma gondii is a ubiquitous protozoan parasite of vertebrates. Infection can lead to a wide spectrum of disease states, ranging from altered behaviour to severe, often fatal illness. Virulence depends, in part, upon the genetotype of the parasite. Although T. gondii has been identified in Australian marsupials, there have been few studies of the prevalence of the parasite or its genetic characteristics in natural populations. We obtained tissue samples from five different organs of 16 adult kangaroos from arid rangeland in Western Australia. Samples were screened for T. gondii by DNA extraction and direct sequencing. There were three very surprising results. First, all 16 kangaroos were infected, which indicates either heavy environmental contamination or substantial verical transmission of the parasite. Second, multiple infections with different genotypes of T. gondii were found in all of the kangaroos, which suggests that sexual reproduction occurs regularly in the life cycle of the parasite in Australia. Finally, 88% of the genotypes of T. gondii that were detected were different to the three common strains found in domestic transmission cycles in other parts of the world, which suggests that T. gondii in Australian wildlife may exhibit a wide range of virulence states.

One of the largest mammal translocations in Australia took place earlier this year, providing a unique opportunity for an in depth investigation into the population ecology, parasitology and survival of relocated animals. 183 golden bandicoots (Isoodon auratus) from Barrow Island and 144 boodies (Bettongia lesueur) were translocated from 2 different source populations (Barrow Island and Dryandra captive breeding centre) and released in central Western Australia, at Lorna Glen. This study aims to assess important factors in determining the translocation success at an individual and population level, and understand the population ecology of parasites and stressed hosts. The animals were closely monitored for parasitological, reproductive and condition status before and after the relocation at six-week intervals. Half of the population were treated with a topical antiparasitic treatment in order to experimentally manipulate the transmission of blood parasites and reduce overall parasite load. We expect to see an effect on survival, fecundity and condition of the animals according to their treatment groups and population origin. This project addresses the need for more quantitative science and experimentation in translocations in order to promote greater successes.

Prior to European settlement, the Woylie (or brush-tailed bettong) Bettongia penicillata, had a distribution over much of Australia. Over the next 180 years, the woylie distribution was reduced and became restricted to three principal areas in south-west Australia, namely Upper Warren, Tatanning and Dryandra. As part of the recovery plan, fox control and woylie relocations were initiated and by 1996 the woylie became the first Australian mammal to have its conservation status downgraded. However, since 2001 the number of woylies has declined rapidly, with capture rates indicating a 70–80% reduction in population sizes over a 5 year period. During the investigation into the recent decline, a distinct species of Trypanosoma was identified at high prevalence and studies have shown a correlation between parasite prevalence, high parasitaemia and woylie decline. In efforts to further understand this vector-borne parasite, sampling of haematophagic arthropods has focused on Tabanids, Sandflies, Fleas, Ticks and Midges in the Upper Warren and Karakamia regions. It is hoped that this understanding of the vector and its distribution will provide baseline data for future woylie relocation programs, safe-guarding against the inadvertent introduction of disease into naive populations or naive animals into infected populations, thus increasing the chances for success.

Woylie or brush-tailed bettong (Bettongia pencillata) populations are undergoing a major decline in southwest Western Australia. Through collaboration with the Department of Environment and Conservation (DEC) it has been possible to examine the parasite fauna of the declining population since the decline commenced in 2006. Only two potential pathogens have been identified, Trypanosoma and Toxoplasma, which, when compared with healthy woylie populations, are associated with the decline. Although it appears unlikely that the parasites are solely responsible for the decline in woylie population size, they may predispose woylies to increased mortality. Molecular characterisation has revealed how little we know about the phylogenetic relationships and ecology of both Trypanosoma and Toxoplasma in Australian native wildlife raising questions about transmission and control. The parasitological investigation of woylies has demonstrated the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents in terms of wildlife health, parasite biodiversity and ecology.

Since 2006 veterinarians from Perth Zoo have been working collaboratively with the Department of Environment and Conservation and Murdoch University to investigate the causes of dramatic population declines in free-ranging woylies in south west Western Australia. Investigation to date has included examining and collecting diagnostic samples from free-ranging and captive woylies, and from conspecific free-living vertebrate species. Blood samples are analysed for haematologic and biochemical parameters, along with evidence for specific pathogens (including Macropod Herpes Virus, Trypanosomes, Toxoplasmosis and Piroplasmosis). Obviously diseased animals undergo thorough veterinary examination and treatment at Perth Zoo. Full post mortem examinations are conducted on deceased animals, including histological examination of tissues, by university pathologists. Historical information from previous disease outbreaks or population declines in woylies has been collated. Temporal and spatial analysis of the complex data sets collected during field investigations facilitates understanding of the factors influencing population dynamics.

Australian native fauna have long been recognised for their susceptibility to infection with Toxoplasma, often suffering serious clinical consequences. There have also been anecdotal suggestions that Toxoplasma could have caused die-offs of native fauna in the past, and that the source of such infections was likely to be cats introduced by Europeans. However, much of our understanding relates to the consequences of infection in captive animals. There are few data on the prevalence and impact of Toxoplasma in free ranging wildlife. We present data suggesting that Toxoplasma infection is associated with the decline of woylies in the south-west of Western Australia. Further, the molecular characterisation of Toxoplasma isolates from marsupials has demonstrated the occurrence of novel ‘trains’ thus questioning the origin of Toxoplasma in Australia.

A PCR-based method capable of the direct detection of the enteric protozoan Blastocystis in faeces was used to detect the parasite from various hosts from the Perth Zoo and West Australian native wildlife. Overall, 48% and 2% of animals from the Perth Zoo and native wildlife were positive for Blastocystis, respectively. This is the first report of Blastocystis found in the southern hairy nosed wombat, quokka, elephant and giraffe. Novel isolates were also found in the elephant, giraffe and white cheeked gibbon. Most of the primates at the Perth Zoo are harbouring Blastocystis. Seven isolates from five different primate hosts are identical and belong to Subtype 1, which may be of zoonotic significance. Two of these primate hosts harboured mixed infections of Subtypes 1 and 2. Also, a brushtail possum isolate belonged to Subtype 4. The significance of these findings will be discussed.

Due to the endangered status of African painted dogs (Lycaon pictus) it is important to understand what parasitic diseases they are exposed to and what effect these are having on the rapidly declining wild populations. Conversely, zoo collections of these animals are under different pressures due to their captive lifestyle such as stress, nutrition, inbreeding and intensive housing. Faecal samples were collected from captive populations housed at Perth Zoo, Monarto and Adelaide Zoos and DeWildt Wildlife Trust in South Africa. Wild populations have been sampled from Zambia and Namibia with further sampling to be undertaken Zimbabwe and South Africa. Samples have been analysed via microscopy and parasites observed identified to genus. Giardia cysts and Spirometra sp. were detected in captive populations while parasite eggs of Taeniidae, Ancylostomatidae and Sarcocystis were detected in the wild populations. Molecular characterisation was then conducted in order to characterise those parasites found. Of particular interest is the zoonotic potential of the Giardia sp. detected in captive animals and the determination of Echinococcus sp. from the Taeniid ova found. Further sampling will add statistical rigour in order to quantify faunal structure.

Ectoparasite biodiversity across a range of Western Australian threatened mammals is being described as part of a larger project examining the presence and impact of parasites in fauna. Fleas, ticks, mites and lice are collected in animals that are trapped across the State as part of the Department of Environment and Conservation's threatened mammal monitoring programs, an Australian Research Council funded project and the Woylie Conservation Research Programme. Most of the published work on ectoparasite biodiversity was done in the first half of the last century and is based on drawings of morphological features. These monographs have been found to be inadequate and many rare mammals have no records of their parasite fauna described. New methods utilising PCR and scanning electron microscopes are being used to help describe species of ectoparasite. A tick found on the woylie may prove to be a new species. PCR is also being used to examine the role of ectoparasites as vectors of disease. The presence of introduced ectoparasites such as the rat flea Xenopsylla cheopis underlines the risks to Australian fauna of novel vector-borne diseases. Results to date will be discussed and it is hoped that the work will contribute to wildlife management decisions as well as biodiversity research.