Amanda Duarte Barbosa

Blood-borne and enteric protozoan parasites belonging to the genera Trypanosoma, Babesia, Theileria, Cryptosporidium and Giardia are responsible for severe animal and human illnesses worldwide. In addition, parasites of the genus Hepatozoon have been associated with animal morbidity and mortality. Despite recent research and improved knowledge of the taxonomy and distribution of native Australian protozoan parasites, still relatively little is known about their epidemiology, genetic diversity and pathogenicity. The overarching aim of this thesis was to determine the prevalence, molecular characterisation and potential clinical impact of protozoan pathogens of potential conservation and zoonotic importance in Australian native mammals. A total of 465 blood samples, 167 faecal samples and 91 ticks were collected from mammals belonging to seven target species: common brush-tailed possums (Trichosurus vulpecula), northern brown bandicoots (Isoodon macrourus), northern quolls (Dasyurus hallucatus), brush-tailed rabbit-rats (Conilurus penicillatus), koalas, a little red flying fox (Pteropus scapulatus) and grey-headed flying foxes (Pteropus poliocephalus). The sampling was undertaken across four states/territories in Australia: the Northern Territory (NT), Queensland (Qld), New South Wales (NSW) and South Australia (SA). Molecular and morphological analyses were utilised to identify and characterise Trypanosoma, Babesia, Theileria, Hepatozoon, Cryptosporidium and Giardia. The potential clinical impact of the parasites identified was investigated by associating clinical, haematological and biochemical parameters, whenever available, with presence or absence of infection. A molecular survey was conducted in the NT to investigate the prevalence, genetic diversity and potential pathogenicity of protozoan parasites in common brush-tailed possums, northern brown bandicoots, northern quolls, and brush-tailed rabbit-rats. Overall, 22.5% (95% confidence interval (CI): 17.0-28.8%) of the animals tested were positive for haemoprotozoans by Polymerase Chain Reaction (PCR) targeting the 18S ribosomal RNA (rRNA) gene. Trypanosoma vegrandis and T. noyesi were found in 26.6% (95% CI: 18.7-35.7%) of the bandicoots and in 23.7% (95% CI: 11.4-40.2%) of the possums, respectively. Babesia spp. and Hepatozoon spp. were identified in bandicoots only, both at a prevalence of 5.3% (95% CI: 2.7-9.3%). Hepatozoon gamonts were detected using light microscopy in two out of 11 animals positive for this parasite by PCR. Faecal samples were tested for Cryptosporidium spp. at the 18S rRNA locus, and for Giardia spp. at the glutamate dehydrogenase (gdh) and 18S rRNA loci. The total prevalence of intestinal protozoan parasites observed was relatively low (3%; 95% CI: 1.0-6.9%). No clear signs of major morbidity were observed in infected animals, however bandicoots positive for Trypanosoma exhibited a significantly lower packed cell volume (PCV) compared to negative bandicoots (p = 0.046). The first report of T. vegrandis in koalas using morphology and sequence analysis of the 18S rRNA gene is also described. The prevalence of T. vegrandis in koalas was (13.6%; 95% CI: 5.2-27.4%). In addition, a novel next-generation sequencing (NGS)-based assay for Trypanosoma, developed during the present study, revealed that mixed infections with up to five trypanosome species in koalas were significantly more prevalent (27.4%; 95% CI: 21-35%) than single trypanosome infections (4.8%; 95% CI: 2-9%). Infections with T. gilletti, T. irwini, T. copemani and T. vegrandis were identified. Additionally, T. noyesi was detected for the first time in koalas, although at a low prevalence (0.6%; 95% CI: 0-3.3%), and a novel species (Trypanosoma sp. AB- 2017) was identified at a prevalence of 4.8% (95% CI: 2.1-9.2%). Overall, a considerably higher proportion (79.7%) of the Trypanosoma sequences isolated from koala blood were identified as T. irwini, suggesting this was the dominant species. The study also employed the NGS methodology to profile trypanosome communities within Ixodes holocyclus and I. tasmani ticks removed from koala hosts. Co-infections involving T. gilletti, T. irwini, T. copemani, T. vegrandis and Trypanosoma sp. AB- 2017 were also detected in the ticks, with T. gilletti and T. copemani being the dominant species within the invertebrate hosts. This thesis also characterised a novel trypanosome species in a little red flying fox with clinical signs of trypanosomiasis, using morphology and molecular analyses at the 18S rRNA and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loci. Morphological comparison showed that trypomastigotes of the novel species were significantly different from those of Trypanosoma pteropi and T. hipposideri, two species previously described from Australian bats for which genetic information was unfortunately not available. Phylogenetic analysis revealed that the novel species was genetically distinct and clustered with other bat-derived trypanosome species within the Trypanosoma cruzi clade. The discovery of a new bat-derived trypanosome species in Australia prompted the screening of an additional 87 blood samples from grey-headed flying foxes, which were negative for Trypanosoma 18S rDNA. In summary, this research provides new insights on the prevalence, spatial distribution, inter- and intra-specific genetic diversity and the potential negative effects of bloodborne and enteric protozoan parasites on the health of Australian mammals. Furthermore, the identification of trypanosome polyparasitism in koalas and two species of native ticks will inform future epidemiological conservation studies. The outcomes of this thesis may be used to inform wildlife management and zoonotic disease programs.