Characterisation of native Trypanosomes and other protozoans in the Australian marsupials the Quokka (Setonix Brachyurus) and the Gilbert's Potoroo (Potorus Gilbertii)

Trypanosomes are blood-borne parasites that can cause severe disease in both humans and animals, resulting in very large economic losses worldwide. In contrast to the wealth of information on pathogenic species such as Trypanosoma cruzi and Trypanosoma brucei, little is known of the pathogenicity, prevalence and life-cycles of trypanosomes in native Australian mammals. The aim of this thesis was to characterise trypanosomes and other protozoans from the critically endangered Gilbert’s potoroo (Potorous gilbertii) and the quokka (Setonix brachyurus) from Western Australia using morphological and molecular analysis. A novel Trypanosoma species, Trypanosoma copemani was identified in Gilbert’s potoroos and quokkas using molecular and morphological analysis. Further molecular characterisation of T. copemani in quokkas at both the 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loci identified multiple T. copemani genotypes revealing that the parasite is genetically variable. Given the phylogenetic grouping of T. copemani as a stercorarian (requiring faecal transmission by vector), the fact that ticks are common ectoparasites of Gilbert’s potoroos and quokkas and the previous identification of trypanosomes in ticks, ticks were examined as potential vectors of T. copemani. Motile trypanosomes were detected in both the haemolymph and midgut sections of Ixodes australiensis ticks removed from quokkas and Gilbert’s potoroos and stained trypanosomes were detected within a faecal smear. Morphologically, the tick trypanosomes resembled in vitro forms of T. copemani, representing epimastigotes and slender trypomastigote stages, with dividing stages detected within the midgut region. Molecular analysis of the tick isolates, showed 100% sequence identity to T. copemani at the 18S rRNA locus, suggesting that the tick is a putative vector for T. copemani. Investigation of the life-cycle stages of native Australian trypanosomes using microscopy and in vitro culture of blood from quokkas and Gilbert’s potoroos revealed that native Australian trypanosomes are highly polymorphic, with three different trypomastigote blood stream forms detected within blood films, resembling slender, medium and broad stages. In addition, promastigote, sphaeromastigote and amastigote stages were observed directly within blood films and this is the first report of these stages in the circulatory system of Australian marsupials. Three novel trypanosome life-cycle forms representing an oval form, an extremely thin form and an adherent form were also identified both in vitro and in vivo, while a novel tiny form and a novel circular form were only detected in culture. Trypanosoma vegrandis was also detected for the first time in one quokka isolate (Q1340) and confirmed using species-specific primers. As tourists and quokkas on Rottnest Island have a close relationship, the zoonotic potential of T. copemani was investigated by determining the relative susceptibility of T. copemani to human serum using the blood incubation infectivity test. Trypanosoma copemani was observed by microscopy in all human blood cultures from day 5 to day 14 post inoculation. The mechanism for normal human serum resistance in T. copemani is not known. The results of this thesis show that at least one native Australian trypanosome species may have the potential to infect humans. In addition to blood, quokka faecal samples were also collected to investigate the prevalence of Eimeria by PCR screening of faecal samples from three quokka populations, Two Peoples Bay, Bald Island and Rottnest Island, respectively. The PCR prevalence of Eimeria was 62.5%, 85.0% and 78.3% for these three locations respectively. Two Eimeria species were identified based on morphometric analysis from sporulated oocysts, Eimeria quokka and Eimeria setonicis, with the majority of quokkas co-infected with both species. Singular infections, however, were identified in a few individuals allowing molecular analysis to be performed using both the 18S rRNA and cytochrome c oxidase subunit 1 genes. Phylogenetic analysis grouped E. quokka and E. setonicis within the Eimeria marsupial clade. This study is the first one to characterise E. quokka and E. setonicis by molecular analyses, enabling more extensive resolution of evolutionary relationships among marsupial-derived Eimeria species. Overall this study has characterised the novel T. copemani in both the quokka and the Gilbert’s potoroo and has shown T. copemani to be both morphologically and genetically variable. This is a stercorarian trypanosome, transmitted through vector faecal contamination and may potentially be infectious to humans. The findings of this thesis highlight the complexity of Australian trypanosomes and provide insights into the prevalence, potential pathogenicity and human infectivity of this novel parasite. Further research is required to assess the role of trypanosome infection on marsupial population dynamics and the implications for management and conservation.