Genotypic and phenotypic diversity of trypanosomes infecting Australian marsupials and their association with the population decline of the brush-tailed bettong or woylie (Bettongia penicillata)

Trypanosomes are flagellated blood parasites that are capable of infecting virtually all classes of vertebrates. They range from non-pathogenic species to those that are highly pathogenic and are the causative agents of many diseases of medical and veterinary importance. While much is known of their impact on human health or economic development, a great deal less is known of those associated with wildlife. Within Australia, trypanosomes have been found naturally infecting a wide range of native marsupials, most of which are considered threatened or endangered. However, their research has largely been confined to the description of trypanosome morphology in blood, and a complete lack of information regarding their life cycle, virulence, and pathogenicity is evident. This study therefore, aimed to investigate the genotypic and phenotypic diversity of Trypanosoma spp. infecting Western Australia marsupials and to determine their potential pathogenicity with particular emphasis in the critically endangered marsupial, the woylie (Bettongia penicillata). The genotypic characterisation was achieved using a combination of sequencing and phylogenetic analysis of trypanosomes in the blood and tissues of nine different marsupial species, as well as the sequencing of partial fragments of the minicircles of the kinetoplast DNA of trypanosomes isolated in culture. The phenotypic characterisation involved a combination of histology, microscopy techniques, and in vitro experiments of cell infection and drug susceptibility. Results revealed that eight different genotypes belonging to three different Trypanosoma species: T. copemani, T. vegrandis, and T. sp H25 were found infecting woylies (Bettongia penicillata), quendas (Isoodon obesulus), quokkas (Setonix brachyurus), tammar wallabies (Macropus eugenii), banded hare wallabies (Lagostrophus fasciatus), boodies (Bettongia lesueur), Chuditches (Dasyurus geoffroii), common brush tailed possums (Trichosurus vulpecula), and western grey kangaroos (Macropus fuliginosus). However, the woylie was the only marsupial species where single individuals and single tissues were co-infected with genotypes belonging to the three different Trypanosoma species. Furthermore, T. copemani G2, the predominant trypanosome in the declining population of woylies, was shown to be able to infect tissue cells and generate a strong immune response characterised by tissue degeneration and necrosis in vital organs, suggesting an association between these infections and the decline of the woylie. Comparative analysis between T. copemani G2 and the pathogenic T. cruzi showed not only similarities in their capacity to infect tissue cells, but also in drug susceptibility and kinetoplast DNA organisation. In summary, this study not only contributes valuable information towards directing management decisions for endangered species where trypanosomes are known to be present at high prevalence levels, but also provides new knowledge about the evolutionary biology and relationships that Australian trypanosomes have with the exotic and pathogenic T. cruzi.