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Prevalence and molecular characterisation of Trypanosoma spp. in two wild koala populations; Moreton Bay, Queensland and Mount Lofty, South Australia

Howard, Eliza May (2022) Prevalence and molecular characterisation of Trypanosoma spp. in two wild koala populations; Moreton Bay, Queensland and Mount Lofty, South Australia. Honours thesis, Murdoch University.

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The koala (Phascolarctos cinereus) is an iconic Australian marsupial that is under threat of extinction across two thirds of its range, with populations recently listed as ‘endangered’ in Queensland (QLD), New South Wales (NSW) and the Australian Capital Territory (ACT). Many risk factors have been implicated in the koala population decline, including habitat loss, vehicle collisions, dog attack and infectious diseases such as chlamydiosis and koala immune deficiency syndrome caused by koala retrovirus (KoRV).

Trypanosomes are blood-borne protozoan parasites that can infect all classes of mammals and are known to cause serious disease in humans and domestic livestock worldwide. Recent studies have identified numerous Trypanosoma species in a range of Australian marsupials, including the koala which is known to harbour up to six species in either single or mixed infections: Trypanosoma irwini, Trypanosoma gilletti, Trypanosoma copemani, Trypanosoma vegrandis, Trypanosoma noyesi and Trypanosoma sp. AB-2017. Importantly, preliminary data from analyses of hospitalised koalas in QLD suggest that trypanosome infections (alone or with concurrent diseases) may adversely affect koala health and survival.

Whilst a large number of studies have been conducted on chlamydia and KoRV, there is still a paucity of research investigating the prevalence, diversity and clinical impact of trypanosomes in koalas. In particular, there is a dearth of research comprising random, representative samples from various wild koala populations across Australia, including more stable populations from South Australia (SA).

This descriptive cross-sectional study utilised nested PCR, targeting partial fragments of the nuclear 18S ribosomal RNA (18S rRNA) gene, to screen blood samples from wild-caught koalas for the presence of trypanosomes. Samples were randomly collected from koalas belonging to two distinct wild populations; Moreton Bay, Queensland (QLD) (n= 72) and Mount Lofty, South Australia (SA) (n= 89). The overall prevalence of Trypanosoma in both populations was 47.2% (76/161; 95% CI: 39.3-55.2%). The prevalence of trypanosomes in koalas from Moreton Bay was 80.6% (58/72; 95% CI: 69.5-88.9%), whereas the prevalence in koalas from Mount Lofty was significantly lower: 20.2% (18/89; 95% CI: 12.4-30.1%). Sanger sequencing of PCR positive products was performed and phylogenetic analysis conducted on the partial 18S rDNA fragments obtained. A total of 35 Trypanosoma isolates from Moreton Bay koalas were identified as Trypanosoma irwini (n= 36), with intra-specific genetic variations ranging from 0% - 2.99%. Remaining QLD isolates (n=16) were identified as Trypanosoma gilletti, with genetic distances ranging from 0% - 1.20%. These results are similar to findings from previous studies of hospitalised koalas from QLD and NSW.

All Trypanosoma isolates from the Mount Lofty population (n = 18) formed a unique, highly diverse clade within the Trypanosoma cruzi clade of trypanosomes. These novel sequences displayed a high genetic variation amongst each other (genetic distances = 0% - 7.04%) and from their most closely related species (T. sp 1EA-2008) (genetic distances = 1.90% - 7.73%). To the best of the author’s knowledge, this is the first report of trypanosomes in koalas from SA. The unique phylogenetic position of the isolates identified, associated with a relatively high genetic distance from their most closely related known Trypanosoma sp., suggests that they may potentially represent novel Trypanosoma spp.. Further analyses of full-length 18S sequences and additional loci are required to confirm this finding and reliably delimit the species.

Sanger sequencing of seven PCR positive isolates from Moreton Bay koalas revealed mixed chromatograms and were excluded from phylogenetic analyses. Further analyses using next-generation metabarcoding are required to identify and characterise mixed trypanosome infections in all positive samples detected in the present study, particularly those that produced mixed Sanger sequencing chromatograms.

This study provides valuable novel baseline data which will contribute to the growing knowledge base of Australian trypanosomes, and future studies on the potential impact of Trypanosoma spp. (with and without concurrent infectious diseases) on the health and conservation of koalas.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary Medicine
Supervisor(s): Barbosa, Amanda and Austen, Jill
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