Trypanosomes from native Australian mammals: novel genotypes from the platypus (Ornithorhynchus anatinus)

Trypanosomes are vector-borne parasitic haemoprotozoa that infect all classes of vertebrates, and are the etiological agents of severe diseases accompanied by a range of clinical signs including fatigue, fever, anaemia, and death, in both animals and humans1 . Little is known about the prevalence and pathogenesis of trypanosomes in Australian marsupials and monotremes, and few genetic characterisation studies have been conducted. During this study, using molecular and microscopic methods, we screened peripheral blood (n=27) and ectoparasites (n=8, from 7 animals) collected from wild Tasmanian platypuses (Ornithorhynchus anatinus), for the presence of trypanosomatid-specific DNA and trypanosomes. The genes for the small ribosomal subunit RNA (18S rDNA) and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) were amplified and sequenced, prior to examining phylogenetic relationships by the maximum likelihood (ML) and neighbour joining (NJ) methods. Based on molecular results, parasite 18S rDNA prevalence was 37% (n=10/27) in blood samples. Additionally, one tick out of eight provided positive amplification and specific sequencing products, at the same locus. This apparently high prevalence was confirmed by the microscopy results, consistent with high parasitemia and the presence of abundant trypomastigotes in the blood films. Phylogenetic analyses at the 18S locus revealed the existence of multiple trypanosomatid-like genotypes, similar but distinct from the previously described Trypanosoma binneyi Mackerras, 19592 . The novel, statistically-supported clade (>85%), included new platypus-derived genotypes with 0.4-0.9% genetic distance from known T. binneyi sequences. The phylogenetic reconstructions of the trypanosomatid 18S rDNA, isolated from monotremes and marsupials3 , reveal a significant genetic diversity of the parasites associated with these unique hosts. The presence of various native Australian mammalian hosts, on multiple branches of the 18Sr rDNA tree of the monophyletic genus Trypanosoma, confirms the long evolutionary history of the parasite-host system, within the mammalian lineage