James MacGregor

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.

Piroplasms belonging to genera Theileria and Babesia (Phylum: Apicomplexa) are vector-borne protozoan haemoparasites with similar phenotype that infect erythrocytes of domesticated mammals and wildlife including birds. In addition, piroplasms of Theileria have an exoerythrocytic life cycle stage within the host’s white blood cells. Observations of intraerythrocytic piroplasms have been made sporadically in peripheral blood films of Australian native mammals since the first report was made nearly a Century ago and the advent of PCR has stimulated renewed research interest and descriptions of the molecular phylogeny of these organisms in a variety of marsupial hosts. A piroplasm (Theileria ornithorhynchi) has been described in previous studies of the platypus (Ornithorhynchus anatinus). As part of a study into the health and ecology of this iconic monotreme, blood samples were collected for haematological and biochemical analysis from wild-trapped platypuses in Tasmania. A subset of these samples, together with their ectoparasites, was evaluated for piroplasm infections by blood film microscopy and molecular analysis of the 18S ribosomal RNA gene, and phylogenetic analysis was performed with NJ trees using BIONJ. Estimates of evolutionary divergence between sequences were calculated by MEGA 5. A total of 27 blood samples and eight ectoparasites (seven ticks, one leech) were examined from 27 platypuses. All ticks were identified as Ixodes ornithorhynchi. Piroplasm infections were highly prevalent in the population studied (27/27; 100% infected); organisms were pleomorphic with infrequent tetrads and intra-leukocytic forms, thought to be Theileria schizonts, were occasionally observed. Anaemia was not detected. Many platypuses were co-infected with trypanosomes. All blood samples and three ectoparasites were positive for piroplasm DNA and phylogenetic analysis suggested the presence of more than one piroplasm species. This study emphasises the importance of combining traditional parasitological techniques (e.g. microscopy and tick examination) with molecular techniques if the life cycle of organism is to be understood.

The order Piroplasmida (Phylum: Apicomplexa) includes three main genera, Babesia, Theileria, and Cytauxzoon that are vector-borneprotozoan haemoparasites, some of which have clinical relevance both in humans and animals. With exceptions, Babesia is typically differentiated from Theileria based largely on morphology, serology, and several life-cycle peculiarities, within the tick vectors and vertebrate hosts. Despite these differences, and their global socio-economic importance, piroplasms often present conflicting or uncertain molecular classifications, nomenclature, and intergenic relationships. For instance, based on the small ribosomal subunit RNA gene (18S rDNA), the taxon Piroplasmida includes at least nine mono- and para-phyletic clades, which are sometimes populated by relatively distant members of the same genus, or by closely related species of different genera1 . As part of a study into the health and ecology of the platypus (Ornithorhynchus anatinus; Order: Monotremata), blood samples were collected for haematological and biochemical analysis, from wild-trapped individuals in Tasmania. Blood, together with ectoparasites removed from each individual, was also evaluated for piroplasm infections by blood film microscopy, and molecular analysis of the 18S rDNA. Moreover, phylogenetic analyses were performed on a subset of samples. Pleomorphic organisms with occasional tetrads and intra-leukocytic forms, thought to be Theileria schizonts, were observed by microscopy, and were phenotypically consistent with Theileria ornithorhynchi, a piroplasm of the platypus named in previous studies2 . However, molecularly, the parasite-derived DNA belonged to two potentially novel piroplasm species, forming one monophyletic clade, clearly separated from other known marsupial-derived Theileria spp.3 . The high prevalence (100%), apparent lack of clinical signs, and distinct phylogenetic position of the parasite, likely reflect the unique ecology and evolutionary history of its ancient vector-host system.