Peter Irwin

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.

The first Australian case of human babesiosis was recently reported (Senanayake et al., 2012), and the infection is believed to have been locally acquired. Intra-erythrocytic parasites were identified at the Camberra Hospital, by microscopic blood film-examinations, in a 56YO man from NSW. Piroplasms (Apicomplexa) are protozoa comprising the (mainly) tick-transmitted genus Babesia. Human babesiosis, is caused predominantly by B. microti (USA) and B. divergens (Europe), and has recently been found in Asia, Africa and South America, but never before in Australia. Objective. To investigate the genes for β-tubulin and 18S ribosomal RNA (18S rDNA), and confirm the phenotypic diagnosis of babesiosis in this patient. To reconstruct strain phylogeny and tentatively infer its origin, using multiple phylogenetic markers. Method. An 18S rDNA consensus sequence of the parasite was obtained from the patient’s blood using several available PCR primers. A BLAST-search identified, in GenBank, sequences of B. microti resembling the query. An alignment of available B. microti sequences, was used to design five novel PCR primers sets, specific for the β-tubulin gene. Results. All the novel β-tubulin primers provided strong amplification, which allowed successful sequencing and confirmation of the blood sample’s positivity for B. microti. A phylogenetic tree revealed clustering with North American B. microti isolates derived from a tick, humans and voles. Conclusions. The five novel β-tubulin primer sets proved useful for confirming the 18S rDNA-based identification using a second locus. This is the first step towards unravelling the intriguing questions about the origin and epidemiology of this organism in Australia.

Protozoa of the genus Babesia (Piroplasmida, Apicomplexa) are the second most common blood-borne parasites of mammals after the borne parasites of mammals after the trypanosomes. Over 100 species have been identified, infecting many mammalian and some avian species. Babesias are the aetiological agents of babesiosis, a tick-transmitted disease causing substantial losses of livestock and companion animals worldwide. Human babesiosis is a globally emerging zoonosis. Its clinical symptoms include: fever, fatigue, headache, chills, neck stiffness, accompanied by erythrocytelysis, and possibly organ failure. With exceptions, human babesiosis, is caused predominantly by B. microti (USA) and B. divergens (Europe). Babesiamicroti has a Holarctic (N.Hemisphere) distribution and has never before been identified in Australia.

Three series of severe pet food associated toxicoses were diagnosed during 2009 in Western Australia. Five cases of clinical hepatotoxicity were diagnosed in dogs fed a commercial elimination diet of camel meat and sweet potato for atopic skin disease. These dogs all had detectable blood levels of indospicine, a toxic non-protein amino acid, isolated from Indigofera linnaei, a plant grazed by camels. Histological lesions in these dogs were consistent with previously reported lesions in dogs fed horse meat concentrated with indospicine. Four dogs exclusively fed a commercial pet diet available at supermarkets presented with marked neurological signs and were diagnosed with thiamine deficiency due to sulphur preservatives in the food. Two cats exclusively fed a commercial cat food diet available at supermarkets, were diagnosed with hypercalcaemia due to excessive vitamin D. The pet food was tested, and found to have high vitamin D concentrations. This diet was exclusively fed to the cats, with no other potential source of toxicity. Conclusion: These cases highlight that not all animals’ fed a diet will develop clinical disease and that an accurate description of an animal’s diet and feeding plan are a mandatory component of disease investigation. It is also possible that a significant number of pet food toxicities go unrecognized. Pet food should be considered as a potential source of toxins and a cause of clinical disease, and these cases support tighter regulation of the pet food industry.

Protozoan parasites remain ubiquitous pathogens for both humans and animals and continue to cause significant morbidity and mortality. Currently, there is a lack of fundamental knowledge on protozoan prevalence and impact on marsupial populations. Here, ectoparasites, blood, and faecal samples were collected from different marsupials and screened by PCR for Giardia, Cryptosporidium, Babesia, Theileria, and trypanosomes. Samples were collected from three locations in Western Australia and Queensland: Kanyana Wildlife Rehabilitation Centre, WA (n=41), Jarrah Forrest near Dwellingup, WA (n=63), Australian Wildlife Hospital, QLD (n=71). About 10% of the wild animals from WA were infected with trypanosomes and Babesia, while Theileria and Cryptosporidium were only detected in about 4% of the sheltered marsupials from Kanyana. Giardia was detected in all groups, at frequencies ranging from 6 to 17%. Sequencing of amplified DNA revealed the presence of novel as well as previously identified protozoan species, some of which were detected in critically endangered species. Phylogenetic trees were constructed based on 18S rDNA (Babesia, Theileria, Cryptosporidium, and Giardia), and 18S rDNA/ GAPDH (trypanosomes). One sheltered boodie (Bettongia lesueur) from Kanyana had a novel Theileria sp., related to T. bicornis (92% identity), which is associated with mortality in black rhinoceros (Diceros bicornis). Seven woylies (Bettongia penicillata) were infected with a novel Babesia sp., most closely related (96% identity) to Babesia species from cattle and an antelope. 18S rDNA/GAPDH trypanosome sequences amplified from woylies and brushtail possums (Trichosurus vulpecula) were 99% similar to sequences previously found in kangaroos. Cryptosporidium macropodum was identified in faecal samples of two sheltered joeys, and Giardia duodenalis assemblage A was detected in various hosts, including koalas and threatened species such as chuditchs, bilbies, and boodies. A better understanding of protozoan parasites infecting marsupials is essential to successfully maintain and manage our marsupial populations, and limit the spread of zoonotic diseases.