Una Ryan

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.

Cyanobacteria are ubiquitous, pleomorphic, photosynthetic microalgae. Traditionally, they have been identified and classified through morphological methods, however this process is highly subjective as distinctive phenotypic characteristics may vary within species and growth phase, or be lost as a result of environmental or culture conditions. Pleomorphism during long-term cultivation has also resulted in strains being misidentified. Hence, studies into cyanobacteria identification and diversity have increasingly incorporated both molecular and morphological methods into the process. To determine how well these methods correlate, 37 cyanobacteria isolates were characterized both morphologically, by one or more experienced taxonomists, and by DNA sequence analysis at the 16S, rpoC1 and phycocyanin loci. Of the 37 isolates, 17 were examined by 2 independent taxonomists and results revealed that 23.5% were in agreement to the species level, 47.1% to the genus level, and the remaining 17.6% to the family or order level. Amplification was successful for 27 isolates for 2 or more loci. Phylogenetic analysis showed that between loci, there was 25.9% agreement at the species level, 63.0% to the genus level, and 22.2% to the order level. Between morphology and phylogeny, there was only 21.6% agreement to the species level and 45.9% agreement at the genus level. These data highlight the difficulties in identification of cyanobacteria species and the importance of using multiple loci for molecular characterization. Another important issue is the paucity of fully characterized sequences available in GenBank, which impairs the ability to accurately identify new cyanobacteria isolates.