Deep-sequencing to resolve complex diversity of apicomplexan parasites in platypuses and echidnas: Proof of principle for wildlife disease investigation.

The short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus) are iconic egg-laying monotremes (Mammalia: Monotremata) from Australasia. The aim of this study was to demonstrate the utility of diversity profiles in disease investigations of monotremes. Using small subunit (18S) rDNA amplicon deep-sequencing we demonstrated the presence of apicomplexan parasites and confirmed by direct and cloned amplicon gene sequencing Theileria ornithorhynchi, Theileria tachyglossi, Eimeria echidnae and Cryptosporidium fayeri. Using a combination of samples from healthy and diseased animals, we show a close evolutionary relationship between species of coccidia (Eimeria) and piroplasms (Theileria) from the echidna and platypus. The presence of E. echidnae was demonstrated in faeces and tissues affected by disseminated coccidiosis. Moreover, the presence of E. echidnae DNA in the blood of echidnas was associated with atoxoplasma-like stages in white blood cells, suggesting Hepatozoon tachyglossi blood stages are disseminated E. echidnae stages. These next-generation DNA sequencing technologies are suited to material and organisms that have not been previously characterised and for which the material is scarce. The deep sequencing approach supports traditional diagnostic methods, including microscopy, clinical pathology and histopathology, to better define the status quo. This approach is particularly suitable for wildlife disease investigation.