Diagnostics and molecular epidemiology of the Sarcoptes scabiei mite infesting Australian wildlife

Parasitic infestations have always been a noteworthy topic for human and animal health globally, with many considered a result of spill-over and zoonosis. One such parasite, Sarcoptes scabiei, is known to infest over 300 million humans per year and has been documented in over 104 mammals. It has recently been classified as a neglected tropical disease and is among the top 50 most prevalent human diseases. With uncertainty over (i) the global epidemiology of S. scabiei and (ii) the reliability of current diagnostics methods, much still needs to be understood if stakeholders are to successfully develop strategies to control this parasite. The overall objective of this thesis was to study the molecular epidemiology and genetic typing of S. scabiei infesting Australian animals and assess the diagnostic methods for sarcoptic mange. At the global scale, numerous genetic studies have attempted to reveal how the host species and host geographic location influence S. scabiei phylogenetics. By performing an analysis of the global literature (Chapter 2), I was able to reveal that there were inconsistencies in gene loci and phylogenetic conclusions used in these previous studies. Furthermore, by executing a contemporary analytical approach employing molecular markers on existing S. scabiei sequences, it was apparent that (i) new S. scabiei samples, (ii) appropriate gene loci targets, and (iii) advanced phylogenetic approaches are necessary to more confidently comprehend the origins of mange in Australia. As there were only a limited number of Australian marsupial-derived S. scabiei sequences, and that three of the most commonly used gene loci used for typing are located within the mitochondria, I performed mitochondrial genome sequencing of mites collected from koalas and wombats (Chapter 3). It was revealed that there is a high sequence similarity not just within marsupial S. scabiei mites, but also to the only human-derived S. scabiei mitochondrial genome. Furthermore, by examining individual gene phylogenies, I concluded that cox1 is the most informative gene as the cox1 phylogeny inferred was consistent with the complete mitochondrial genome phylogeny with the highest resolution of ancestral lineages. Building on the identification of cox1 as an informative gene target, I greatly expanded the molecular typing of S. scabiei within Australia (Chapter 4). I identified that mites collected from koalas, wombats, foxes and dogs across five states of Australia were unable to be phylogenetically separated by their host or location. Thus, I considered it highly plausible that multiple spill-over events may have occurred in Australia, as many haplotypes are identical to European and non-European sequences. Furthermore, I suggested that it is likely that canids are the source for transmission of mange throughout Australian wildlife as dogs and foxes share identical haplotypes to wombats and koalas. Finally, I detected a distinguishable human-specific lineage, distinct from the dominant mixed animal clade. Clinical diagnosis of mange/scabies typically involves the collection of skin scrapings followed by microscopic detection of the mite. This method yields results with a high risk of false negatives, however. I performed the first comparative S. scabiei diagnostic study on a unique sample set collected from bare-nosed wombats. Here, I assessed a variety of putatively useful approaches including observational scoring, microscopy, PCR on skin scraping DNA and PCR on skin swab DNA (Chapter 5). I concluded that: (i) observational scoring positively correlated with counts from microscopy, however this approach tended to under-diagnose early mange; (ii) species-specific S. scabiei PCR enhanced the sensitivity of mite detection in relation to microscopy and; (iii) swabs as a method for sample collection is questionable due to inadequate host cell uptake and likelihood of producing false negatives. Finally, I sought to improve the use of molecular techniques for S. scabiei diagnosis (Chapter 6). I developed a novel rapid diagnostic tool using a Loop Mediated Isothermal Amplification assay, which I demonstrated to be specific to S. scabiei and able to produce a rapid diagnostic result within 30 minutes. Since this method can be performed without advanced laboratory equipment, this development has potential direct roles as an ancillary method with microscopy at the point-of-care to reduce the number of potential S. scabiei false-negative results obtained by microscopy alone in both human and veterinary settings. In summary, this thesis has contributed to: (i) the expansion of S. scabiei phylogeny by highlighting the high genetic variability of the single mite species; (ii) suggested multiple spill-over events may be the consequence of inadequate screening of imports/exports possibly globally and; (iii) has demonstrated the incompatible variety of different diagnostic methods for S. scabiei which may be supplemented with the development of a new highly sensitive and specific molecular technique. The contributions I have made in S. scabiei research will aid in future conservation efforts to aid in understanding transmission risks to threatened populations and enhance diagnostic procedures in clinical, field and remote settings.