Diversity, impacts and diagnosis of pathogenic parasites in sea turtles from Queensland, Australia

Parasitism is a significant cause of stranding and mortality in endangered sea turtles. Twoparasites in particular (or groups thereof) have been noted for their impacts on green seaturtle populations. The first are the spirorchiid blood flukes, which inhabit the circulatorysystems of their host. The second is the eimeriid coccidian Caryospora cheloniae, asystemic parasite affecting a number of organs. Diversity among these organisms is poorlyexplored, and is almost certainly greater than currently recognised. The parasites and theirassociated diseases fluctuate both temporally and spatially in terms of prevalence andseverity, however, the factors driving these fluctuations are poorly understood. A variety ofquestions remain unanswered in terms of the epidemiology and relative pathogenicity ofthe different parasite species. Of particular interest are brain lesions, which have beenassociated with both parasites and in some cases lead to neurological deficiency andsubsequent mortality. Difficulties in finding answers to these questions is compounded bya lack of reliable, fast and quantitative ante- and post-mortem diagnostic tools for eitherparasite, which restricts their investigation.In order to catalogue local species and assess diversity, adult spirorchiid flukes werecollected during post-mortem examination of deceased sea turtles and characterised bymolecular and morphological means. Eleven distinct species or genotypes were identified,comprising a mix of previously described and novel species or variants. Samples werealso collected from green sea turtles that died during a coccidiosis related mass mortalityevent in south east Queensland and northern New South Wales. While only one species ofcoccidian (C. cheloniae) had been previously described, molecular characterisation of theorganism implicated in the outbreak revealed two distinct coccidian genotypes, which hassignificant implications for diagnosis and management.Spirorchiid ova have been associated with granulomatous lesions in a wide range of hosttissues. However, it has not been possible to identify spirorchiids beyond the genus levelbased on morphology, which has frustrated any attempts to identify the species or variantsresponsible for severe lesions. In this thesis, a molecular approach was used and aterminal restriction fragment length polymorphism (T-RFLP) assay was developed todetect and identify individual species within the often mixed assemblages of ova in turtletissues. This assay proved to be a more specific and sensitive alternative to traditional microscopic detection methods. Through correlation with histopathology and grosspathology, the tissue tropisms, relative occurrence and pathogenicity of each species wereinvestigated. The most common species (Neospirorchis Genotype 2) was found in 96% ofsamples, encompassing tissues from all organs sampled. On average, a greater numberof spirorchiid species were detected in tissues where lesions were present, and numbersincreased along with the severity of lesions. The age, sex or body condition of the host didnot show an effect, however, age was found to be a significant factor in the diversity ofspirorchiid infections in some other organs.Distinct tissue tropisms were evident for the two coccidian genotypes. The first and mostcommon genotype was found in gastrointestinal, brain and lung tissues, with associatedencephalitis and enteritis. The second was detected in kidney and thyroid tissue, againwith an accompanying inflammatory response. The two species are therefore likely to havedifferent impacts on their host, and this must be considered in epidemiologicalinvestigations and development of diagnostic tools.In order to explore options for future ante- and post-mortem diagnostics, TaqMan qPCRassays were developed to detect and quantify both Neospirorchis spp. and Caryosporaspp. infections. These assays were able to be run as a single tube multiplex reaction, andreliably detected both parasites in tissue samples. This provided an efficient and costeffective means of differentiating between two parasitic infections that may present withsimilar neurological signs. Given that the data collected using the T-RFLP assay indicatedthat spirorchiids (most notably the Neospirorchis) are almost universally present instranded turtles, the relative quantitative data provided by these assays will be central inascertaining the factors that induce severe inflammatory responses to infections.This study has uncovered diversity among important turtle parasites, investigated theepidemiology and pathology associated with infections, and has contributed two importantdiagnostic tests to the investigation and management of these significant causes ofdisease and mortality in green sea turtles. Moving in to the future, these tests will makeimportant contributions to the investigation of disease outbreaks, understanding of diseaseepidemiology and pathology, and the relationship between disease and environmentalfactors – objectives that have been beyond the capability of previously existing methodsand tools.