Sea turtles disease risk analysis and discovery of the first Australian green turtle (Chelonia mydas) papillomavirus

Sea turtle populations are in decline, which has been attributed to threats such as predation, bycatch, unsustainable harvesting, habitat degradation, and pollution. The impact of these threats has resulted in the listing of six out of seven sea turtles in the IUCN red list of endangered animals. Infectious diseases are listed among the top five reasons for global species extinctions, but these have not been thoroughly evaluated in sea turtles. Suggested sea turtle conservation plans, based on risk and threat matrices, reported diseases of infectious aetiology with the least priority. Pollution and pathogens have been surveyed in less than half of regional management units (RMUs) and as such these threats have been identified as areas of data deficiency, warranting future investigations. In this thesis, I have addressed the issue of increasing incidence of disease in sea turtles by carrying out a disease risk analysis (DRA) based on the published literature and with the contributions of a group of experts representing more than 20 regions around the globe. In Chapter three of the thesis a comprehensive list of health hazards is provided for all seven species of sea turtles. The risk these hazards possibly pose to the health of sea turtles were assessed and "One Health" aspects of interacting with sea turtles were also investigated. The general result of the DRA was the distinct lack of knowledge regarding a link between the presence of pathogens (either bacteria, fungi, parasites or viruses) and diseases of sea turtles. Among all pathogens, viruses were studied the least, although the debilitating disease of sea turtles, fibropapillomatosis (FP) is suggested to have a viral aetiology: Chelonid alphaherpesvirus 5 (ChHV5). The results of the DRA inspired efforts to improve methods for viral discovery in sea turtles. Chapter four of this thesis is focused on methods establishment. Three main virological methods: cell culture, molecular methods and histopathologial analysis, were used for this project. While the first two were developed and validated as part of this thesis, pathological analyses were done by a qualified veterinary pathologist. The aim was to increase the chance of finding new viruses using a combination of culture and non-culture methods. Sea turtle primary cell lines were established from different stages of green turtle embryos to provide a broad range of host tissues for viral isolation and propagation. PCR assays were designed to detect four DNA viruses previously described in chelonians: herpes, papilloma, adeno and iridoviruses. The methods developed were instrumental in identifying a papillomavirus associated with FP tumours in green turtles and led to the discovery of the first Australian Chelonia mydas papillomavirus (CmPV). First, cytopathic effects were observed in primary cell lines inoculated with homogenates from eight FP tumours and subsequently PCR assays of the affected cultures and the original tissues confirmed presence of CmPV. The Australian CmPV isolates were partially characterised and were examined further in Chapter five. Primer walking and Sanger sequencing revealed some features of the Australian CmPV isolates comparable to green turtle and loggerhead (Caretta caretta) turtle papillomaviruses from Florida, CmPV-1 and CcPV-1 respectively. The E1 gene partial sequencing and the L1 gene full sequencing suggested that Australian CmPV isolates are 100% similar to CmPV-1 and are whithin the same species as CcPV-1. Chapter six is focused on investigating the relationship between CmPV and FP, as the eight CmPVpositive samples were found only in FP tumours of green turtles but not in normal skin, blood and cloacal samples of the same animals. To investigate the possibility of concurrent infection with ChHV5 and CmPV in sea turtles with FP, the samples were screened for both viruses. DNA from 131 tumour tissues and 36 normal skin samples from 89 green turtles afflicted with FP tumours and also 47 normal skin samples from asymptomatic green turtles were extracted and used for the survey. These samples were collected from different regions of Queensland, Australia. Out of 89 FP green turtle samples, 77.52% tested positive for ChHV5, 51.68% for CmPV, 46.06% for both viruses and samples from 15.73% turtles tested negative for both. From 36 normal tissues tested for presence of ChHV5 and CmPV, 50% samples reacted in PCR for ChHV5, 27.77% for CmPV, 8.33% for both viruses and 30.55% samples did not react for either of these viruses. Six samples were collected from loggerhead turtles with FP and assessed for the presence of CcPV, CmPV and ChHV5. The samples were positive for CcPV and ChHV5 and negative for CmPV. The green turtle samples that were positive for CmPV, were negative for CcPV. Papillomaviruses of loggerhead and green turtles appear to be species specific, however further analyses on these samples and possibly more loggerhead samples are required to make sound conclusions about these findings. The high level of co-infection of ChHV5 and CmPV in both tumour tissues and normal skins of sea turtles reported here, challenges the general opinion in the scientific literature for the past 30 years about the role of ChHV5 in FP