Characterisation of ranaviral infection and its management in Australian lizards

Background Reptiles are considered to be one of the most evolutionary and ecologically remarkable groups of living organisms, having successfully inhabited most of the planet including the oceans. Despite this, reptile species worldwide are on the decline due to threats such as residential and commercial development, agriculture and aquaculture, climate change, and introduction of invasive species and diseases. Approximately 19% of all assessed reptiles globally are listed as 'threatened' by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Infectious diseases are listed as one of the top five causes of global species extinctions and one of the biggest causes of morbidity and mortality in reptiles. Ranaviruses (family Iridoviridae) have been identified as emerging pathogens of ecological significance in ectothermic vertebrates due to their expanding host and geographic range. This group of viruses infects over 175 species of ectothermic vertebrates worldwide and is listed as notifiable to the The World Organization for Animal Health (Office International des Epizootics, OIE) in amphibians and fish. The majority of ranaviral research has been conducted in amphibians with only a few surveys targeting wild reptiles despite several reported mortality events in captive lizards and turtles. Hence the aims of this thesis were to investigate the susceptibility and pathogenesis of Ranavirus sp. in juvenile eastern water dragons (Intellagama lesueurii lesueurii); determine if Ranavirus sp. is present in Australian lizards; and to identify and understand Australian reptile owners experience and management of disease in captive reptile collections. The susceptibility of an Australian semi-aquatic lizard to Bohle iridovirus In Chapter 2 we investigated the susceptibility of juvenile eastern water dragons to a local ranavirus isolate (Bohle iridovirus, BIV) via oral inoculation, intramuscular injection, and cohabitation with orally infected lizards. This lizard species was investigated as they share habitat with several fish, amphibians and reptiles shown to be susceptible to BIV. A range of tissues (spleen, kidney, lung, liver, kidney, gastrointestinal tract, heart, tongue, brain, and bone marrow) were collected for histopathology, and liver and kidney samples were also collected for viral isolation and polymerase chain reaction (PCR). The outcome of this study demonstrated that juvenile eastern water dragons are susceptible to BIV via all exposure methods and have the ability to infect naïve individuals. These findings add another ectotherm to the list of species susceptible to ranavirus. The pathogenesis of Bohle iridovirus infection in juvenile eastern water dragons In order to investigate the pathogenesis of BIV in this host, juvenile eastern water dragons were orally infected with BIV and euthanized at pre-determined time-points (Chapter 3). Tissue samples were collected for histopathology, immunohistochemistry (ISH), in-situ hybridization (ISH), viral isolation and PCR. The findings from this study identified the progression of BIV infection which appeared to start in the spleen, followed by the liver, then the other organs. Ranaviral DNA was detected by PCR in liver, kidney and cloacal swabs at 3 days post infection, suggesting cloacal swabs could be a reliable source of diagnostic sampling in BIV-infected lizards. Histopathology changes were observed in the liver and tongue at 3 days post infection and IHC identified viral antigen in the spleen at 6 days post infection. The ISH labelling of skin, bone marrow, liver, pancreas, stomach, intestine and spleen matched the location and pattern detected by IHC. Infection was well underway before clinical signs were observed. Molecular detection of Ranavirus sp. in captive and wild Australian lizards Wild and captive Australian lizards from northern Queensland, New South Wales and Australian Capital Territory were surveyed for ranaviral DNA using combined oral-cloacal swabs and PCR (Chapter 4). Ranaviral DNA was detected in samples from 4/123 asymptomatic captive lizards and 5/63 asymptomatic wild lizards. These PCR-positive samples belonged to three central bearded dragons (Pogona vitticeps) and one frilled neck lizard (Chlamydosaurus kingii) from two different captive collections, and five wild eastern water dragons from Paluma Range National Park, Queensland. Amplicons from this study shared 100% nucleotide identity with the cognate regions of BIV and four other ranaviruses and were only one base different to the cognate region of epizootic haematopoietic necrosis virus, an Australian ranavirus that affects fish and is listed as notifiable to the OIE. The detection of ranavirus in asymptomatic lizards in both captivity and in the wild introduces the possibility of carrier lizards and highlights importance of disease management strategies (e.g. quarantine). The health and wellbeing of Australian pet reptiles An online survey (SurveyMonkey®) of Australian reptile owners was conducted between November and December 2017 (Chapter 5). This cross-sectional study consisted of multiple choice and open-ended questions. Quantitative data were analysed descriptively using frequencies, mean, median, standard deviation, range, and interquartile range. Open-ended question responses were analysed thematically and grouped into themes. The average age of participants was 34 years old with snakes and lizards the most popular reptile kept in captivity. Most participants cleaned enclosures weekly, disinfected enclosures monthly, and used UVA/UVB lights, heat lamps and multivitamin supplements to prevent health problems within their collection. Quarantine periods were employed by 72% of participants for an average of 4 weeks, with only 30% physically isolating the animal. Disease knowledge was limited to non-infectious diseases such as metabolic bone disease. Barriers to seeking veterinary assistance for unwell reptiles included cost and perceived lack of knowledge/experience on the veterinarians' part. Findings from this survey identified the need for more readily available resources for Australian reptile keepers including access to information on diseases and experienced veterinarians. Outcomes This research has identified eastern water dragons as a susceptible species to ranaviral infection and provides further evidence of the ability of ranaviruses to infect a wide range of ectothermic vertebrates. The detection of ranavirus in asymptomatic wild and captive lizards suggests the possibility that ranavirus is circulating in the wild and is part of the normal microflora of Australian lizards. This also identifies lizards as a potential host that can spread and amplify ranaviruses in the wild. Further investigation is required to characterize the ranavirus found in this study, and molecular and serum surveys of wild and captive populations. Furthermore, the detection of ranavirus in captive lizards combined with the results from the survey of Australian reptile owners highlights the need for more readily available resources on disease identification, prevention, and treatment