Metagenomic and Molecular Detection of Novel Fecal Viruses in Free-Ranging Agile Wallabies.

The agile wallaby (Notamacropus agilis) is one of the most abundant marsupial species in northern Queensland and a competent host for the zoonotic Ross River virus. Despite their increased proximity and interactions with humans, little is known about the viruses carried by these animals, and whether any are of conservation or zoonotic importance. Metagenomics and molecular techniques were used in a complementary manner to identify and characterize novel viruses in the fecal samples of free-ranging agile wallabies. We detected a variety of novel marsupial-related viral species including agile wallaby atadenovirus 1, agile wallaby chaphamaparvovirus 1-2, agile wallaby polyomavirus 1-2, agile wallaby associated picobirnavirus 1-9, and a known macropod gammaherpesvirus 3. Phylogenetic analyses indicate that most of these novel viruses would have co-evolved with their hosts (agile wallabies). Additionally, non-marsupial viruses that infect bacteria (phages), plants, insects, and other eukaryotes were identified. This study highlighted the utility of non-invasive sampling as well as the integration of broad-based molecular assays (consensus PCR and next generation sequencing) for monitoring the emergence of potential pathogenic viruses in wildlife species. Furthermore, the novel marsupial viruses identified in this study will enrich the diversity of knowledge about marsupial viruses, and may be useful for developing diagnostics and vaccines.

Correction: Trace element concentrations in forage seagrass species of Chelonia mydas along the Great Barrier Reef.

[This corrects the article DOI: 10.1371/journal.pone.0269806.].

Green Turtle (Chelonia mydas) Blood and Scute Trace Element Concentrations in the Northern Great Barrier Reef.

Marine turtles face numerous anthropogenic threats, including that of chemical contaminant exposure. The ecotoxicological impact of toxic metals is a global issue facing Chelonia mydas in coastal sites. Local investigation of C. mydas short-term blood metal profiles is an emerging field, while little research has been conducted on scute metal loads as potential indicators of long-term exposure. The aim of the present study was to investigate and describe C. mydas blood and scute metal profiles in coastal and offshore populations of the Great Barrier Reef. This was achieved by analyzing blood and scute material sampled from local C. mydas populations in five field sites, for a suite of ecologically relevant metals. By applying principal component analysis and comparing coastal sample data with those of reference intervals derived from the control site, insight was gleaned on local metal profiles of each population. Blood metal concentrations in turtles from coastal sites were typically elevated when compared with levels recorded in the offshore control population (Howick Island Group). Scute metal profiles were similar in Cockle Bay, Upstart Bay, and Edgecumbe Bay, all of which were distinct from that of Toolakea. Some elements were reported at similar concentrations in blood and scutes, but most were higher in scute samples, indicative of temporal accumulation. Coastal C. mydas populations may be at risk of toxic effects from metals such as Co, which was consistently found to be at concentrations magnitudes above region-specific reference intervals. Environ Toxicol Chem 2023;42:2375-2388. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Draft Genome Sequence of a Novel Adenovirus Recovered from the Metagenome of Agile Wallabies.

Here, we report the draft genome sequence of a novel agile wallaby adenovirus that was detected in the fecal metagenome of agile wallabies. The genome is 31,512 bp long, with a G+C content of 34.4%. Currently, the pathogenic and zoonotic potential of this novel virus is unknown.

Development of subfamily-based consensus PCR assays for the detection of human and animal herpesviruses.

Consensus PCR assays that can be used to sensitively detect several herpesvirus (HV) species across the different subfamilies were developed in this study. Primers containing degenerate bases were designed to amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs, and the glycoprotein B (gB) gene of beta-HVs in a singleplex, non-nested touchdown PCR format. The singleplex touchdown consensus PCR (STC-PCR) was used to amplify the DNA of eight human and 24 animal HVs. The assay was able to detect the lowest DNA dilution of 10-5 for alpha-HVs and 10-3 for beta- and gamma-HVs. In comparison, lowest detection limits of 10-5, 10-3, and 10-2 were obtained for alpha-, beta-, and gamma-HVs respectively when a nested PCR was used. The findings in this study suggest that the STC-PCR assays can be employed for the molecular surveys and clinical detection of novel and known HVs.

A Standardized Protocol for Measuring Bioelectrical Impedance in Green Turtles (Chelonia mydas).

AbstractBioelectrical impedance analysis (BIA) is gaining popularity in wildlife studies as a portable technology for immediate and nondestructive predictions of body composition components, such as fat-free and fat masses. Successful application of BIA for field-based research requires the identification and control of potential sources of error, as well as the creation of and adherence to a standardized protocol for measurement. The aim of our study was to determine sources of error and to provide a standardization protocol to improve measurement precision of BIA on juvenile green turtles (Chelonia mydas; n=35). We assessed the effects of altered environmental temperature (20°C-30°C), postprandial state (2-72 h), and time out of the water (2 h) on five impedance parameters (resistance at infinite frequency [Rinf], resistance at zero frequency [R0], resistance at 50 kHz [R50], phase angle at 50 kHz [PhA50], and intracellular resistance [Ri]) using a bioimpedance spectroscopy device. Technical reproducibility of measurements and interanimal variability were also assessed. We found an inverse exponential relationship between change in environmental temperature and impedance parameters Rinf, R0, and R50. Postprandial state significantly increased Rinf and Ri 72 h after feeding. BIA measurements were reproducible within individual juvenile green turtles at temperatures from 20°C to 30°C. Significant variation in impedance values was found between animals at all temperatures, sampling times, and postprandial states, but the relative differences (%) were small in magnitude. Our study suggests that measurement precision is improved by measuring animals at consistent environmental temperatures close to their preferred thermal range. We propose a standardized protocol of measurement conditions to facilitate laboratory and field use of BIA for body composition assessment studies in turtles.

Pathogenesis of Bohle iridovirus infection in Krefft's freshwater turtle hatchlings (Emydura macquarii krefftii).

Ranaviruses have been detected in over 12 families of reptiles including many genera of turtles, tortoises, and terrapins, but the pathogenesis of these infections is still poorly understood. Krefft's river turtle hatchlings (N = 36; Emydura macquarii krefftii) were inoculated intramuscularly with Bohle iridovirus (BIV, Ranavirus, isolate) or saline, and euthanized at 9 timepoints (3 infected and 1 control per timepoint) over a 24-day period. Samples of lung, liver, kidney, and spleen were collected for quantitative polymerase chain reaction (PCR); internal organs, skin, and oral cavity samples were fixed for histopathological examination. The earliest lesions, at 8 days postinoculation (dpi), were lymphocytic inflammation of the skin and fibrinoid necrosis of regional vessels at the site of inoculation, and mild ulcerative necrosis with lymphocytic and heterophilic inflammation in the oral, nasal, and tongue mucosae. Fibrinonecrotic foci with heterophilic inflammation were detected in spleen and gonads at 16 dpi. Multifocal hepatic necrosis, heterophilic inflammation, and occasional basophilic intracytoplasmic inclusion bodies were observed at 20 dpi, along with ulcerative lymphocytic and heterophilic tracheitis and bronchitis. Tracheitis, bronchitis, and rare bone marrow necrosis were present at 24 dpi. Of the viscera tested for ranaviral DNA by PCR, the liver and spleen had the highest viral loads throughout infection, and thus appeared to be major targets of viral replication. Testing of whole blood by qPCR was the most-effective ante-mortem method for detecting ranaviral infection compared with oral swabs. This study represents the first time-dependent pathogenesis study of a ranaviral infection in turtles.

Field-based adipose tissue quantification in sea turtles using bioelectrical impedance spectroscopy validated with CT scans and deep learning.

Loss of adipose tissue in vertebrate wildlife species is indicative of decreased nutritional and health status and is linked to environmental stress and diseases. Body condition indices (BCI) are commonly used in ecological studies to estimate adipose tissue mass across wildlife populations. However, these indices have poor predictive power, which poses the need for quantitative methods for improved population assessments. Here, we calibrate bioelectrical impedance spectroscopy (BIS) as an alternative approach for assessing the nutritional status of vertebrate wildlife in ecological studies. BIS is a portable technology that can estimate body composition from measurements of body impedance and is widely used in humans. BIS is a predictive technique that requires calibration using a reference body composition method. Using sea turtles as model organisms, we propose a calibration protocol using computed tomography (CT) scans, with the prediction equation being: adipose tissue mass (kg) = body mass - (-0.03 [intercept] - 0.29 * length2/resistance at 50 kHz + 1.07 * body mass - 0.11 * time after capture). CT imaging allows for the quantification of body fat. However, processing the images manually is prohibitive due to the extensive time requirement. Using a form of artificial intelligence (AI), we trained a computer model to identify and quantify nonadipose tissue from the CT images, and adipose tissue was determined by the difference in body mass. This process enabled estimating adipose tissue mass from bioelectrical impedance measurements. The predictive performance of the model was built on 2/3 samples and tested against 1/3 samples. Prediction of adipose tissue percentage had greater accuracy when including impedance parameters (mean bias = 0.11%-0.61%) as predictor variables, compared with using body mass alone (mean bias = 6.35%). Our standardized BIS protocol improves on conventional body composition assessment methods (e.g., BCI) by quantifying adipose tissue mass. The protocol can be applied to other species for the validation of BIS and to provide robust information on the nutritional and health status of wildlife, which, in turn, can be used to inform conservation decisions at the management level.

Trace element concentrations in forage seagrass species of Chelonia mydas along the Great Barrier Reef.

Toxic metal exposure is a threat to green sea turtles (Chelonia mydas) inhabiting and foraging in coastal seagrass meadows and are of particular concern in local bays of the Great Barrier Reef (GBR), as numerous sources of metal contaminants are located within the region. Seagrass species tend to bioaccumulate metals at concentrations greater than that detected in the surrounding environment. Little is known regarding ecotoxicological impacts of environmental metal loads on seagrass or Chelonia mydas (C. mydas), and thus this study aimed to investigate and describe seagrass metal loads in three central GBR coastal sites and one offshore site located in the northern GBR. Primary seagrass forage of C. mydas was identified, and samples collected from foraging sites before and after the 2018/2019 wet season, and multivariate differences in metal profiles investigated between sites and sampling events. Most metals investigated were higher at one or more coastal sites, relative to data obtained from the offshore site, and cadmium (Cd), cobalt (Co), iron (Fe) and manganese (Mn) were found to be higher at all coastal sites. Principle Component Analysis (PCA) found that metal profiles in the coastal sites were similar, but all were distinctly different from that of the offshore data. Coastal foraging sites are influenced by land-based contaminants that can enter the coastal zone via river discharge during periods of heavy rainfall, and impact sites closest to sources. Bioavailability of metal elements are determined by complex interactions and processes that are largely unknown, but association between elevated metal loads and turtle disease warrants further investigation to better understand the impact of environmental contaminants on ecologically important seagrass and associated macrograzers.

Haematological and biochemical reference intervals for wild green turtles (Chelonia mydas): a Bayesian approach for small sample sizes.

Animal health is directly linked to population viability, which may be impacted by anthropogenic disturbances and diseases. Reference intervals (RIs) for haematology and blood biochemistry are essential tools for the assessment of animal health. However, establishing and interpreting robust RIs for threatened species is often challenged by small sample sizes. Bayesian predictive modelling is well suited to sample size limitations, accounting for individual variation and interactions between influencing variables. We aimed to derive baseline RIs for green turtles (Chelonia mydas) across two foraging aggregations in North Queensland, Australia, using Bayesian generalized linear mixed-effects models (n = 97). The predicted RIs were contained within previously published values and had narrower credible intervals. Most analytes did not vary significantly with foraging ground (76%, 22/29), body mass (86%, 25/29) or curved carapace length (83%, 24/29). Length and body mass effects were found for eosinophils, heterophil:lymphocyte ratio, alkaline phosphatase, aspartate transaminase and urea. Significant differences between foraging grounds were found for albumin, cholesterol, potassium, total protein, triglycerides, uric acid and calcium:phosphorus ratio. We provide derived RIs for foraging green turtles, which will be helpful in future population health assessments and conservation efforts. Future RI studies on threatened species would benefit from adapting established veterinary and biomedical standards.

Importance of health assessments for conservation in noncaptive wildlife.

Wildlife health assessments help identify populations at risk of starvation, disease, and decline from anthropogenic impacts on natural habitats. We conducted an overview of available health assessment studies in noncaptive vertebrates and devised a framework to strategically integrate health assessments in population monitoring. Using a systematic approach, we performed a thorough assessment of studies examining multiple health parameters of noncaptive vertebrate species from 1982 to 2020 (n = 261 studies). We quantified trends in study design and diagnostic methods across taxa with generalized linear models, bibliometric analyses, and visual representations of study location versus biodiversity hotspots. Only 35% of studies involved international or cross-border collaboration. Countries with both high and threatened biodiversity were greatly underrepresented. Species that were not listed as threatened on the International Union for Conservation of Nature Red List represented 49% of assessed species, a trend likely associated with the regional focus of most studies. We strongly suggest following wildlife health assessment protocols when planning a study and using statistically adequate sample sizes for studies establishing reference ranges. Across all taxa blood analysis (89%), body composition assessments (81%), physical examination (72%), and fecal analyses (24% of studies) were the most common methods. A conceptual framework to improve design and standardize wildlife health assessments includes guidelines on the experimental design, data acquisition and analysis, and species conservation planning and management implications. Integrating a physiological and ecological understanding of species resilience toward threatening processes will enable informed decision making regarding the conservation of threatened species.

Importancia de los exámenes diagnósticos para la conservación de fauna silvestre Resumen Los exámenes diagnósticos de fauna silvestre ayudan a identificar poblaciones en riesgo por desnutrición, enfermedades infecciosas y disminución poblacional, causadas por impactos antropogénicos. Revisamos los estudios disponibles que llevaron a cabo exámenes diagnósticos en fauna silvestre y diseñamos un marco de trabajo para integrar dichos exámenes en monitoreos poblacionales. Empleando un enfoque sistemático, evaluamos aquellos estudios que examinaban múltiples indicadores de salud en vertebrados no cautivos entre 1982 y 2020 (n = 261 estudios). Cuantificamos las tendencias estadísticas, clasificadas por taxones, del diseño del estudio y de los métodos diagnósticos usando modelos lineales generalizados, análisis bibliométricos y representaciones visuales del lugar de estudio versus los hotspots (puntos calientes) de biodiversidad. Sólo el 35% de los estudios incluían colaboraciones internacionales o transfronterizas, y los países ricos en biodiversidad y especies amenazadas estaban gravemente subrepresentados. Las especies no clasificadas como amenazadas en la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza representaban el 49% de las especies examinadas; una tendencia posiblemente asociada al enfoque regional de la mayoría de los estudios. Recomendamos encarecidamente seguir protocolos diagnósticos y manuales de técnicas del estudio de la fauna silvestre, además de usar tamaños muestrales estadísticamente adecuados al establecer rangos de referencia. Los métodos diagnósticos más comunes para todos los taxones fueronanálisis sanguíneos (89%), evaluaciones de composición corporal (81%), exámenes físicos (72%) y análisis fecales (24% de los estudios). Presentamos un marco conceptual para mejorar y estandarizar los exámenes diagnósticos en estudios de fauna silvestre; dicho marco incluye guías para el diseño experimental, para la obtención y el análisis de datos, y para elaborar planes de acción para especies amenazadas. La combinación de conocimientos fisiológicos y ecológicos, relacionados con la resiliencia biológica de especies amenazadas, facilitará una toma de decisiones eficiente para el manejo y para la conservación de la biodiversidad.

Nidoviruses in Reptiles: A Review.

Since their discovery in 2014, reptile nidoviruses (also known as serpentoviruses) have emerged as significant pathogens worldwide. They are known for causing severe and often fatal respiratory disease in various captive snake species, especially pythons. Related viruses have been detected in other reptiles with and without respiratory disease, including captive and wild populations of lizards, and wild populations of freshwater turtles. There are many opportunities to better understand the viral diversity, species susceptibility, and clinical presentation in different species in this relatively new field of research. In captive snake collections, reptile nidoviruses can spread quickly and be associated with high morbidity and mortality, yet the potential disease risk to wild reptile populations remains largely unknown, despite reptile species declining on a global scale. Experimental studies or investigations of disease outbreaks in wild reptile populations are scarce, leaving the available literature limited mostly to exploring findings of naturally infected animals in captivity. Further studies into the pathogenesis of different reptile nidoviruses in a variety of reptile species is required to explore the complexity of disease and routes of transmission. This review focuses on the biology of these viruses, hosts and geographic distribution, clinical signs and pathology, laboratory diagnosis and management of reptile nidovirus infections to better understand nidovirus infections in reptiles.

Testudines as Sentinels for Monitoring the Dissemination of Antibiotic Resistance in Marine Environments: An Integrative Review.

Dissemination of antibiotic resistance (AR) in marine environments is a global concern with a propensity to affect public health and many ecosystems worldwide. We evaluated the use of sea turtles as sentinel species for monitoring AR in marine environments. In this field, antibiotic-resistant bacteria have been commonly identified by using standard culture and sensitivity tests, leading to an overrepresentation of specific, culturable bacterial classes in the available literature. AR was detected against all major antibiotic classes, but the highest cumulative global frequency of resistance in all represented geographical sites was against the beta-lactam class by a two-fold difference compared to all other antibiotics. Wastewater facilities and turtle rehabilitation centres were associated with higher incidences of multidrug-resistant bacteria (MDRB) accounting for an average of 58% and 49% of resistant isolates, respectively. Furthermore, a relatively similar prevalence of MDRB was seen in all studied locations. These data suggest that anthropogenically driven selection pressures for the development of AR in sea turtles and marine environments are relatively similar worldwide. There is a need, however, to establish direct demonstrable associations between AR in sea turtles in their respective marine environments with wastewater facilities and other anthropogenic activities worldwide.

Herpesviruses in Reptiles.

Since the 1970s, several species of herpesviruses have been identified and associated with significant diseases in reptiles. Earlier discoveries placed these viruses into different taxonomic groups on the basis of morphological and biological characteristics, while advancements in molecular methods have led to more recent descriptions of novel reptilian herpesviruses, as well as providing insight into the phylogenetic relationship of these viruses. Herpesvirus infections in reptiles are often characterised by non-pathognomonic signs including stomatitis, encephalitis, conjunctivitis, hepatitis and proliferative lesions. With the exception of fibropapillomatosis in marine turtles, the absence of specific clinical signs has fostered misdiagnosis and underreporting of the actual disease burden in reptilian populations and hampered potential investigations that could lead to the effective control of these diseases. In addition, complex life histories, sampling bias and poor monitoring systems have limited the assessment of the impact of herpesvirus infections in wild populations and captive collections. Here we review the current published knowledge of the taxonomy, pathogenesis, pathology and epidemiology of reptilian herpesviruses.

The Concurrent Detection of Chelonid Alphaherpesvirus 5 and Chelonia mydas Papillomavirus 1 in Tumoured and Non-Tumoured Green Turtles.

Characterised by benign tumours, fibropapillomatosis (FP) is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of histological and molecular evidence has associated FP tumours with Chelonid alphaherpesvirus 5 (ChHV5). However, a recent study which detected both ChHV5 and Chelonia mydas papillomavirus 1 (CmPV1) DNA in FP tumour tissues has challenged this hypothesis. The present study aimed to establish a probe-based qPCR to assess the wider prevalence of CmPV1 and co-occurrence with ChHV5 in 275 marine turtles foraging in waters adjacent to the east coast of Queensland, Australia: three categories: Group A (FP tumours), Group B (non-tumoured skin from FP turtles) and Group C (non-tumoured skin from turtles without FP). Concurrent detection of ChHV5 and CmPV1 DNA is reported for all three categories, where Group A had the highest rate (43.5%). ChHV5 viral loads in Group A were significantly higher than loads seen in Group B and C. This was not the case for CmPV1 where the loads in Group B were highest, followed by Group A. However, the mean CmPV1 load for Group A samples was not significantly different to the mean load reported from Group B or C samples. Collectively, these results pivot the way we think about FP; as an infectious disease where two separate viruses may be at play.

Characterisation and comparison of the mucosa-associated bacterial communities across the gastrointestinal tract of stranded green turtles, Chelonia mydas.

Chelonia mydas are primarily herbivorous long-distance migratory sea turtles that contribute to marine ecosystems. Extensive research has been conducted to restore the populations of green turtles. Little is known about their gut microbiota which plays a vital role in their health. We investigated the mucosa-associated bacterial communities across the gastrointestinal (GI) tract of a total four (3, juvenile and 1, adult) stranded green turtles. Samples taken from four GI regions including oesophagus, stomach, small intestine and large intestine were analysed by high-throughput sequencing targeting hypervariable V1-V3 regions of the bacterial 16S rRNA gene. Bacterial diversity and richness decreased longitudinally along the GI tract from oesophagus to the small intestine of stranded turtles. The large intestine showed a higher bacterial diversity and richness compared to small intestine. The bacterial community of green turtles' GI tract was largely dominated by Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria. Aerobic and facultative anaerobic bacteria prevailed primarily in the oesophagus while anaerobes (Lachnospiraceae, Peptostreptococcaceae and Ruminococcaceae) constituted the bulk of large intestinal microbiota. Firmicutes dominated the GI tract except within the small intestine where Proteobacteria prevailed. At the OTU level, six percent of the total OTUs (>1% relative abundance) were common in all GI regions. This is a comprehensive characterisation of bacterial microbiota across the GI tract in green turtles which will provide a reference for future studies on turtle gut microbiome and their metabolism to improve their health and nutrition during rehabilitation.

Disease risk analysis in sea turtles: A baseline study to inform conservation efforts.

The impact of a range of different threats has resulted in the listing of six out of seven sea turtle species on the IUCN Red List of endangered species. Disease risk analysis (DRA) tools are designed to provide objective, repeatable and documented assessment of the disease risks for a population and measures to reduce these risks through management options. To the best of our knowledge, DRAs have not previously been published for sea turtles, although disease is reported to contribute to sea turtle population decline. Here, a comprehensive list of health hazards is provided for all seven species of sea turtles. The possible risk these hazards pose to the health of sea turtles were assessed and "One Health" aspects of interacting with sea turtles were also investigated. The risk assessment was undertaken in collaboration with more than 30 experts in the field including veterinarians, microbiologists, social scientists, epidemiologists and stakeholders, in the form of two international workshops and one local workshop. The general finding of the DRA was the distinct lack of knowledge regarding a link between the presence of pathogens and diseases manifestation in sea turtles. A higher rate of disease in immunocompromised individuals was repeatedly reported and a possible link between immunosuppression and environmental contaminants as a result of anthropogenic influences was suggested. Society based conservation initiatives and as a result the cultural and social aspect of interacting with sea turtles appeared to need more attention and research. A risk management workshop was carried out to acquire the insights of local policy makers about management options for the risks relevant to Queensland and the options were evaluated considering their feasibility and effectiveness. The sea turtle DRA presented here, is a structured guide for future risk assessments to be used in specific scenarios such as translocation and head-starting programs.

Cutaneous Lesions in Freshwater Turtles (Emydura macquarii krefftii and Myuchelys latisternum) in a Rainforest Creek in North Queensland, Australia.

Freshwater turtles inhabit most rivers and creeks on the east coast of Australia, but some species are only found in specific catchments, which makes them vulnerable to extinction. During annual fieldtrips to Alligator Creek, North Queensland, the resident population of Myuchelys latisternum and Emydura macquarii krefftii in a natural pond, just outside Bowling Green National Park, have been surveyed for a number of years and demographic data recorded against tagged turtles. Rounded, cutaneous lesions on individual animals were first noted in August 2016, three years after the first survey of the population. Turtles living in the upstream sections of the creek were not affected. An initial investigation into the cause of the lesions ruled out pollutants and although the bacterial communities appeared to be different on turtles with lesions, a causative agent was not identified. Attempts to isolate virus in culture was not successful and specific PCRs for ranavirus, papillomavirus, adenovirus and herpesvirus did not identify their presence. Blood biochemical parameters, body condition and activity levels were not significantly different between affected turtles and those without lesions. The turtles in this pond were monitored regularly over the following three years with 249 M. latisternum and 192 E. m. krefftii captured, tagged and released. The prevalence of the lesions fluctuated with season from 0 to 77 and 68% respectively, but did not vary significantly between species or sex in adults. There was a tendency for larger animals to be more likely to have lesions. The position of the lesions on the turtles was mostly on dorsal surfaces, distally on the legs and proximal on the tales of males, indicating that the initial lesion may have been associated with a behaviourally induced trauma. Recaptured animals (n = 43) during this period, provided records of lesion progression over time and while some healed up between capture events, others persisted for up to 24 months. Some turtles were repeatedly captured without lesions. Intra-species aggression associated with seasonal behaviours could potentially be the primary cause of skin trauma, followed by a secondary invasion of an unusual pathogen present in the environment.

COCCIDIOSIS IN GREEN TURTLES (CHELONIA MYDAS) IN AUSTRALIA: PATHOGENESIS, SPATIAL AND TEMPORAL DISTRIBUTION, AND CLIMATE-RELATED DETERMINANTS OF DISEASE OUTBREAKS.

An epizootic of coccidiosis in free-ranging green turtles (Chelonia mydas) occurred in Australia in 1991 and the parasites were thought to be Caryospora cheloniae. Recurring outbreaks over an increased geographic range followed. We used medical records and temporal and spatial data of turtles diagnosed with coccidiosis between 1991 and 2014 to characterize the disease and factors associated with outbreaks. Most affected animals were subadults or older. Neurologic signs with intralesional cerebral coccidia were observed. Coccidia associated with inflammation and necrosis were predominantly found in the intestine, brain, kidney, and thyroid. Cases occurred in the spring and summer. Three major outbreaks (1991, 2002, and 2014) were concentrated in Port Stephens, New South Wales (NSW) and Moreton Bay, Queensland, but cases occurred as far south as Sydney, NSW. Coccidiosis cases were more likely during, or 1 mo prior to, El Niño-like events. Molecular characterization of the 18S rRNA locus of coccidia from tissues of 10 green turtles collected in 2002 and 2004 in Port Stevens and Sydney imply that they were Schellackia-like organisms. Two genotypes were identified. The Genotype 3 sequence was most common (in eight of 10 turtles), with 98.8% similarity to the 18S sequence of Schellackia orientalis. The Genotype 4 sequence was less common (in two of 10 turtles) with 99.7% similarity to the 18S sequence of the most common genotype (Genotype 1) detected in turtles from the 2014 Moreton Bay outbreak. Our study will help with the identification and management of future outbreaks and provide tools for identification of additional disease patterns in green turtles.

Dose-dependent morbidity of freshwater turtle hatchlings, Emydura macquarii krefftii, inoculated with Ranavirus isolate (Bohle iridovirus, Iridoviridae).

Ranaviral infections cause mass die-offs in wild and captive turtle populations. Two experimental studies were performed to first determine the susceptibility of an Australian turtle species (Emydura macquarii krefftii) to different routes of infection and second examine the effect of viral titre on the morbidity in hatchlings. All inoculation routes (intracoelomic, intramuscular and oral) produced disease, but the clinical signs, histopathology and time to onset of disease varied with the route. The median infectious and lethal doses for intramuscularly inoculated hatchlings were 102.52 (1.98-2.93) and 104.43 (3.81-5.19) TCID50 ml-1, respectively. Clinical signs began 14 to 29 days post-inoculation and the median survival time was 22 days (16-25) across all dose groups. For every 10-fold increase in dose, the odds of developing any clinical signs or severe clinical signs increased by 3.39 [P<0.01, 95 % confidence interval (CI): 1.81-6.36] and 3.71 (P<0.01, 95 % CI: 1.76-7.80), respectively. Skin lesions, previously only reported in ranaviral infection in lizards, were observed in the majority of intramuscularly inoculated hatchlings that developed ranaviral disease. The histological changes were consistent with those in previous reports for reptiles and consisted of necrosis at or near the site of injection, in the spleen, liver and oral cavity. Systemic inflammation was also observed, predominantly affecting necrotic organs. The estimates reported here can be used to model ranaviral disease and quantify and manage at-risk populations.