Techniques to assess reproductive status in adult female American alligators (Alligator mississippiensis) using laparoscopic examination.

This protocol describes a minimally invasive surgical technique and approach to successfully examine the gonads of live female American alligators as part of a reproductive examination used in conservation medicine and biology. Best practices are based on examination of over 80 American alligators in the last two years adapting principles derived from other reptilian megafauna species. This protocol is designed for appropriately qualified veterinarians and biologists working in the field. We show likely reproductive tract presentations with respect to breeding status and environmental cues to help guide interpretation of observations. The laparoscopic approach and findings presented here provide tools to safely clinically examine animals in a welfare-oriented way that will advance our understanding of crocodilian reproduction. This technique has not previously been described in this species.

Use of soybean as an alternative protein source for welfare-orientated production of American alligators (Alligator mississippiensis).

Soybean meal based diets have been suggested to cause gastrointestinal issues in certain species when used as a protein alternative. Using a randomized design, we tested 1,728 alligators in one of 13 communal pens offered one of two diets (seven pens (n = 928) of soybean-based protein diets and six pens (n = 800) of animal-based protein diets) to determine if soybeans would negatively affect the growth, hide quality, behavior and health, when compared with an otherwise identical diet using animal-based protein. Both feeds were nearly identical in composition for protein and fat percentages and identical for all minerals and elements. Crude protein was a minimum of 50%, crude fat a minimum of 12%, crude fiber a minimum of 4%, and phosphorous was maintained at 1%. From this information we estimated the One Welfare of using soy as a protein in commercial diets. Although there was a statistically significant decrease in belly width (p = 0.0009; harvested hide size) for alligators fed soybean-based protein diets, all other measured parameters of soybean vs. animal-based protein diets were comparable, suggesting this environmentally sustainable alternative protein source warrants consideration as a feed base. Weight was not significantly different suggesting either diet would yield similar volumes of meat. Total length was significantly affected by diet. Hide quality was not negatively impacted by protein type, with both diets producing high quality hides free of defects (assessed at the salted hide stage prior to tanning). Behaviors were not influenced by the feed type, with animals fed either diet using the pen structures the same. Further, feeding times were the same suggesting the soybean-based protein diet was equally easy to eat and palatable as the animal-based protein diet. Behavior and feeding suggested soy-based diets do not alter time budgets or activities. There were no differences in the frequency or severity of pathologies for animals fed either diet. Respiratory (lung and trachea as a proxy to measure dust inhalation), gastrointestinal (small intestine as a proxy to measure digestive disturbances), and renal (kidney as a proxy for excretory stress) histopathology demonstrated neither diet was causing overt problems. One Welfare conclusions were feeding a soybean-based protein diet did not cause production or welfare issues. Further, soybean protein-based diets may be an environmentally sustainable alternative to currently used animal-based diets. Research examining different soybean protein concentrations and sources is warranted.

Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020-2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly, becoming a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter the virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g., shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g., pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n = 76 individuals), free-ranging mink (n = 57), multiple species of bats (n = 59), and other wildlife in addition to domestic cats (n = 275) and pigs (n = 184). In total, we tested 792 individual animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing the re-emergence of SARS-CoV-2 in humans or animals.

Refinement of water-based foam depopulation procedures for finisher pigs during field conditions: Welfare implications and logistical aspects.

Water-based foam (WBF) depopulation is currently being researched as an alternative for rapid destruction of swine populations under emergency circumstances. Appropriate guidelines are needed to maintain method reliability and depopulation efficacy while minimizing animal distress under field conditions. Finisher pigs were depopulated using WBF with a 7.5-minute dwell time in two trials to evaluate the effect of; trial 1) foam fill level (1.5, 1.75, or 2.0 times the pig's head height) and trial 2) foam fill rate (slow, medium, or fast) on aversive pig responses (surface breaks, vocalization, and escape attempts) and time to cessation of cardiac activity. Activity and cardiac activity were recorded using subcutaneous bio-loggers for swine in trial 2. The average time to cessation of movement (COM) from the start of foam filling was compared for the foam fill rate groups using a generalized linear mixed effect model under Poisson distribution. Foam rate group was used as an independent variable, and replicates as a random effect. For trial 1, the average (mm:ss ± SD) time to fill completion was 01:18 ± 00:00, 00:47 ± 00:05, and 00:54 ± 00:05, for 1.5, 1.75, and 2.0 times the pig's head height, respectively. For trial 2, the average time to fill completion was 03:57 ± 00:32, 01:14 ± 00:23 and 00:44 ± 00:03, and the average time (mm:ss ± SE) to COM was 05:22 ± 00:21, 03:32 ± 00:14, and 03:11 ± 00:13 for slow, medium, and fast fill rate groups, respectively. A higher number of aversive pig responses were observed for the lowest foam fill level and slowest foam fill rate compared to increased fill levels and faster fill rates. For trial 2 the median (mm:ss ± IQR) time to fatal arrhythmia was 09:53 ± 02:48, 11:19 ± 04:04, and 10:57 ± 00:47 post-foam initiation for fast, medium, and slow foam rate groups, respectively. Time to cessation of cardiac activity was significantly shorter for the fast foam rate group compared to medium and slow foam rates groups (P = 0.04). For both trials, vocalizations were absent, and all pigs were unconscious following the 7.5-minute dwell time and no pigs needed a secondary euthanasia method. This WBF study showed that slower fill rates and low foam fill levels may extend the time until cessation of cardiac activity in swine during depopulation. A conservative recommendation with consideration of swine welfare during an emergency scenario would be a minimum foam fill level twice the pig's head height and a foam fill rate capable of covering pigs in foam within 60 s to minimize aversive responses and expedite cessation of cardiac activity.

Using ecosystem health and welfare assessments to determine impacts of wild collection for public aquariums.

Aquatic ecosystems are currently facing a multitude of stressors from anthropogenic impacts, including climate change, pollution, and overfishing. Public aquariums positively contribute to ecosystems through conservation, education, and scientific advancement; but may also negatively detract from these systems through collection of animals from the wild and sourcing from commercial suppliers. Changes within the industry have occurred, although evidence-based assessments of 1) how aquariums collect and maintain their populations to determine sustainability of the environment they have harvested; and 2) the welfare of these harvested animals once within the aquariums are still needed. The objectives of this study were to assess the ecosystem health of locations aquariums frequently visit to collect fish from the wild, and then evaluate the wellbeing of fishes at aquariums after extended periods in captivity. Assessments included use of chemical, physical, and biological indicators at field sites, and use of a quantitative welfare assessment at aquariums for comparison to species reared through aquaculture. Anthropogenic pressures at field sites were observed, but no evidence of high degradation or compromised health of animals were found. Welfare assessments of aquarium exhibit tanks produced high-positive scores overall (> 70/84), demonstrating that both wild collected (avg. score 78.8) and aquaculture fishes (avg. score 74.5) were coping appropriately within their environments. Although findings indicated that fish can be taken from the wild at low-moderate rates without any deleterious impact on the environment and cope equally well in aquarium settings, alternatives such as aquaculture should be considered as a strategy to reduce pressure on known stressed aquatic environments or where significant numbers of fishes are being taken.

PLASMA BIOCHEMISTRY PROFILES OF JUVENILE GREEN TURTLES (CHELONIA MYDAS) FROM THE BAHAMAS WITH A POTENTIAL INFLUENCE OF DIET.

Plasma biochemistry profiles aid health assessment of marine turtles, but knowledge of the influence of regional biological factors (e.g., habitat, diet) on marine turtle blood plasma values is limited. To investigate the influence of diet on plasma biochemistry values in juvenile green turtles (Chelonia mydas), we used carbon and nitrogen stable isotopes to provide a quantitative estimate of forage items in green turtles feeding at two distinct areas (Bonefish Hole and South Flats) in Bimini, Bahamas. Plasma samples were obtained from 13 turtles in Bonefish Hole (a mangrove tidal estuary) and 15 turtles in South Flats (an open water seagrass bed) in 2018. All turtles appeared outwardly healthy. Sessile filter feeders contributed the largest proportion of diet in Bonefish Hole, and seagrass contributed the highest proportion of diet in South Flats. Turtles at Bonefish Hole presented significantly lower cholesterol, total protein, phosphorus, triglycerides, and aspartate transaminase compared to South Flats. Across all turtles, those feeding primarily on red algae presented the highest uric acid and alkaline phosphatase, and turtles with a seagrass-dominated diet had the highest cholesterol. Understanding dietary influence on plasma biochemistry may help explain variances seen in local health and nutritional evaluations, and the trends reported can aid the interpretation of plasma analyte values in marine turtles.

Pharmacokinetics and pharmacodynamics of alfaxalone after a single intramuscular or intravascular injection in mallard ducks (Anas platyrhynchos).

Pharmacokinetics and pharmacodynamics of alfaxalone was performed in mallard ducks (Anas platyrhynchos) after single bolus injections of 10 mg/kg administered intramuscularly (IM; n = 10) or intravenously (IV; n = 10), in a randomized cross-over design with a washout period between doses. Mean (±SD) Cmax following IM injection was 1.6 (±0.8) µg/ml with Tmax at 15.0 (±10.5) min. Area under the curve (AUC) was 84.66 and 104.58 min*mg/ml following IV and IM administration, respectively. Volume of distribution (VD ) after IV dose was 3.0 L/kg. The mean plasma clearance after 10 mg/kg IV was 139.5 (±67.9) ml min-1  kg-1 . Elimination half-lives (mean [±SD]) were 15.0 and 16.1 (±3.0) min following IV and IM administration, respectively. Mean bioavailability at 10 mg/kg IM was 108.6%. None of the ducks achieved a sufficient anesthetic depth for invasive procedures, such as surgery, to be performed. Heart and respiratory rates measured after administration remained stable, but many ducks were hyperexcitable during recovery. Based on sedation levels and duration, alfaxalone administered at dosages of 10 mg/kg IV or IM in mallard ducks does not induce clinically acceptable anesthesia.

Spirorchiidiasis in marine turtles: the current state of knowledge.

Blood flukes of the family Spirorchiidae are important disease agents in marine turtles. The family is near cosmopolitan in distribution. Twenty-nine marine species across 10 genera are currently recognized, but taxonomic problems remain and it is likely that more species will be discovered. Spirorchiids infect the circulatory system, where they and their eggs cause a range of inflammatory lesions. Infection is sometimes implicated in the death of the turtle. In some regions, prevalence in stranded turtles is close to 100%. Knowledge of life cycles, important for control and epidemiological studies, has proven elusive until recently, when the first intermediate host identifications were made. Recent molecular studies of eggs and adult worms indicate that a considerable level of intrageneric and intraspecific diversity exists. The characterization of this diversity is likely to be of importance in exploring parasite taxonomy and ecology, unravelling life cycles, identifying the differential pathogenicity of genotypes and species, and developing antemortem diagnostic tools, all of which are major priorities for future spirorchiid research. Diagnosis to date has been reliant on copromicroscopy or necropsy, which both have significant limitations. The current lack of reliable antemortem diagnostic options is a roadblock to determining the true prevalence and epidemiology of spirorchiidiasis and the development of effective treatment regimes.

Monitoring the health of green turtles in northern Queensland post catastrophic events.

Between 2014 and 2017, the Rivers to Reef to Turtles (RRT) project examined the health of green turtles at two coastal sites impacted by urban and agricultural human activities (Cleveland and Upstart Bays) and one proposed pristine site (Howick Group of Reefs) in northern Queensland, Australia, through blood biochemistry and haematology, plasma protein electrophoresis, and clinical assessments including body condition and barnacle counts. Furthermore, cases of mortality were subjected to comprehensive postmortem examination. In an attempt to advance diagnostics, associations between specific contaminants and health of turtles in this region were tested. No comprehensive health assessments had been conducted at these sites prior to this study. The coastal Cleveland and Upstart Bays both demonstrated effects likely to be in response to stressors suspected to be anthropogenic in origin (elevated total white cell counts and creatinine kinase levels across the populations, respectively). This was associated with a suite of trace elements, in particular cobalt. While these indicators of stress resolved by the final year of the study, a chronic stressor was suspected to be persisting with ongoing low albumin: globulin. Necropsies did not elucidate any specific diseases. Although body condition index did not closely correlate with site health, barnacle counts in juvenile turtles may prove a reliable indicator of site health. Based on previously established indicators of poor health, barnacle counts showed that 10% of the population was in poor health at Upstart Bay and nearly 20% of the population at Cleveland Bay. This is above what would be expected for a normal population. Overall, the health component of this study suggested that the pristine turtle population was healthy and the coastal turtle populations were under active stressors, possibly caused by anthropogenic effectors such as chemical pollutants, when initially examined in 2014. These stressors resolved by the conclusion of the study in 2017; but chronic stressors remained absent in the pristine site and present within each of the studied coastal populations.

Evaluating internal exposure of sea turtles as model species for identifying regional chemical threats in nearshore habitats of the Great Barrier Reef.

Marine megafauna that forage in proximity to land can be exposed to a diverse mixture of chemicals that - individually or combined - have the potential to affect their health. Characterizing such complex exposure and examining associations with health still poses considerable challenges. The present study summarizes the development and application of novel approaches to identifying chemical hazards and their potential impacts on the health of coastal wildlife, using green sea turtles as model species. We used an epidemiological study approach to collect blood and keratinized scute samples from free-ranging turtles foraging in nearshore areas and an offshore control site. These were analyzed using a combination of non-targeted, effect-based and multi-chemical analytical screening approaches to assess internal exposure to a wide range of chemicals. The screening phase identified a suite of elements (essential and non-essential) as priority for further investigation. Many of these elements are not commonly analyzed in marine wildlife, illustrating that comprehensive screening is important where exposure is unknown or uncertain. In particular, cobalt was present at highly elevated concentrations, in the order of those known to elicit acute effects across other vertebrate species. Several trace elements, including cobalt, were correlated with clinical indicators of impaired turtle health. In addition, biomarkers of oxidative stress (e.g. 3-indolepropionic acid and lipid peroxidation products) identified in the blood of turtles showed significant correlations with clinical health markers (particularly alkaline phosphatase and total bilirubin), as well as with cobalt. To assist interpretation of trace element blood data in the absence of sufficient information on reptile toxicity, we established exposure reference intervals using a healthy control population. In addition, trace element exposure history was investigated by establishing temporal exposure indices using steady-state relationships between blood and scute. Overall, the data provide a strong argument for the notion that trace element exposure is having an impact on the health of coastal sea turtle populations.

Comparative health assessment of urban and non-urban free-ranging mule deer (Odocoileus hemionus) in southeastern British Columbia, Canada.

BACKGROUND: The provincial wildlife management agency, British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development, performed a translocation to control the urban mule deer (Odocoileus hemionus; uMD) overpopulation and supplement the declining non-urban mule deer (nuMD) population in the Kootenay region, British Columbia, Canada. The objectives of this cross-sectional study were to evaluate the health of the urban and nuMD populations by comparing pathogen exposure, body condition scores (BCS) and pregnancy rates, to characterize the health risks associated with the translocation and to investigate the role of infectious diseases in the decline of the nuMD deer population. METHODS: Two hundred free-ranging mule deer were captured in urban and non-urban environments in the Kootenay region from 2014 to 2017. BCS and morphometric examinations were performed for each deer. Blood samples collected from each deer were tested for exposure to selected pathogens and pregnancy status. RESULTS: Body condition scores averaged 3.4 on a five-point scale, was greater in nuMD, and significantly differed between years. Antibodies were detected for adenovirus hemorrhagic disease virus (38.4% (uMD 43.7%, nuMD 33.3%)), bluetongue virus (0.6% (uMD 1.2%, nuMD 0%)), bovine respiratory syncytial virus (8.4% (uMD 4.6%, nuMD 12.1%)), bovine viral diarrhea virus (1.1% (uMD 0%, nuMD 2.2%)), bovine parainfluenza-3 virus (27.0% (uMD 27.6%, nuMD 26.4%)), Neospora caninum (22.1% (uMD 24.4%, nuMD 19.7%)) and Toxoplasma gondii (8.2% (uMD 12.3%, nuMD 3.9%)). No antibodies against epizootic hemorrhagic disease virus were detected. Pregnancy rates did not differ between the two deer populations (90.7% (uMD 90.6%, nuMD 90.9%)). Exposure to N. caninum was associated with a reduced probability of being pregnant. uMD were more likely to be exposed to T. gondii than nuMD. DISCUSSION: Comparison of BCS, pregnancy rates and pathogen exposure of uMD and nuMD showed that the health of the two populations did not significantly differ, suggesting uMD translocations do not pose a severe risk of pathogen transmission between mule deer populations and that these selected pathogens do not factor in the decline of the nuMD population. However, inclusion of additional health indicators and creation of a robust predictive disease model are warranted to further characterize the health of mule deer and the health risks associated with uMD translocations. These results should be considered as part of a formal risk assessment for future uMD translocations in southeastern British Columbia.

The impact of environmental factors on marine turtle stranding rates.

Globally, tropical and subtropical regions have experienced an increased frequency and intensity in extreme weather events, ranging from severe drought to protracted rain depressions and cyclones, these coincided with an increased number of marine turtles subsequently reported stranded. This study investigated the relationship between environmental variables and marine turtle stranding. The environmental variables examined in this study, in descending order of importance, were freshwater discharge, monthly mean maximum and minimum air temperatures, monthly average daily diurnal air temperature difference and rainfall for the latitudinal hotspots (-27°, -25°, -23°, -19°) along the Queensland coast as well as for major embayments within these blocks. This study found that marine turtle strandings can be linked to these environmental variables at different lag times (3-12 months), and that cumulative (months added together for maximum lag) and non-cumulative (single month only) effects cause different responses. Different latitudes also showed different responses of marine turtle strandings, both in response direction and timing.Cumulative effects of freshwater discharge in all latitudes resulted in increased strandings 10-12 months later. For latitudes -27°, -25° and -23° non-cumulative effects for discharge resulted in increased strandings 7-12 months later. Latitude -19° had different results for the non-cumulative bay with strandings reported earlier (3-6 months). Monthly mean maximum and minimum air temperatures, monthly average daily diurnal air temperature difference and rainfall had varying results for each examined latitude. This study will allow first responders and resource managers to be better equipped to deal with increased marine turtle stranding rates following extreme weather events.

Postmortem examination of Australian sea snakes ( Hydrophiinae): Anatomy and common pathologic conditions.

There is limited published information about disease in wild sea snakes and no standardized guideline for postmortem examination of sea snakes. Identifying causes of morbidity and mortality of marine vertebrate species has been pivotal to understanding disease factors implicated in stranding events and assisting with the formulation of conservation plans. Additionally, postmortem findings can provide valuable information on life history traits and the ecology of these reclusive species. Sick, moribund, or dead sea snakes are intermittently washed ashore along Australian and international beaches and provide an opportunity to examine a subset of the population and identify causes of population decline. We present an illustrated description of sea snake anatomy and describe a systematic approach to postmortem examination of sea snakes. We describe common pathologic conditions identified from clinical and postmortem examinations of stranded Australian sea snakes from southeast Queensland. Notable pathologic conditions include traumatic injury, inflammatory conditions, parasitic infections, and neoplasia.

Status of marine turtle rehabilitation in Queensland.

Rehabilitation of marine turtles in Queensland has multifaceted objectives. It treats individual animals, serves to educate the public, and contributes to conservation. We examined the outcome from rehabilitation, time in rehabilitation, and subsequent recapture and restranding rates of stranded marine turtles between 1996 and 2013 to determine if the benefits associated with this practice are cost-effective as a conservation tool. Of 13,854 marine turtles reported as stranded during this 18-year period, 5,022 of these turtles were stranded alive with the remainder verified as dead or of unknown condition. A total of 2,970 (59%) of these live strandings were transported to a rehabilitation facility. Overall, 1,173/2,970 (39%) turtles were released over 18 years, 101 of which were recaptured: 77 reported as restrandings (20 dead, 13 alive subsequently died, 11 alive subsequently euthanized, 33 alive) and 24 recaptured during normal marine turtle population monitoring or fishing activities. Of the turtles admitted to rehabilitation exhibiting signs of disease, 88% of them died, either unassisted or by euthanasia and 66% of turtles admitted for unknown causes of stranding died either unassisted or by euthanasia. All turtles recorded as having a buoyancy disorder with no other presenting problem or disorder recorded, were released alive. In Queensland, rehabilitation costs approximately $1,000 per animal per year admitted to a center, $2,583 per animal per year released, and $123,750 per animal per year for marine turtles which are presumably successfully returned to the functional population. This practice may not be economically viable in its present configuration, but may be more cost effective as a mobile response unit. Further there is certainly benefit giving individual turtles a chance at survival and educating the public in the perils facing marine turtles. As well, rehabilitation can provide insight into the diseases and environmental stressors causing stranding, arming researchers with information to mitigate negative impacts.

Molecular epidemiology and pathology of spirorchiid infection in green sea turtles (Chelonia mydas).

Spirorchiid blood fluke infections affect endangered turtle populations globally, and are reported as a common cause of mortality in Queensland green sea turtles. Both the flukes and their ova are pathogenic and can contribute to the stranding or death of their host. Of particular interest are ova-associated brain lesions, which have been associated with host neurological deficits. Accurate estimations of disease frequency and the relative effect of infection relating to different spirorchiid species are made difficult by challenges in morphological identification of adults of some genera, and a lack of species-level identifying features for ova. A new specifically designed molecular assay was used to detect and identify cryptic spirorchiids and their ova in Queensland green sea turtle tissues collected from 2011 to 2014 in order to investigate epidemiology, tissue tropisms and pathology. Eight spirorchiid genotypes were detected in 14 distinct tissues, including multiple tissues for each. We found no evidence of a characteristic pathway of the eggs to the exterior; instead the results suggest that a high proportion of eggs become lost in dead-end tissues. The most common lesions observed were granulomas affecting most organs with varying severity, followed by arteritis and thrombi in the great vessels. The number of spirorchiid types detected increased with the presence and severity of granulomatous lesions. However, compared with other organs the brain showed relatively low levels of spirorchiid diversity. An inverse relationship between host age and spirorchiid diversity was evident for the liver and kidneys, but no such relationship was evident for other organs. Molecular data in this study, the first of its kind, provides the first species-level examination of spirorchiid ova and associated pathology, and paves the way for the future development of targeted ante-mortem diagnosis of spirorchiidiasis.

Development of a Humane Slaughter Device for Green Turtles for Use by Traditional Owners in the Torres Strait Islands, Australia.

Marine turtles are caught and slaughtered for consumption as part of traditional indigenous community harvest in Australia as well as in many countries in which marine turtles can be found. However, changes to the Animal Care and Protection Act 2001 in 2012 resulted in Australian indigenous hunters becoming potentially liable to prosecution for using traditional practices to slaughter marine turtles. To provide indigenous hunters with an alternative scientifically tested method to hunt, we developed and tested a humane method as an option to use in indigenous communities. Between 2012 and 2015, a device was developed, tested on 11 carcasses to determine effectiveness and repeatability, used on 5 anaesthetised animals independently diagnosed as candidates for euthanasia, and ultimately used on 2 healthy, conscious animals as part of normal indigenous community subsistence harvesting under observation before being left with the communities for use. Feedback was sought from the communities on the suitability and potential adoption of the device. The device effectively ablated the hind brain and severed the spinal cord when deployed in 81% (9/11) of the tested carcasses, with death in 100% (5/5) of turtles, on average, within 78 seconds of deployment on anaesthetised turtles and death in 100% (2/2) of turtles, on average, within 144 seconds when deployed on healthy turtles within community. Failure to ablate the hindbrain and sever the spinal cord in the cadaver cases was due to incorrect deployment of the device. This device showed promise as an alternative euthanasia method available to indigenous communities of the Torres Straits. Further work is required to encourage acceptance by hunters.

Terminal Restriction Fragment Length Polymorphism for the Identification of Spirorchiid Ova in Tissues from the Green Sea Turtle, Chelonia mydas.

Blood flukes are among the most common disease causing pathogens infecting vertebrates, including humans and some of the world's most globally endangered fauna. Spirorchiid blood flukes are parasites of marine turtles, and are associated with pathology, strandings and mortalities worldwide. Their ova embolize in tissues and incite significant inflammatory responses, however attempts to draw correlations between species and lesions are frustrated by difficulties in identifying ova beyond the genus level. In this study, a newly developed terminal restriction fragment length polymorphism (T-RFLP) method was validated as a tool for differentiating between mixed spirorchiid ova in turtle tissue. Initially, a multiplex PCR was used to differentiate between the five genera of spirorchiid flukes. Following this, PCR was performed using genus/genera-specific fluorescently tagged primer pairs and PCR products digested analysis using restriction endonucleases. Using capillary electrophoresis, this T-RFLP method could differentiate between twelve species and genotypes of spirorchiid flukes in turtles. It was applied to 151 tissue samples and successfully identified the spirorchiid species present. It was found to be more sensitive than visual diagnosis, detecting infections in 28 of 32 tissues that were negative on histology. Spirorchiids were present in 96.7% of tissues tested, with Neospirorchis genotype 2 being the most prevalent, present in 93% of samples. Mixed infections were common, being present in 60.7% of samples tested. The method described here is, to our knowledge, the first use of the T-RFLP technique on host tissues or in an animal ecology context, and describes a significant advancement in the clinical capacity to diagnose a common cause of illness in our environment. It is proven as a sensitive, specific and cost-efficient means of identifying spirorchiid flukes and ova in turtles, with the potential to contribute valuable information to epidemiological and pathological studies as well as future diagnostics for this poorly understood disease.

Potential spawn induction and suppression agents in Caribbean Acropora cervicornis corals of the Florida Keys.

The enhanced ability to direct sexual reproduction may lead to improved restoration outcomes for Acropora cervicornis. Gravid fragments of A. cervicornis were maintained in a laboratory for two sequential trials in the seven days prior to natural spawning in the Florida Keys. Ten replicates of five chemicals known to affect spawning in various invertebrate taxa were tested. Hydrogen peroxide at 2 mM (70%) and L-5-hydroxytryptophan (5-HTP) at 5 (40%) and 20 µM (30%) induced spawning within 15.4 h, 38.8 h and 26.9 h of dosing at or above the rate of release of the control (30%) within 14.6 h. Serotonin acetate monohydrate at 1 µM (20%) and 10 µM (20%), naloxone hydrochloride dihydrate at 0.01 µM (10%) and potassium phosphate monobasic at 0.25 µM (0%) induced spawning at rates less than the control. Although the greatest number of fragments spawned using hydrogen peroxide, it was with 100% mortality. There was a significantly higher induction rate closer to natural spawn (Trial 2) compared with Trial 1 and no genotype effect. Mechanisms of action causing gamete release were not elucidated. In Caribbean staghorn corals, 5-HTP shows promise as a spawning induction agent if administered within 72 h of natural spawn and it will not result in excessive mortality. Phosphate chemicals may inhibit spawning. This is the first study of its kind on Caribbean acroporid corals and may offer an important conservation tool for biologists currently charged with restoring the imperiled Acropora reefs of the Florida Keys.

Ultrastructural and Molecular Characterisation of an Heterosporis-Like Microsporidian in Australian Sea Snakes (Hydrophiinae).

Four sea snakes (two Hydrophis major, one Hydrophis platurus, one Hydrophis elegans) were found washed ashore on different beaches in the Sunshine Coast region and Fraser Island in Queensland, Australia between 2007-2013. Each snake had multiple granulomas and locally extensive regions of pallor evident in the hypaxial and intercostal musculature along the body. Lesions in two individuals were also associated with vertebral and rib fractures. Histological examination revealed granulomas scattered throughout skeletal muscle, subcutaneous adipose tissue and fractured bone. These were composed of dense aggregates of microsporidian spores surrounded by a mantle of macrophages. Sequences (ssrRNA) were obtained from lesions in three sea snakes and all revealed 99% similarity with Heterosporis anguillarum from the Japanese eel (Anguillarum japonica). However, ultrastructural characteristics of the organism were not consistent with those of previous descriptions. Electron microscopic examination of skeletal muscle revealed large cysts (not xenomas) bound by walls of fibrillar material (Heterosporis-like sporophorocyst walls were not detected). The cysts contained numerous mature microsporidian spores arranged in small clusters, sometimes apparently within sporophorous vesicles. The microspores were monomorphic, oval and measured 2.5-3.0 µm by 1.6-1.8 µm. They contained isofilar polar filaments with 11 (infrequently 9-12) coils arranged in two ranks. This is the first published report of a microsporidian infection in hydrophiid sea snakes. This discovery shows microsporidia with molecular affinities to Heterosporis anguillarum but ultrastructural characters most consistent with the genus Pleistophora (but no hitherto described species). Further studies are required to determine whether the microsporidian presented here belongs to the genus Heterosporis, or to a polymorphic species group as suggested by the recognition of a robust Pleistophora/Heterosporis clade by molecular studies. The gross and histological pathology associated with these infections are described.

Molecular Characterization of Coccidia Associated with an Epizootic in Green Sea Turtles (Chelonia mydas) in South East Queensland, Australia.

In the spring of 2014, mass mortalities among wild green sea turtles occurred off the coast of south-east Queensland, Australia. The suspected causative agent was Caryospora cheloniae, an eimeriid coccidian implicated in previous epizootics. Necropsies were undertaken on a subset of 11 dead turtles, with subsequent histopathology and molecular analyses. All turtles returned positive PCR results for coccidial infection in various tissues; these included the brain, gastrointestinal tract, lung, kidney and thyroid. Granulomatous encephalitis was consistently observed, as well as enteritis and, less frequently, thyroiditis and nephritis. Sequencing and phylogenetic analyses indicated the presence of two distinct coccidian genotypes, presumably separate species-one associated with the brain, gastrointestinal tract and lung, and the second with the thyroid and kidney. Maximum likelihood and Bayesian inference analyses placed the first genotype closest to the lankesterellid genus Schellackia, rather than in the Eimeriidae, while the second was paraphyletic to the eimeriids. Presence of coccidial stages in extra-intestinal tissues of the primary host raises questions about the potential presence of intermediate or paratenic hosts within the life cycles, as well as their current placement relative to the genus Caryospora. This study represents the first genetic characterization of this emerging disease agent in green sea turtles, an endangered species, and has relevance for life-cycle elucidation and future development of diagnostics.