Detection and population genomics of sea turtle species via noninvasive environmental DNA analysis of nesting beach sand tracks and oceanic water.

Elusive aquatic wildlife, such as endangered sea turtles, are difficult to monitor and conserve. As novel molecular and genetic technologies develop, it is possible to adapt and optimize them for wildlife conservation. One such technology is environmental (e)DNA - the detection of DNA shed from organisms into their surrounding environments. We developed species-specific green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle probe-based qPCR assays, which can detect and quantify sea turtle eDNA in controlled (captive tank water and sand samples) and free ranging (oceanic water samples and nesting beach sand) settings. eDNA detection complemented traditional in-water sea turtle monitoring by enabling detection even when turtles were not visually observed. Furthermore, we report that high throughput shotgun sequencing of eDNA sand samples enabled sea turtle population genetic studies and pathogen monitoring, demonstrating that noninvasive eDNA techniques are viable and efficient alternatives to biological sampling (e.g., biopsies and blood draws). Genetic information was obtained from sand many hours after nesting events, without having to observe or interact with the target individual. This greatly reduces the sampling stress experienced by nesting mothers and emerging hatchlings, and avoids sacrificing viable eggs for genetic analysis. The detection of pathogens from sand indicates significant potential for increased wildlife disease monitoring capacity and viral variant surveillance. Together, these results demonstrate the potential of eDNA approaches to ultimately help understand and conserve threatened species such as sea turtles.

Pharmacokinetics of ponazuril after administration of a single oral dose to green turtles (Chelonia mydas).

The coccidian protozoan, Caryospora cheloniae, has been associated with severe enteritis and encephalitis in immature farm-raised green turtles (Chelonia mydas) in the Cayman Islands, immature green turtles off the coast of Florida, and immature stranded sea turtles in Australia. An effective anti-coccidial drug that is both orally absorbed and well-distributed throughout the body is needed for treatment of turtles diagnosed with coccidiosis in rehabilitation facilities. Ponazuril is a triazine antiprotozoal drug that is approved in the USA for the treatment of another Apicomplexan, Sarcocystis neurona, and has also been successfully used in the therapy of other coccidian parasites. The objective of this study was to perform an oral dose-ranging pilot study (10-100 mg/kg of body weight ponazuril) in green turtles (N = 9), followed by oral administration of ponazuril at 100 mg/kg body weight (N = 8) to assess its disposition. Another goal of this study was to optimize the method of oral drug administration to green turtles. Plasma ponazuril concentrations were quantified using high performance liquid chromatography (HPLC). Standard compartmental models were fit to the data. Ponazuril was absorbed after oral administration at 100 mg/kg BW, with a maximum plasma concentration of 3.3 µg/ml. Dose-dependent pharmacokinetic parameters only weakly correlated with the dose rate, apparently due to considerable pharmacokinetic variability observed between turtles. Administration of ponazuril in gelatin capsules using a balling gun was deemed the least variable and most successful method of drug administration. Further studies are needed to evaluate the safety and efficacy of ponazuril in sea turtles with coccidiosis.

Red blood cell osmotic fragility in healthy loggerhead and green sea turtles.

Loggerhead (Caretta caretta; Cc) and green sea (Chelonia mydas; Cm) turtles admitted to rehabilitation facilities may require blood transfusions for supportive treatment of disorders resulting in life-threatening anemia, but, considering the unique erythrocyte chemistry of sea turtles, standardized donor red blood cell (RBC) storage protocols have not been established. Prolonged cold storage and the effects of various anticoagulant-preservative solutions have been associated with increased RBC osmotic fragility across a broad range of species. Increased RBC fragility in stored RBC products has been associated with acute transfusion reactions. The osmotic fragility test is used to measure erythrocyte resistance to hemolysis while being exposed to a series of dilutions of a saline solution. We obtained baseline measurements for osmotic fragility in healthy Cc and Cm. Osmotic fragility testing was performed on samples from 10 Cc to 10 Cm. Fifty percent (50%) RBC hemolysis was identified at a mean NaCl concentration of 0.38% in both species. Results of our study will help guide future studies evaluating optimal storage solutions for sea turtle blood products.

Tumor re-growth, case outcome, and tumor scoring systems in rehabilitated green turtles with fibropapillomatosis.

Fibropapillomatosis (FP) is an infectious, neoplastic disease of major concern in sea turtle rehabilitation facilities. Rehabilitating sea turtles that undergo tumor removal surgery often have tumor regrowth and may experience mortality. We evaluated tumor score, removal, and regrowth in rehabilitating green sea turtles with FP in 4 rehabilitation facilities in the southeastern USA during 2009-2017. Of 756 cases, 312 (41%) underwent tumor removal surgery, 155 (50%) of those had tumor regrowth within an average of 46 ± 45 d, and 85 (27%) had multiple (>1) regrowth events. Of 756 turtles with FP, 563 (75%) did not survive after admission into a rehabilitation facility, including 283 (37%) that were euthanized and 280 that died without euthanasia (37%), and 193 survived, including 186 (25%) released and 7 (1%) placed in permanent captive care. Tumor removal surgery increased the odds of tumor regrowth but also enhanced survivorship, whereas tumor regrowth was not a significant predictor of case outcome. Three FP tumor scoring systems were used to assign tumor scores to 449 cases, and differing results emphasize that tumor scoring systems should be applied to the situations and/or location(s) for which they were intended. FP tumor score was not a significant predictor for the event or extent of FP tumor regrowth after surgical excision. Under current rehabilitation regimes, outcomes of rehabilitation for tumored turtles have a low probability of success. The results of this study may be used to help guide clinical decision-making and determine prognoses for rehabilitating sea turtles with FP.

Caryospora-Like Coccidia Infecting Green Turtles (Chelonia mydas): An Emerging Disease With Evidence of Interoceanic Dissemination.

Protozoa morphologically consistent with Caryospora sp. are one of the few pathogens associated with episodic mass mortality events involving free-ranging sea turtles. Parasitism of green turtles (Chelonia mydas) by these coccidia and associated mortality was first reported in maricultured turtles in the Caribbean during the 1970s. Years later, epizootics affecting wild green turtles in Australia occurred in 1991 and 2014. The first clinical cases of Caryospora-like infections reported elsewhere in free-ranging turtles were from the southeastern US in 2012. Following these initial individual cases in this region, we documented an epizootic and mass mortality of green turtles along the Atlantic coast of southern Florida from November 2014 through April 2015 and continued to detect additional, sporadic cases in the southeastern US in subsequent years. No cases of coccidial disease were recorded in the southeastern US prior to 2012 despite clinical evaluation and necropsy of stranded sea turtles in this region since the 1980s, suggesting that the frequency of clinical coccidiosis has increased here. Moreover, we also recorded the first stranding associated with infection by a Caryospora-like organism in Hawai'i in 2018. To further characterize the coccidia, we sequenced part of the 18S ribosomal and mitochondrial cytochrome oxidase I genes of coccidia collected from 62 green turtles found in the southeastern US and from one green turtle found in Hawai'i. We also sequenced the ribosomal internal transcribed spacer regions from selected cases and compared all results with those obtained from Caryospora-like coccidia collected from green turtles found in Australia. Eight distinct genotypes were represented in green turtles from the southeastern US. One genotype predominated and was identical to that of coccidia collected from the green turtle found in Hawai'i. We also found a coccidian genotype in green turtles from Florida and Australia with identical 18S and mitochondrial sequences, and only slight inter-regional differences in the internal transcribed spacer 2. We found no evidence of geographical structuring based on phylogenetic analysis. Low genetic variability among the coccidia found in green turtle populations with minimal natural connectivity suggests recent interoceanic dissemination of these parasites, which could pose a risk to sea turtle populations.

ADVANCING TRANSFUSION MEDICINE IN SEA TURTLES: OPTIMIZATION OF A CROSS-MATCHING PROTOCOL.

Whole blood transfusions are an essential treatment modality during rehabilitation of stranded sea turtles, however, standardized protocols for transfusions are not available in reptile medicine. The objective of this study was to optimize a cross-matching protocol for sea turtle blood transfusions. Fresh venous blood samples from 15 turtles (n = 14 green turtles, Chelonia mydas [Cm]; n = 1 loggerhead sea turtle, Caretta caretta [Cc]) were tested using a temperature-appropriate (i.e., reflecting body temperature), time-sensitive protocol in 26 reactions using two procedures for cross-match evaluation at 30 and 60 min at ambient air and water bath temperature. There were no significant differences between both protocols at 30- and 60-min incubation times or between microscopic evaluations at 2 or 5 min. The major cross-match identified 7/22 incompatible Cm-Cm reactions as observed by microscopic erythrocyte agglutination. Minor cross-matches resulted in 6/22 incompatible Cm-Cm reactions. About half of all Cm-Cm reactions (12/22) were compatible by major and minor cross-match. All Cc-Cm reactions (4/4) were incompatible. A higher than expected proportion of incompatible Cm cross-matching reactions suggests preexisting antibodies to nonself erythrocyte antigens in this species, or other factors promoting erythrocyte aggregation or agglutination. Preliminary data across Cm and Cc suggests cross-species incompatibility. These results indicate that sea turtles may react to donor erythrocytes even at the first transfusion. Concurrent major and minor cross-matching using the proposed protocol at 30-min incubation at room temperature should be considered a necessary and effective way to test for patient and donor incompatibilities in sea turtles.

Plasma Bleomycin Concentrations during Electrochemotherapeutic Treatment of Fibropapillomas in Green Turtles Chelonia mydas.

Fibropapillomatosis of sea turtles is traditionally treated with surgical debulking techniques that are often associated with prolonged healing and tumor recurrence. Electrochemotherapy was recently described for green turtles Chelonia mydas and can be an alternative to surgery and even general anesthesia. The objectives of this study were to replicate an electrochemotherapy protocol from a previous report and add plasma bleomycin analysis to the treatment. After bleomycin injection into similarly sized tumors of two green turtles and immediate electroporation at two time points, plasma bleomycin reached detectable concentrations that were considerably lower than those found in human studies. At 3 months posttherapy, no healing complications or recurrences were encountered and only scar tissue remained. This study adds further support that electrochemotherapy with bleomycin has the potential to be used as an effective alternative treatment for this complex disease.

Sea turtle fibropapilloma tumors share genomic drivers and therapeutic vulnerabilities with human cancers.

Wildlife populations are under intense anthropogenic pressures, with the geographic range of many species shrinking, dramatic reductions in population numbers and undisturbed habitats, and biodiversity loss. It is postulated that we are in the midst of a sixth (Anthropocene) mass extinction event, the first to be induced by human activity. Further, threatening vulnerable species is the increased rate of emerging diseases, another consequence of anthropogenic activities. Innovative approaches are required to help maintain healthy populations until the chronic underlying causes of these issues can be addressed. Fibropapillomatosis in sea turtles is one such wildlife disease. Here, we applied precision-medicine-based approaches to profile fibropapillomatosis tumors to better understand their biology, identify novel therapeutics, and gain insights into viral and environmental triggers for fibropapillomatosis. We show that fibropapillomatosis tumors share genetic vulnerabilities with human cancer types, revealing that they are amenable to treatment with human anti-cancer therapeutics.