Assessing Karenia brevis red tide as a mortality factor of sea turtles in Florida, USA.

Data on Karenia brevis red tides (≥105 cells l-1) and on dead or debilitated (i.e. stranded) Kemp's ridleys Lepidochelys kempii, loggerheads Caretta caretta, green turtles Chelonia mydas, hawksbills Eretmochelys imbricata, and leatherbacks Dermochelys coriacea documented in Florida during 1986-2013 were evaluated to assess red tides as a sea turtle mortality factor. Unusually large numbers of stranded sea turtles were found coincident with red tides primarily along Florida's Gulf coast but also along a portion of Florida's Atlantic coast. These strandings were mainly adult and large immature loggerheads and Kemp's ridleys, and small immature green turtles and hawksbills. Unusually large numbers of stranded leatherbacks never coincided with red tide. For the 3 most common species, results of stranding data modeling, and of investigations that included determining brevetoxin concentrations in samples collected from stranded turtles, all indicated that red tides were associated with greater and more frequent increases in the numbers of stranded loggerheads and Kemp's ridleys than in the number of stranded green turtles. The mean annual number of stranded sea turtles attributed to K. brevis red tide was 80 (SE = 21.6, range = 2-338). Considering typical stranding probabilities, the overall mortality was probably 5-10 times greater. Red tide accounted for a substantial portion of all stranded loggerheads (7.1%) and Kemp's ridleys (17.7%), and a smaller portion of all stranded green turtles (1.6%). Even though K. brevis red tides occur naturally, the mortality they cause needs to be considered when managing these threatened and endangered species.

INTRAVENOUS LIPID EMULSION TREATMENT REDUCES SYMPTOMS OF BREVETOXICOSIS IN TURTLES (TRACHEMYS SCRIPTA).

Harmful algal blooms (HABs) occur when excess nutrients allow dinoflagellates to reproduce in large numbers. Marine animals are affected by blooms when algal toxins are ingested or inhaled. In the Gulf of Mexico, near annual blooms of Karenia brevis release a suite of compounds (brevetoxins) that cause sea turtle morbidity and mortality. The primary treatment at rehabilitation facilities for brevetoxin-exposed sea turtles is supportive care, and it has been difficult to design alternative treatment strategies without an understanding of the effects of brevetoxins in turtles in vivo. Previous studies using the freshwater turtle as a model species showed that brevetoxin-3 impacts the nervous and muscular systems, and is detoxified and eliminated primarily through the liver, bile, and feces. In this study, freshwater turtles (Trachemys scripta) were exposed to brevetoxin (PbTx-3) intratracheally at doses causing clear systemic effects, and treatment strategies aimed at reducing the postexposure neurological and muscular deficits were tested. Brevetoxin-exposed T. scripta displayed the same behaviors as animals admitted to rehabilitation centers for toxin exposure, ranging from muscle twitching and incoordination to paralysis and unresponsiveness. Two treatment regimes were tested: cholestyramine, a bile acid sequestrant; and an intravenous lipid emulsion treatment (Intralipidt) that provides an expanded circulating lipid volume. Cholestyramine was administered orally 1 hr and 6 hr post PbTx-3 exposure, but this regime failed to increase toxin clearance. Animals treated with Intralipid (100 mg/kg) 30 min after PbTx-3 exposure had greatly reduced symptoms of brevetoxicosis within the first 2 hr compared with animals that did not receive the treatment, and appeared fully recovered within 24 hr compared with toxin-exposed control animals that did not receive Intralipid. The results strongly suggest that Intralipid treatment for lipophilic toxins such as PbTx-3 has the potential to reduce morbidity and mortality in HAB-exposed sea turtles.

PLASMA COLLOID OSMOTIC PRESSURE IN CHRONICALLY DEBILITATED LOGGERHEAD SEA TURTLES (CARETTA CARETTA).

Colloid osmotic pressure (COP) is an important component of the forces that manage capillary filtration and is determined by circulating plasma proteins. Patients affected by conditions resulting in hypoproteinemia often suffer severe hemodynamic derangements, including decreased COP. Because chronically debilitated sea turtles (CDT) present with severe hypoproteinemia, the objectives of this study were to 1) determine differences in plasma COP and blood analyte data (packed cell volume [PCV], sodium, chloride, plasma protein fractions) in CDT at admission compared with data from apparently healthy rehabilitated turtles at time of release (HRT) admitted from various stranding causes, and 2) to investigate correlations of COP with these selected blood analytes. COP, PCV, and most plasma protein fractions (excluding pre-albumin and γ-globulins) were significantly lower in CDT upon admission as compared with HRT. Sodium and chloride did not significantly differ between CDT and HRT. A significant increase was observed with PCV and all plasma protein fractions as COP increased. Of all protein fractions tested, albumin contributed the most toward COP (r2 = 0.88, P < 0.001). The results of this study suggest that COP is significantly lower in CDT as compared with HRT, providing insight into the complexity of this critical clinical condition and a small step in advancing the understanding of associated hemodynamic imbalances. Although COP analysis is not readily available as a diagnostic test, this preliminary baseline data suggests that additional research studies are warranted, given the potential for optimization of fluid therapy during rehabilitation of CDT.

Ingested Micronizing Plastic Particle Compositions and Size Distributions within Stranded Post-Hatchling Sea Turtles.

From July 2015 to November 2016, 96 post-hatchling sea turtles were collected from 118 km of the Atlantic coastline in Florida, USA, including loggerhead, green, and hawksbill sea turtle species. Forty-five of the recovered turtles were rehabilitated and released, but the remaining 52 died and were frozen. At necropsy, the gastrointestinal tracts of most the turtles contained visible plastic, and collected particles of 27 individuals were chemically characterized by Raman microscopy as polyethylene, polypropylene, polyethylene terephthalate, and polystyrene. Mesoparticle plastic fragments 1.0-8.7 mm, microparticle fragments 20-1000 µm, and nanoparticles 5-169 nm were identified in the turtles. Polyethylene and polypropylene were the most common plastics ingested from specimens representing 54.1 and 23.7% of the total observed mesoparticles and 11.7 and 21.0% of the total observed microparticles, respectively. A plastic-to-body mass ratio of 2.07 mg/g was determined for this group. The authors suggest that ingestion of micronizing plastic by post-hatchling sea turtles is likely a substantial risk to survival of these endangered and threatened species. This study also provides some of the first evidence for the formation of nanoscopic plastic particles that we theorize forms in the post-hatchling and juvenile environment and are present post-ingestion.

Evaluation of the use of metallothionein as a biomarker for detecting physiological responses to mercury exposure in the bonnethead, Sphyrna tiburo.

Previous studies have demonstrated that sharks, perhaps more so than any other fishes, are capable of bioaccumulating the non-essential toxic metal mercury (Hg) to levels that threaten the health of human seafood consumers. However, few studies have explored the potential effects of Hg accumulation in sharks themselves. Therefore, the goal of this study was to examine if physiological effects occur in sharks in response to environmentally relevant levels of Hg exposure. To address this goal, the relationship between muscle Hg concentrations and muscle/hepatic levels of metallothionein (MT), a widely used protein biomarker of toxic metal exposure in fish, was examined in bonnetheads, Sphyrna tiburo, from three Florida estuaries. Total Hg concentrations in bonnethead muscle, as determined using thermal decomposition and atomic absorption spectrometry, ranged from 0.22 to 1.78 µg/g wet weight and were correlated with animal size. These observations were consistent with earlier studies on Florida bonnetheads, illustrating that they experience bioaccumulation of Hg, often to levels that threaten the health of these animals or consumers of their meat. However, despite this, MT concentrations measured using Western blot analysis were not correlated with muscle Hg concentrations. These results suggest that either environmentally relevant levels of Hg exposure and uptake are below the physiological threshold for inducing effects in sharks or MT is a poor biomarker of Hg exposure in these fishes. Of these two explanations, the latter is favored based on a growing body of evidence that questions the use of MTs as specific indicators of Hg exposure and effects in fish.

Dehydration as an effective treatment for brevetoxicosis in loggerhead sea turtles (Caretta caretta).

Harmful algal blooms are known to cause morbidity and mortality to a large number of marine and estuarine organisms worldwide, including fish and marine mammals, birds, and turtles. The effects of these algal blooms on marine organisms are due to the various toxins produced by the different algal species. In southwest Florida, frequent blooms of the dinoflagellate Karenia brevis, which produces neurotoxins known as brevetoxins, cause widespread fish kills and affect many marine animals. In 2005-2007, numerous sea turtles of several species underwent treatment for brevetoxicosis at the Sea Turtle Rehabilitation Hospital. In green sea turtles, Chelonia mydas, and Kemp's ridley sea turtles, Lepidochelys kempii, symptoms associated with brevetoxicosis were limited to neurologic signs, such as the inability to control the head (head bobbing) and nervous twitching. For these turtles, treatment involved removing the turtles from the environment containing the toxins and providing short-term supportive care. In loggerhead sea turtles, Caretta caretta, symptoms were more generalized; thus, a similar approach was unsuccessful, as was routine treatment for general toxicosis. Loggerhead sea turtles had more extreme neurologic symptoms including coma, and other symptoms that included generalized edema, conjunctival edema, and cloacal or penile prolapse. Treatment of brevetoxicosis in loggerhead sea turtles required a therapeutic regimen that initially included dehydration and systemic antihistamine treatment followed by supportive care.

Tissue enzyme activities in the loggerhead sea turtle (Caretta caretta).

The loggerhead sea turtle, Caretta caretta, one of the seven species of threatened or endangered sea turtles worldwide, is one of the most commonly encountered marine turtles off the eastern coast of the United States and Gulf of Mexico. Although biochemical reference ranges have been evaluated for several species of sea turtles, tissue specificity of the commonly used plasma enzymes is lacking. This study evaluated the tissue specificity of eight enzymes, including amylase, lipase, creatine kinase (CK), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in 30 tissues from five stranded loggerhead sea turtles with no evidence of infectious disease. Amylase and lipase showed the greatest tissue specificity, with activity found only in pancreatic samples. Creatine kinase had high levels present in skeletal and cardiac muscle, and moderate levels in central nervous system and gastrointestinal samples. Gamma-glutamyl transferase was found in kidney samples, but only in very low levels. Creatine kinase, ALP, AST, and LDH were found in all tissues evaluated and ALT was found in most, indicating low tissue specificity for these enzymes in the loggerhead.

Brevetoxin in blood, biological fluids, and tissues of sea turtles naturally exposed to Karenia brevis blooms in central west Florida.

In 2005 and 2006, the central west Florida coast experienced two intense Karenia brevis red tide events lasting from February 2005 through December 2005 and August 2006 through December 2006. Strandings of sea turtles were increased in the study area with 318 turtles (n = 174, 2005; n = 144, 2006) stranding between 1 January 2005 and 31 December 2006 compared to the 12-yr average of 43 +/- 23 turtles. Live turtles (n = 61) admitted for rehabilitation showed clinical signs including unresponsiveness, paresis, and circling. Testing of biological fluids and tissues for the presence of brevetoxin activity by enzyme-linked immunosorbent assay found toxin present in 93% (52 of 56) of live stranded sea turtles, and 98% (42 of 43) of dead stranded sea turtles tested. Serial plasma samples were taken from several live sea turtles during rehabilitation and toxin was cleared from the blood within 5-80 days postadmit depending upon the species tested. Among dead animals the highest brevetoxin levels were found in feces, stomach contents, and liver. The lack of significant pathological findings in the majority of animals necropsied supports toxin-related mortality.

NONPIGMENTED VERSUS PIGMENTED: HEALTH VARIABLES AND GENETICS OF ALBINO FLORIDA GREEN TURTLE (CHELONIA MYDAS) HATCHLINGS COMPARED WITH NORMALLY PIGMENTED HATCHLINGS FROM THE SAME CLUTCH.

At the time of hatchling emergence from a nest laid on Juno Beach, Florida, US, by a normally pigmented green turtle (Chelonia mydas), 23 albino hatchlings and 75 normally pigmented hatchlings were observed. This condition is rarely seen in sea turtles, and little is known about blood analytes and genetics of albino wildlife to date. Therefore, the objective of our study was to assess and compare morphometric measurements (mass, minimum straight carapace length, body condition index), carapacial scute anomalies, a suite of hematologic and plasma biochemical analytes, and two glucose analysis methodologies (glucometer and dry chemistry analysis) in albino (n=20) versus normally pigmented (n=24) hatchlings from this nest. Genetic analyses were completed to identify paternal contributions of hatchlings and to test Mendelian inheritance assumptions. Although morphometric measurements, scute anomalies, and leukocyte morphology were similar between albino and normally pigmented hatchlings, several differences were observed in blood analyte data: immature erythrocytes, packed cell volume, heterophil:lymphocyte ratio, and glucose concentrations (by both methodologies) were significantly higher, whereas absolute immature heterophils, absolute lymphocytes, number of erythrocyte micronuclei, sodium, and chloride were significantly lower in albino hatchlings compared with normally pigmented hatchlings. Considerations for these differences include a stress response from sampling (e.g., timing of procedures or possibly from photosensitivity or reduced visual acuity in albinos) and different osmoregulation, which may reflect physiologic variations or stress. There was a small positive bias (0.10 mmol/L) with glucose by glucometer, similar to reports in other sea turtle species and confirming its suitability for use in hatchlings. All albino hatchlings analyzed (n=10) were from the same father, but the normally pigmented hatchlings (n=24) were from two other fathers. These findings provide insight into the physiology and genetics of albinism in sea turtles.

Morphologic and physiologic characteristics of green sea turtle (Chelonia mydas) hatchlings in southeastern Florida, USA.

The ability of sea turtle hatchlings to survive into adulthood is related, in part, to their individual health status. Documenting a variety of health data is essential for assessing individual and population health. In this study, we report health indices for 297 green sea turtle (Chelonia mydas) hatchlings that emerged from 32 nests deposited on Juno Beach, Florida, USA in June-July, 2017. Results of physical examination, morphometrics, and infectious disease testing (chelonid alphaherpesvirus 5, ChHV5), and blood analyte reference intervals (hematology, plasma protein, glucose) are presented. Carapacial scute abnormalities were observed in 36% (108/297) of all hatchlings, including abnormal vertebral (86/297, 29%), lateral (72/297, 24%), and both vertebral and lateral (50/297, 17%) scutes. Hatchlings from nests laid in July, which was ~ 1.6 °C warmer than June, had significantly shorter incubation periods, and higher body mass, straight carapace length, body condition index, packed cell volume, and heterophil:lymphocyte ratios compared to hatchlings from nests laid in June. These results suggest that incubation temperatures are linked to hatchling developmental factors and size, nutritional and/or hydration status, and/or blood cell dynamics. Blood samples from all 297 hatchlings tested negative for ChHV5 DNA via quantitative PCR, including 86 hatchlings from the nests of 11 adult females that tested positive for ChHV5 via qPCR or serology in a separate study, lending support to the hypothesis that ChHV5 is horizontally (rather than vertically) transmitted among green turtles. Information resulting from this study represents a useful dataset for comparison to future health assessment and population monitoring studies of green turtle hatchlings in the northwestern Atlantic Ocean.

Cultivating epizoic diatoms provides insights into the evolution and ecology of both epibionts and hosts.

Our understanding of the importance of microbiomes on large aquatic animals-such as whales, sea turtles and manatees-has advanced considerably in recent years. The latest observations indicate that epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful approach to culture putatively obligate epizoic diatoms without their hosts. That some taxa can be cultured independently from their epizoic habitat raises several questions about the nature of the interaction between these animals and their epibionts. This insight allows us to propose further applications and research avenues in this growing area of study. Analyzing the DNA sequences of these cultured strains, we found that several unique diatom taxa have evolved independently to occupy epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices.

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris).

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra- and inter-assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R(2)≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free-ranging or short-term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long-term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free-ranging, short-term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate.

Use of intravenous lipid emulsion therapy as a novel treatment for brevetoxicosis in sea turtles.

The southwest coast of Florida experiences annual red tides, a type of harmful algal bloom that results from high concentrations of Karenia brevis. These dinoflagellates release lipophilic neurotoxins, known as brevetoxins, that bind to sodium channels and inhibit their inactivation, resulting in a variety of symptoms that can lead to mass sea turtle strandings. Traditional therapies for brevetoxicosis include standard and supportive care (SSC) and/or dehydration therapy; however, these treatments are slow-acting and often ineffective. Because red tide events occur annually in Florida, our objective was to test intravenous lipid emulsion (ILE) as a rapid treatment for brevetoxicosis in sea turtles and examine potential impacts on toxin clearance rates, symptom reduction, rehabilitation time, and survival rates. Sea turtles exhibiting neurological symptoms related to brevetoxicosis were brought to rehabilitation from 2018-2019. Upon admission, blood samples were collected, followed by immediate administration of 25 mg ILE/kg body mass (Intralipid® 20%) at 1 mL/min using infusion pumps. Blood samples were collected at numerous intervals post-ILE delivery and analyzed for brevetoxins using enzyme-linked immunosorbent assays. In total, nine (four subadults, one adult female, four adult males) loggerheads (Caretta caretta), five (four juvenile, one adult female) Kemp's ridleys (Lepidochelys kempii), and four juvenile green turtles (Chelonia mydas) were included in this study. We found that plasma brevetoxins declined faster compared to turtles that received only SSC. Additionally, survival rate of these patients was 94% (17/18), which is significantly higher than previous studies that used SSC and/or dehydration therapy (47%; 46/99). Nearly all symptoms were eliminated within 24-48 h, whereas using SSC, symptom elimination could take up to seven days or more. The dosage given here (25 mg/kg) was sufficient for turtles in this study, but the use of a higher dosage (50-100 mg/kg) for those animals experiencing severe symptoms may be considered. These types of fast-acting treatment plans are necessary for rehabilitation facilities that are already resource-limited. Intravenous lipid emulsion therapy has the potential to reduce rehabilitation time, save resources, and increase survival of sea turtles and other marine animals experiencing brevetoxicosis.

Plasma proteomics of green turtles (Chelonia mydas) reveals pathway shifts and potential biomarker candidates associated with health and disease.

Evaluating sea turtle health can be challenging due to an incomplete understanding of pathophysiologic responses in these species. Proteome characterization of clinical plasma samples can provide insights into disease progression and prospective biomarker targets. A TMT-10-plex-LC-MS/MS platform was used to characterize the plasma proteome of five, juvenile, green turtles (Chelonia mydas) and compare qualitative and quantitative protein changes during moribund and recovered states. The 10 plasma samples yielded a total of 670 unique proteins. Using ≥1.2-fold change in protein abundance as a benchmark for physiologic upregulation or downregulation, 233 (34.8%) were differentially regulated in at least one turtle between moribund and recovered states. Forty-six proteins (6.9%) were differentially regulated in all five turtles with two proteins (0.3%) demonstrating a statistically significant change. A principle component analysis showed protein abundance loosely clustered between moribund samples or recovered samples and for turtles that presented with trauma (n = 3) or as intestinal floaters (n = 2). Gene Ontology terms demonstrated that moribund samples were represented by a higher number of proteins associated with blood coagulation, adaptive immune responses and acute phase response, while recovered turtle samples included a relatively higher number of proteins associated with metabolic processes and response to nutrients. Abundance levels of 48 proteins (7.2%) in moribund samples significantly correlated with total protein, albumin and/or globulin levels quantified by biochemical analysis. Differentially regulated proteins identified with immunologic and physiologic functions are discussed for their possible role in the green turtle pathophysiologic response and for their potential use as diagnostic biomarkers. These findings enhance our ability to interpret sea turtle health and further progress conservation, research and rehabilitation programs for these ecologically important species.

Comprehensive health assessment and blood analyte reference intervals of gopher tortoises (Gopherus polyphemus) in southeastern FL, USA.

The gopher tortoise (Gopherus polyphemus), a keystone species, is declining throughout its geographic range. Lack of knowledge with respect to the potential infectious diseases present within wild populations creates a dilemma for wildlife biologists, conservationists and public policy makers. The objective of this study was to conduct a health assessment of two previously unstudied gopher tortoise aggregations located at two sites in southeastern FL. Samples were collected from 91 tortoises (48 adults, 35 juveniles, 8 hatchlings) captured at Florida Atlantic University's Harbor Branch Oceanographic Institute, in Fort Pierce, FL, USA in 2019, and Loggerhead Park in Juno Beach, FL, USA, during 2018-2019. Samples of blood, nasal swabs and oral/cloacal swabs were analyzed for hematology, plasma protein electrophoretic profiles and infectious disease testing including Mycoplasma spp. serology and polymerase chain reaction (PCR) assays for Ranavirus, Herpesvirus and Anaplasma spp. Hematological and plasma protein electrophoresis reference intervals are presented for adult and juvenile tortoises from both sites combined. Clinical signs consistent with upper respiratory tract disease (URTD) were observed in 18/91 (20%) tortoises, and antibodies to Mycoplasma agassizii were detected in 33/77 (42.9%) tortoises. Adult tortoises were significantly more likely than juveniles to have URTD clinical signs, and statistically significant, positive relationships were observed between the presence of antibodies to Mycoplasma spp. and carapace length, packed cell volume and plasma globulin concentrations. Anaplasma spp. inclusions were observed in 8/82 (10%) tortoises, but PCR detected Anaplasma sp. in 21/83 (25%) tortoises. Herpesvirus and Ranavirus were not detected in any blood or swab samples. This work contributes important baseline information on the health of gopher tortoises toward the southern end of the species' range.

Spirorchiidiasis in stranded loggerhead Caretta caretta and green turtles Chelonia mydas in Florida (USA): host pathology and significance.

Spirorchiid trematodes are implicated as an important cause of stranding and mortality in sea turtles worldwide. However, the impact of these parasites on sea turtle health is poorly understood due to biases in study populations and limited or missing data for some host species and regions, including the southeastern United States. We examined necropsy findings and parasitological data from 89 loggerhead Caretta caretta and 59 green turtles Chelonia mydas that were found dead or moribund (i.e. stranded) in Florida (USA) and evaluated the role of spirorchiidiasis in the cause of death. High prevalence of infection in the stranding population was observed, and most infections were regarded as incidental to the cause of death. Spirorchiidiasis was causal or contributory to death in some cases; however, notable host injury and/or large numbers of parasites were observed in some animals, including nutritionally robust turtles, with no apparent relationship to cause of death. New spirorchiid species records for the region were documented and identified genera included Neospirorchis, Hapalotrema, Carettacola, and Learedius. Parasites inhabited and were associated with injury and inflammation in a variety of anatomic locations, including large arteries, the central nervous system, endocrine organs, and the gastrointestinal tract. These findings provide essential information on the diversity of spirorchiids found in Florida sea turtles, as well as prevalence of infection and the spectrum of associated pathological lesions. Several areas of needed study are identified with regard to potential health implications in the turtle host, and findings caution against over-interpretation in individual cases.

Characterization of progressive keratitis in otariids.

OBJECTIVE: To characterize a form of progressive keratitis that occurs commonly in otariids. MATERIALS AND METHODS: One hundred and thirteen captive otariids were evaluated by ophthalmologic examination and digital photography. RESULTS: Forty-six females and 67 males were in the reference population, average age of 14 years. California sea lions predominated (n = 100); there were also six Steller sea lions, five brown fur seals, one Guadalupe fur seal and one northern fur seal. Three stages of progressive keratitis are described. Overall, 64.6% animals and 142 eyes from 113 animals (62.8%) were affected with one of three stages. The mildest form, Stage 1 keratitis, occurred in 78 of 226 eyes (34.5%); the intermediate Stage 2 keratitis occurred in 30 of 226 eyes (13.3%); and the most severe, Stage 3 keratitis, occurred in 34 of 226 eyes (15%). All but six animals had bilateral disease. Animals with Stage 1 keratitis were significantly younger than those in more advanced stages. DISCUSSION: 'Otariid Keratitis' occurs in all populations of eared seals evaluated. A large-scale epidemiological study is ongoing to identify the risk factors that contribute to this disease. Exposure to chronic sunlight appears to be an important risk factor as shade diminishes clinical signs; animals kept out of sunlight the majority of the time have less severe clinical signs. Age may be important since exposure accumulates with aging. Progression of the disease is also associated with secondary opportunistic bacterial and fungal infections. The surface immune system may be imbalanced contributing to these infections and progression.

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.

Pre-/analytical factors affecting whole blood and plasma glucose concentrations in loggerhead sea turtles (Caretta caretta).

Blood glucose is vital for many physiological pathways and can be quantified by clinical chemistry analyzers and in-house point-of-care (POC) devices. Pre-analytical and analytical factors can influence blood glucose measurements. This project aimed to investigate pre-analytical factors on whole blood and plasma glucose measurements in loggerhead sea turtles (Caretta caretta) by evaluating the effects of storage (refrigeration) up to 48h after sampling and of packed cell volume (PCV) on whole blood glucose analysis by POC glucometer (time series n = 13); and by evaluating the effects of storage (room temperature and refrigeration) on plasma glucose concentrations using a dry slide chemistry analyzer (DCA) at various conditions: immediate processing and delayed plasma separation from erythrocytes at 24h and 48h (time series n = 14). The POC glucometer had overall strong agreement with the DCA (CCC = 0.76, r = 0.84, Cb = 0.90), but consistently overestimated glucose concentrations (mean difference: +0.4 mmol/L). The POC glucometer results decreased significantly over time, resulting in a substantial decline within the first 2h (0.41±0.47 mmol/L; 8±9%) that could potentially alter clinical decisions, thereby highlighting the need for immediate analysis using this method. The effects of PCV on glucose could not be assessed, as the statistical significance was associated with one outlier. Storage method significantly affected plasma glucose measurements using DCA, with room temperature samples resulting in rapid decreases of 3.57±0.89 mmol/L (77±9%) over the first 48h, while refrigerated samples provided consistent plasma glucose results over the same time period (decrease of 0.26±0.23 mmol/L; 6±5%). The results from this study provide new insights into optimal blood sample handling and processing for glucose analysis in sea turtles, show the suitability of the POC glucometer as a rapid diagnostic test, and confirm the reliability of plasma glucose measurements using refrigeration. These findings emphasize the need to consider pre-/analytical factors when interpreting blood glucose results from loggerhead sea turtles.

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.