Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

Interrupted Lives: Welfare Considerations in Wildlife Rehabilitation.

Each year in the United States, thousands of sick, injured, or displaced wild animals are presented to individuals or organizations who have either a federal or state permit that allows them to care for these animals with the goal of releasing them back to the wild. The purpose of this review is to demonstrate the complexity of considerations rehabilitators and veterinarians face while trying to optimize the welfare of wild animals in need of care and rehabilitation. The process of rehabilitation is inherently stressful for wildlife. Maintaining an animal's welfare during the rehabilitation process-from initial contact and tria+ge to the animal's euthanasia, release, or captive placement-requires deliberate, timely and humane decision making. The welfare of wild animals can be improved by preventing human-related causes of admission, providing resources and support for wildlife rehabilitation (almost all rehabilitation in the United States is privately funded and access to veterinary care is often limited); further developing evidence-based wildlife rehabilitation methods and welfare measures, attracting more veterinary professionals to the field, harmonizing regulatory oversight with standards of care, training, and accountability, and increasing public education.

Global diversity of enterococci and description of 18 novel species.

Enterococci are commensal gut microbes of most land animals. They diversified over hundreds of millions of years adapting to evolving hosts and host diets. Of over 60 known enterococcal species, Enterococcus faecalis and E. faecium uniquely emerged in the antibiotic era among leading causes of multidrug resistant hospital-associated infection. The basis for the association of particular enterococcal species with a host is largely unknown. To begin deciphering enterococcal species traits that drive host association, and to assess the pool of Enterococcus-adapted genes from which known facile gene exchangers such as E. faecalis and E. faecium may draw, we collected 886 enterococcal strains from nearly 1,000 specimens representing widely diverse hosts, ecologies and geographies. This provided data on the global occurrence and host associations of known species, identifying 18 new species in the process expanding genus diversity by >25%. The novel species harbor diverse genes associated with toxins, detoxification, and resource acquisition. E. faecalis and E. faecium were isolated from a wide diversity of hosts highlighting their generalist properties, whereas most other species exhibited more restricted distributions indicative of specialized host associations. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades as well as genes associated with range expansion, such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genus Enterococcus, potential threats to human health, and new insights into its evolution.

Phaeohyphomycosis due to Exophiala in Aquarium-Housed Lumpfish (Cyclopterus lumpus): Clinical Diagnosis and Description.

Phaeohyphomycosis caused by Exophiala species represents an important disease of concern for farmed and aquarium-housed fish. The objective of this study was to summarize the clinical findings and diagnosis of Exophiala infections in aquarium-housed Cyclopterus lumpus. Clinical records and postmortem pathology reports were reviewed for 15 individuals from 5 public aquaria in the United States and Canada from 2007 to 2015. Fish most commonly presented with cutaneous ulcers and progressive clinical decline despite topical or systemic antifungal therapy. Antemortem fungal culture of cutaneous lesions resulted in colonial growth for 7/12 samples from 8 individuals. Amplification of the internal transcribed spacer region (ITS) of nuclear rDNA identified Exophiala angulospora or Exophiala aquamarina in four samples from three individuals. Postmortem histopathologic findings were consistent with phaeohyphomycosis, with lesions most commonly found in the integument (11/15), gill (9/15), or kidney (9/15) and evidence of fungal angioinvasion and dissemination. DNA extraction and subsequent ITS sequencing from formalin-fixed paraffin-embedded tissues of seven individuals identified E. angulospora, E. aquamarina, or Cyphellophora sp. in four individuals. Lesion description, distribution, and Exophiala spp. identifications were similar to those reported in farmed C. lumpus. Antemortem clinical and diagnostic findings of phaeohyphomycosis attributable to several species of Exophiala provide insight on the progression of Exophiala infections in lumpfish that may contribute to management of the species in public aquaria and under culture conditions.

PULMONARY AND COELOMIC MYCOSES DUE TO METARHIZIUM AND BEAUVERIA SPECIES IN REPTILES.

This report documents cases of fatal pulmonary mycosis caused by entomopathogenic fungi in the genera Metarhizium and Beauveria (Order Hypocreales) in a loggerhead sea turtle (Caretta caretta), a Chinese alligator (Alligator sinensis), two gopher tortoises (Gopherus polyphemus), a Cuvier's dwarf caiman (Paleosuchus palpebrosus), a false gharial (Tomistoma schlegelii), a green sea turtle (Chelonia mydas), and a Kemp's ridley sea turtle (Lepidochelys kempii), and a case of granulomatous coelomitis in a hawksbill sea turtle (Eretmochelys imbricata). Fungi identified in these cases included Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, Metarhizium robertsii, and one case of infection by a novel Metarhizium species. The animals were either housed at zoos or brought into rehabilitation from the wild. Although the majority of animals had comorbidities, the fungal infections were believed to be the primary cause of death. Fungal susceptibility testing was performed on two Beauveria spp. isolates, and revealed lower minimum inhibitory concentrations for itraconazole and voriconazole when compared to terbinafine and fluconazole. This case series demonstrates that a variety of reptile species from different orders are vulnerable to infection with Metarhizium, and multiple species of sea turtle are susceptible to infection with Beauveria.

Endoscopic sexing in turtles and tortoises: 467 cases (2007-2017).

BACKGROUND: This multi-institutional retrospective study evaluated the feasibility and safety of endoscopic sex identification in 467 turtles and tortoises, representing 10 species. METHODS: Medical records of turtles and tortoises that underwent endoscopic sex identification at the University of Georgia, New England Aquarium and Turtle Conservancy were reviewed for presurgical management, anaesthesia, endoscopic equipment and surgical techniques, endoscopic results and complications. RESULTS: The majority of animals weighed less than 200 g, were fasted and anaesthetised using an injectable combination of ketamine, dexmedetomidine and morphine or hydromorphone, supplemented by local lidocaine at the prefemoral site. Anaesthetic reversal using atipamezole alone or in combination with naloxone was routine. For uncomplicated procedures, mean total anaesthesia, surgery and recovery times were 22, 4 and 18 minutes, respectively. All animals were placed in lateral recumbency for a prefemoral endoscopic approach to the coelom using a rigid telescope and sterile fluid infusion to visualise the gonads. Sex identification was definitive in 99.4% (n = 464) of the animals. Iatrogenic bladder perforation was the most common complication (n = 5), which necessitated extended anaesthesia and surgical time for repair. Only a single anaesthetic-related death was reported, which was associated with human error and drug overdose. CONCLUSIONS: This is the first large-scale study to retrospectively evaluate endoscopic sex identification in multiple chelonian species. Results suggest that endoscopic sexing is a safe, accurate and practical means for sex identification in turtles and tortoises, and represents a valuable tool in their reproductive management.

Correction: Turner et al. Steatitis in Cold-Stunned Kemp's Ridley Sea Turtles (Lepidochelys kempii). Animals 2021, 11, 898.

In the original publication [...].

Observations on the use of passive integrated transponder (PIT) tags in teleosts and elasmobranchs at a public aquarium, 728 cases, 2007-2020.

Between 2007 and 2020 at New England Aquarium, Boston, MA, USA, we implanted passive integrated transponder (PIT) tags into 728 fish representing 105 teleost and elasmobranch species to identify animals as individuals. At the time of retrospective data analysis, mean longevity interval (median, range) after tag placement for animals that remained alive (n = 236) was 4.7 years (4.5, 0.3-13.8). Mean interval (median, range) between tag placement and death (n = 317) was 2.1 years (1.6, 0-11.2); and mean interval (median, range) between tag placement and transfer to other facilities (n = 175) was 2.5 years (3.1, 0.1-9.3). Possible adverse effects of tagging were extremely rare. Using the described methods, the equipment cost for every 10 PIT tag implantations was $2.83. PIT tag implantation in fishes is a safe and cost effective method to identify individuals, providing an opportunity to accumulate valuable data regarding individual longevity, welfare, basic demographics, and outcome of medical management. PIT tag implantation is recommended as a routine aspect of acquisition, quarantine, and medical management of fish under human care.

Osteomyelitis in cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) hospitalized for rehabilitation: 25 cases (2008-2018).

OBJECTIVE: To characterize osteolytic lesions in cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) hospitalized for rehabilitation and describe methods used for the management of such lesions. ANIMALS: 25 stranded, cold-stunned Kemp's ridley sea turtles hospitalized between 2008 and 2018. PROCEDURES: Medical records of sea turtles with a diagnosis of osteolytic lesions were reviewed retrospectively to obtain the date of diagnosis, clinical signs, radiographic findings, microbial culture results, hematologic and plasma biochemical data, cytologic and histologic findings, antimicrobial history, time to first negative culture result, treatment duration, and outcome. RESULTS: Lesions were identified radiographically a median of 50 days after admission and were located within epiphyses or metaphyses of various appendicular joints. Lesions were associated with periarticular swelling (n = 24), lameness (16), lethargy (2), and hyporexia (2). Bacterial culture yielded growth of single organisms (n = 16), multiple organisms (2), or no growth (6). Significant differences in hematologic and biochemical data were detected between the times of diagnosis and convalescence. Cytologic and histologic findings characterized the lesions as osteomyelitis leading to septic arthritis. Sixteen sea turtles were managed medically, and 8 were managed medically and surgically. Surgery resulted in rapid improvement in joint mobility and overall clinical status. Most (22/25 [88%]) sea turtles survived and were released after long-term management. CONCLUSIONS AND CLINICAL RELEVANCE: During rehabilitation, cold-stunned Kemp's ridley sea turtles may be affected by osteomyelitis. Medical management based on antimicrobial susceptibility testing was effective for most turtles. Long term management efforts in turtles are justified by high survival rate.

Fibropapillomatosis and Chelonid Alphaherpesvirus 5 Infection in Kemp's Ridley Sea Turtles (Lepidochelys kempii).

Fibropapillomatosis (FP), a debilitating, infectious neoplastic disease, is rarely reported in endangered Kemp's ridley sea turtles (Lepidochelys kempii). With this study, we describe FP and the associated chelonid alphaherpesvirus 5 (ChHV5) in Kemp's ridley turtles encountered in the United States during 2006-2020. Analysis of 22 case reports of Kemp's ridley turtles with FP revealed that while the disease was mild in most cases, 54.5% were adult turtles, a reproductively valuable age class whose survival is a priority for population recovery. Of 51 blood samples from tumor-free turtles and 12 tumor samples from turtles with FP, 7.8% and 91.7%, respectively, tested positive for ChHV5 DNA via quantitative polymerase chain reaction (qPCR). Viral genome shotgun sequencing and phylogenetic analysis of six tumor samples show that ChHV5 sequences in Kemp's ridley turtles encountered in the Gulf of Mexico and northwestern Atlantic cluster with ChHV5 sequences identified in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles from Hawaii, the southwestern Atlantic Ocean, and the Caribbean. Results suggest an interspecific, spatiotemporal spread of FP among Kemp's ridley turtles in regions where the disease is enzootic. Although FP is currently uncommon in this species, it remains a health concern due to its uncertain pathogenesis and potential relationship with habitat degradation.

Evaluation of the Respiratory Microbiome and the Use of Tracheal Lavage as a Diagnostic Tool in Kemp's Ridley Sea Turtles (Lepidochelys kempii).

Respiratory disease is a common cause of morbidity and mortality in sea turtles, including the Kemp's ridley sea turtle (Lepidochelys kempii). Although culture-dependent methods are typically used to characterize microbes associated with pneumonia and to determine treatment, culture-independent methods can provide a deeper understanding of the respiratory microbial communities and lead to a more accurate diagnosis. In this study, we characterized the tracheal lavage microbiome from cold-stunned Kemp's ridley sea turtles at three time points during rehabilitation (intake, rehabilitation, and convalescence) by analyzing the 16S rRNA gene collected from tracheal lavage samples. We retrospectively developed a radiographic scoring system to grade the severity of lung abnormalities in these turtles and found no differences in diversity or composition of microbial communities based on radiographic score. We also found that the culture isolates from tracheal lavage samples, as well as other previously reported sea turtle pathogens, were present in variable abundance across sequenced samples. In addition to the tracheal microbial community of live turtles, we characterized microbial communities from other segments of the respiratory tract (glottis, trachea, anterior lung, posterior lung) from deceased turtles. We found a high degree of variability within turtles and a high degree of dissimilarity between different segments of the respiratory tract and the tracheal lavage collected from the same turtle. In summary, we found that the pulmonary microbial community associated with pneumonia in sea turtles is complex and does not correlate well with the microbial community as identified by tracheal lavage. These results underscore the limitations of using tracheal lavage for identification of the causative agents of pneumonia in sea turtles.

Characterization of oral and cloacal microbial communities in cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) during the time course of rehabilitation.

Microbial communities of animals play a role in health and disease, including immunocompromised conditions. In the northeastern United States, cold-stunning events often cause endangered Kemp's ridley turtles (Lepidochelys kempii) to become stranded on beaches in autumn. These sea turtles are admitted to rehabilitation facilities when rescued alive and are presumed immunocompromised secondary to hypothermia. To better understand the role that microbes play in the health of cold-stunned sea turtles, we characterized the oral and cloacal microbiome from Kemp's ridley turtles at multiple timepoints during rehabilitation, from admission to pre-release, by using Illumina sequencing to analyze the 16S rRNA gene. Microbial communities were distinct between body sites and among turtles that survived and those that died. We found that clinical parameters such as presence of pneumonia or values for various blood analytes did not correlate with oral or cloacal microbial community composition. We also investigated the effect of antibiotics on the microbiome during rehabilitation and prior to release and found that the type of antibiotic altered the microbial community composition, yet overall taxonomic diversity remained the same. The microbiome of cold-stunned Kemp's ridley turtles gradually changed through the course of rehabilitation with environment, antibiotics, and disease status all playing a role in those changes and ultimately the release status of the turtles.

Steatitis in Cold-Stunned Kemp's Ridley Sea Turtles (Lepidochelys kempii).

The pathogenesis of steatitis that infrequently occurs in cold-stunned Kemp's ridley sea turtles (KRT; Lepidochelys kempii) has been undetermined. The objectives of this study were to investigate the clinical (n = 23) and histologic findings (n = 11) in cold-stunned KRT, and to compare plasma concentrations of α-tocopherol (vitamin E), thiobarbituric acid reactive substances (TBARS), and the TBARS to vitamin E (T/E) ratio (an assessment of oxidative stress) between cold-stunned KRT with clinically and/or histologically confirmed steatitis (n = 10) and free-ranging KRT (n = 9). None of the cold-stunned turtles had clinically detectable steatitis at admission, and the median number of days to diagnosis of steatitis was 71 (range 33­469). Histologic findings of affected adipose tissue included heterophilic (n = 9) and/or histiocytic (n = 5) steatitis, fat necrosis (n = 7), myonecrosis (n = 2), and intralesional bacteria (n = 6). Cold-stunned KRT had significantly lower plasma vitamin E concentrations (median = 3.5 nmol/g), lower plasma TBARS concentrations (median = 1.6 nmol/g), and higher T/E ratios (median = 0.50), than controls (62.3 nmol/g; 2.1 nmol/g; 0.03, respectively). These results suggest a multifactorial etiology for the development of steatitis in KRT during rehabilitation, including tissue injury, septicemia, and various factors resulting in imbalances of anti-/oxidative status. By highlighting the need to provide more effective vitamin E supplementation, and the need to re-assess specific components of the diet, this study may lead to reduced incidence and improved medical management of steatitis in cold-stunned sea turtles.

Venous blood gas and biochemical analysis of wild captured green turtles (Chelonia mydas) and Kemp's ridley turtles (Lepidochelys kempii) from the Gulf of Mexico.

Blood was collected from wild captured green and Kemp's ridley turtles off the west coast of Florida, USA. Blood gases and biochemical values were analyzed using a point of care (POC) device in the field. Analytes include pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), total carbon dioxide (TCO2), bicarbonate (HCO3), base excess (BE), oxygen saturation (sO2), lactate, sodium (Na), potassium (K), chloride (Cl), total carbon dioxide (TCO2), anion gap, ionized calcium, glucose, blood urea nitrogen (BUN), creatinine (Crea), hematocrit (Hct), and hemoglobin (Hb). These are novel data for wild healthy Kemp's ridley turtles, and results for green turtles were generally consistent with past studies of green turtles with exceptions primarily in blood gas values. Ninety percent of the green turtles had fibropapillomatosis (FP), but none of the blood analytes were correlated with disease severity. Only BUN was correlated with weight of green turtles, and there was no correlation between blood parameters and weight of Kemp's ridley turtles. This study provides data that are useful in understanding the physiologic status of sea turtles specific to this region, allowing for comparisons to other populations, life stages, and disease states.

Herpesvirus Encephalitis in a Little Blue Penguin (Eudyptula minor).

An 11-day-old little blue penguin (Eudyptula minor) died unexpectedly. Prior to hatching, the egg experienced trauma and resultant defects were repaired. The chick hatched without complication and was clinically normal prior to death. Necropsy revealed congested lungs. Histologic examination showed moderate nonsuppurative encephalitis with focally extensive neuronal necrosis and intranuclear inclusions in neurons within necrotic foci. Herpesvirus DNA was detected in brain tissue with a generic herpesvirus polymerase chain reaction. Sanger sequencing demonstrated 100% and 98% sequence homology to sphenicid alphaherpesvirus 1 and penguin herpesvirus 2, respectively. In situ hybridization demonstrated large amounts of herpesvirus nucleic acid in intranuclear inclusions and neuronal nuclei. Combined histology, polymerase chain reaction, Sanger sequencing, and in situ hybridization results were most consistent with herpesviral encephalitis, most likely caused by sphenicid alphaherpesvirus 1. To our knowledge, this is the first report of a herpesvirus infection causing encephalitis in a penguin and the first report of herpesvirus in this species.

TEMPORAL STABILITY OF IN VITRO VENOUS BLOOD GAS, pH, AND LACTATE VALUES OF COWNOSE RAYS (RHINOPTERA BONASUS) AND RED-EARED SLIDER TURTLES (PSEUDEMYS SCRIPTA ELEGANS).

This study assessed the in vitro temporal changes that occur in blood pH and lactate concentrations for an elasmobranch species and a chelonian species, as well as blood gases (partial pressures of carbon dioxide [pCO2] and oxygen [pO2]) for a chelonian species, with a portable clinical point-of-care analyzer. Blood samples were collected from 10 cownose rays (Rhinoptera bonasus) and 10 red-eared sliders (Pseudemys scripta elegans), stored on ice, and serially analyzed at six time points up to 90 min postcollection. Results indicate that analysis should be conducted as soon as possible after blood collection for these species, with immediate analysis being preferred. However, if analysis must be delayed, syringes may be capped, placed on ice, and analyzed at a later time. Analysis within 90 min provided clinically acceptable results for pH and lactate in both species and for pCO2 in red-eared sliders, whereas substantial artifactual increases of pO2 were seen in red-eared sliders.

Plasma chemistry in nesting leatherback sea turtles (Dermochelys coriacea) from Florida: Understanding the importance of sample hemolysis effects on blood analytes.

Plasma chemistry is widely used in diagnostic and research settings in sea turtles. However, plasma discolorations such as hemolysis are often not considered in data interpretation. The objectives of this study were to (1) evaluate the effects of moderate hemolysis on plasma electrolytes, minerals, and proteins using dry chemistry analysis (DCA) and protein electrophoresis from nesting leatherback sea turtles (Dermochelys coriacea) from Florida and to (2) establish blood analyte reference intervals. Twenty-six plasma samples with absence of hemolysis were selected and sub-divided into one non-hemolytic aliquot and an aliquot that was experimentally manipulated to mimic moderate hemolysis. Plasma samples were analyzed for hemoglobin using a handheld photometer; sodium, potassium, chloride, magnesium, calcium, phosphorus, and total protein using DCA; and protein electrophoresis. Packed cell volume and hemoglobin were measured in corresponding whole blood samples. Reference intervals were established. All analytes except calcium and pre-albumin were significantly higher and the calcium:phosphorus and albumin:globulin ratios were significantly lower in hemolytic plasma compared to non-hemolytic plasma. Alpha2-globulins and potassium were the analytes most impacted by hemolysis, averaging 3.3- and 2.0-fold higher in hemolyzed samples, respectively, indicating that (1) hemoglobin migrates into the alpha2-globulin region in this species and (2) notable intracellular potassium is released into plasma with hemolysis. Attempted conversion factors for compensation of hemolysis were considered inaccurate for 4 of 16 analytes due to non-significant regression lines. We also report that PCV provides an estimate of hemoglobin (g/L) using the formula: (2.59 × PCV) + 24.59. Given the spurious effects of hemolysis, the degree of this artifact should be reported with biochemistry data, and samples with moderate to severe hemolysis should be excluded from datasets when interpreting electrolyte, mineral, and protein results. This will ensure accurate data interpretation for individual turtles in rehabilitation or research settings and population-level data relevant to conservation-focused projects.

Ameliorating transport-related stress in endangered Kemp's ridley sea turtles (Lepidochelys kempii) with a recovery period in saltwater pools.

Sea turtle rehabilitation clinics and aquaria frequently transport stranded sea turtles long distances out of water, e.g. for release at sites with appropriate water temperatures. Endangered Kemp's ridley turtles (Lepidochelys kempii) are known to exhibit an adrenal stress response during such transports. In an opportunistic study of turtles transported by road from Massachusetts to Georgia for release, we tested whether placing turtles in saltwater pools for short periods after transport would help turtles recover from transport-related stress. Eighteen juvenile Kemp's ridley turtles were examined and blood samples collected (1) immediately pre-transport, (2) immediately post-transport and (3) after a 6 h (n = 9) or 24 h (n = 9) post-transport period in unfamiliar pools, after which all turtles were released to the sea. Blood samples were analyzed for corticosterone, glucose, total white blood cell (WBC) count, heterophil/lymphocyte (H/L) ratio, pH, pO2, pCO2, HCO3 (bicarbonate), sodium, potassium, ionized calcium, lactate and hematocrit. Though the majority of turtles remained in good clinical condition, corticosterone, glucose, WBC and H/L elevated significantly during transport, while potassium declined slightly but significantly. After at least 6 h in a saltwater pool, potassium and glucose returned to pre-transport baselines and corticosterone partially recovered toward baseline. Extending the pool time to 24 h did not markedly enhance the physiological recovery of turtles, and two immune measures (WBC, H/L) remained elevated from the effect of transport. Six hours in a saltwater pool appears to facilitate the recovery of Kemp's ridley sea turtles from transport-related stress and may therefore improve their readiness for release.

Endocrine responses to diverse stressors of capture, entanglement and stranding in leatherback turtles (Dermochelys coriacea).

Leatherback turtles (Dermochelys coriacea) are exposed to many anthropogenic stressors, yet almost no data on stress physiology exist for this species. As a first step toward understanding the physiological responses of leatherback turtles to stress, and with the particular goal of assessment of the effect of capture, we quantified corticosterone (an adrenal stress hormone) and thyroxine (a regulator of metabolic rate, often inhibited by chronic stress) in 17 healthy leatherback turtles captured at sea for scientific study, with comparisons to 15 'distressed' leatherbacks that were found entangled in fishing gear (n = 8), confined in a weir net (n = 1) or stranded on shore (n = 6). Distressed leatherbacks had significantly elevated corticosterone (mean ± SEM 10.05 ± 1.72 ng/ml, median 8.38 ng/ml) and free thyroxine (mean 0.86 ± 0.37 pg/ml, median 0.08 pg/ml) compared with healthy leatherbacks sampled immediately before release (after ∼40 min of handling; corticosterone, mean 4.97 ± 0.62 ng/ml, median 5.21 ng/ml; and free thyroxine, mean 0.05 ± 0.05 pg/ml, median 0.00 pg/ml). The elevated thyroxine in distressed turtles compared with healthy turtles might indicate an energetic burden of entanglement and stranding. Six of the healthy leatherbacks were sampled twice, at ∼25 and ∼50 min after the time of first disturbance. In all six individuals, corticosterone was higher in the later sample (earlier sample, mean 2.74 ± 0.88 ng/ml, median 2.61 ng/ml; later sample, mean 5.43 ± 1.29 ng/ml, median 5.38 ng/ml), indicating that capture and handling elicit an adrenal stress response in this species. However, the corticosterone elevation after capture appeared relatively mild compared with the corticosterone concentrations of the entangled and stranded turtles. The findings suggest that capture and handling using the protocols described (e.g. capture duration <1 h) might represent only a mild stressor, whereas entanglement and stranding might represent moderate to severe stressors.

COCCIDIAL INFECTION OF THE ADRENAL GLANDS OF LEATHERBACK SEA TURTLES (DERMOCHELYS CORIACEA).

Histologic lesions incidental to the cause of death were observed in the adrenal glands of 17 subadult and adult leatherback sea turtles ( Dermochelys coriacea ) found dead or moribund on or near shore in North America. Round bodies, 250-300 µm in diameter composed of an outer capsule and large multinucleated cells surrounding a central mass of acellular material were distributed throughout the affected glands. Protozoal etiology was suspected based on some resemblance to coccidia; however, features diagnostic for coccidial infection were lacking in all but one case, which had a focal area of adrenalitis containing zoites. A novel eucoccidian partial 18S rRNA genetic sequence was consistently detected in adrenal glands with lesions. With the use of quantitative PCR, a specific area of the V4 region of the coccidian 18S gene was quantified in affected adrenal glands and correlated significantly with density of the histologic lesions. A second distinct, but closely related, 18S sequence was also amplified from the adrenal gland of one turtle and from a fecal sample containing unsporulated coccidian oocysts. The two 18S sequences identified from leatherback sea turtles form a clade within the family Eimeriidae. Further investigation is required to understand better the morphology of the life stages, life cycle, and potential effects of this coccidian parasite on adrenal function.