Phylogenetic Variation of Chelonid Alphaherpesvirus 5 (ChHV5) in Populations of Green Turtles Chelonia mydas along the Queensland Coast,Australia

Abstract

Sea turtle fibropapillomatosis (FP) is a disease marked by the proliferation of benign but debilitating cutaneous and occasional visceral tumors, likely to be caused by chelonid alphaherpesvirus 5 (ChHV5). This study presents a phylogeny of ChHV5 strains found on the east coast of Queensland, Australia, and a validation for previously unused primers. Two different primer sets (gB-1534 and gB-813) were designed to target a region including part of the UL27 glycoprotein B (gB) gene and part of UL28 of ChHV5. Sequences obtained from FP tumors found on juvenile green turtles Chelonia mydas (<65 cm curved carapace length) had substantial homology with published ChHV5 sequences, while a skin biopsy from a turtle without FP failed to react in the PCRs used in this study. The resulting sequences were used to generate a neighbor-joining tree from which three clusters of ChHV5 from Australian waters were identified: north Australian, north Queensland, and Queensland clusters. The clusters reflect the collection sites on the east coast of Queensland with a definitive north–south trend.

Received October 22, 2016; accepted May 7, 2017 Published online July 26, 2017     

First Record of Fibropapillomatosis in a Green Turtle Chelonia mydas from the Baja California Peninsula

Fibropapillomatosis (FP) is characterized by multiple fibroepithelial tumors in all parts of the skin and has been reported in sea turtles worldwide. Clinically infected individuals are often emaciated and anemic. In Mexico, however, there are few records of this disease. In this study of green turtles Chelonia mydas in Laguna San Ignacio in Baja California Sur (BCS), we noted one juvenile with multifocal fibropapilloma lesions on the external upper surface of its eyes and hind flippers. Light microscopy revealed hyperkeratosis, epidermal hyperplasia, dermal papillary projections, and fibroblast proliferation. Electron microscopy revealed viral particles. Biopsies of normal skin were done to determine the origin of the turtle through genetic analysis. Its mitochondrial DNA matched that of a haplotype (CMP2) from a Hawaiian green turtle population. Finding FP in a turtle captured in BCS elucidates the need for further monitoring along the west coast of Mexico. Further investigation should include testing tumors to detect and characterize any chelonid herpesviruses and explore any association with FP and other diseases that pose a health risk to other sea turtle species.

Received March 26, 2016; accepted August 3, 2016 Published online October 27, 2016     

Histology and Immunohistochemistry of the Cardiac Ventricular Structure in the Green Turtle (Chelonia mydas)

This study describes the implications of cardiac ventricular microscopy in Chelonia mydas relating to its ability to dive. For this work, 11 specimens of the marine turtle species C. mydas found dead on the coast of Rio Grande do Norte (Northeast Brazil) were used. After necropsy, fragments of the cardiac ventricular wall were fixed in 10% buffered formaldehyde solution for 24 h and then subjected to routine processing for light and scanning electron microscopy (SEM). The ventricle in this species is formed by the epicardium, myocardium and endocardium. The subepicardial layer consists of highly vascularised connective tissue that emits septa to reinforce the myocardium surface. There is an abundant and diffuse subepicardial nerve plexus shown by immunostaining technique. The thickness of the spongy myocardium and the nature of its trabeculae varied between the heart chambers. The endocardium shows no characteristic elements of the heart conduction system. The valves have a hyaline cartilage skeleton, coated by dense irregular connective tissues characterised by elastic fibres. These findings in the green turtle ventricular microscopy are related to hypoxia resistance during diving.     

Pharmacokinetics of ceftriaxone in Green sea turtles (Chelonia mydas) following intravenous and intramuscular administration at two dosages

Green sea turtles are widely distributed in tropical and subtropical waters. Adult green sea turtles face many threats, primarily from humans, including injuries from boat propellers, being caught in fishing nets, pollution, poaching, and infectious diseases. To the best of our knowledge, limited pharmacokinetic information to establish suitable therapeutic plans is available for green sea turtles. Therefore, the present study aimed to describe the pharmacokinetic characteristics of ceftriaxone (CEF) in green sea turtles, Chelonia mydas, following single intravenous and intramuscular administrations at two dosages of 10 and 25 mg/kg body weight (b.w.). Blood samples were collected at assigned times up to 96 hr. The plasma concentrations of CEF were measured by liquid chromatography tandem mass spectrometry. The concentrations of CEF in the plasma were quantified up to 24 and 48 hr after i.v. and i.m. administrations at dosages of 10 and 25 mg/kg b.w., respectively. The C_max values of CEF were 15.43 ± 3.71 μg/ml and 43.48 ± 4.29 μg/ml at dosages of 10 and 25 mg/kg, respectively. The AUC_last values increased in a dose‐dependent fashion. The half‐life values were 2.89 ± 0.41 hr and 5.96 ± 0.26 hr at dosages of 10 and 25 mg/kg b.w, respectively. The absolute i.m. bioavailability was 67% and 108%, and the binding percentage of CEF to plasma protein was ranged from 20% to 29% with an average of 24.6%. Based on the pharmacokinetic data, susceptibility break‐point and PK‐PD index (T > MIC, 0.2 μg/ml), i.m. administration of CEF at a dosage of 10 mg/kg b.w. might be appropriate for initiating treatment of susceptible bacterial infections in green sea turtles.     

Pharmacokinetics of marbofloxacin in Green sea turtles (Chelonia mydas) following intravenous and intramuscular administration at two dosage rates

Limited pharmacokinetic information to establish suitable therapeutic plans is available for green sea turtles. Therefore, the present study was conducted to evaluate the pharmacokinetic characteristics of marbofloxacin (MBF) in the green sea turtle, Chelonia mydas, following single intravenous (i.v.) or intramuscular (i.m.) administration at two dosages of 2 and 4 mg/kg body weight (b.w.). Blood samples were collected at assigned times up to 168 hr. MBF in plasma was extracted using liquid–liquid extraction and analyzed by a validated high-performance liquid chromatography (HPLC). MBF was quantifiable from 15 min to 96 hr after i.v. and i.m. administrations at two dose rates. A noncompartmental model was used to fit the plasma concentration of MBF versus time curve for each green sea turtle. The t_1/2λz value, similar for both the dosages (22–28 hr), indicated that the overall rate of elimination of MBF in green sea turtles is relatively slow. The average i.m. F% ranged 88%–103%. MBF is a concentration-dependent drug and the AUC/MIC ratio is the best PK/PD predictor for its efficacy. The MBF dosage of 4 mg/kg appeared to produce an appropriate value of the PK-PD surrogate that predicts antibacterial success for disease caused by susceptible bacteria. In contrast, i.m. administration of MBF at a dosage of 2 mg/kg b.w. was not found to produce a suitable PK-PD surrogate index. However, further studies of multiple doses and plasma binding proteins are warranted to confirm an appropriate dosage regimen.     

Observations of fibropapillomatosis in green turtles (Chelonia mydas) in Indonesia

Objective To determine the prevalence and manifestations of fibropapillomatosis in green turtles in Indonesia, to identify any relationship between fibropapillomatosis and concurrent parasitic infection, to ascertain the effect of fibropapil-lomatosis on health, and to examine whether environment might have an effect on the prevalence of fibropapillomatosis.
Procedure 4407 green turtles ( Chelonia mydas ) and 401 hawksbill turtles ( Eretmochelys imbricata ) were examined. The occurrence of fibropapillomatosis was correlated with sex, maturity, curved carapace length, body weight/curved carapace length ratio, the number and distribution of tumours on the skin, parasite burdens, some haematological variables and the region of capture.
Results Fibropapillomatosis was seen only in green turtles, and the overall prevalence in these was 21.5%. This prevalence increased with the curved carapace length up to 85 cm. The average number of tumours per affected turtle was 5 SD 4.1 (range, 1 to 29), and was negatively correlated with the body weight/curved carapace length ratio (rs = -0.8; P = 0.001). The red blood cell count in turtles with fibropapilloma was lower than in non-fibropapilloma turtles captured and examined at the same time (P = 0.001). The prevalence of fibropapilloma in turtles captured near densely populated, industrial regions (26.3%) was greater than in turtles from sparsely populated areas (17.7%).
Conclusion Fibropapillomatosis in green sea turtles in Indonesia is of moderate occurrence; young mature turtles (curved carapace length = 85 cm) are most frequently affected. Fibropapilloma adversely affects health of turtles. Fluke infestation seems not to be a causal factor, but viral infection, perhaps with concurrent stress of environmental origin, seems likely.     

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund's complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.     

Gram-negative bacterial infections and cardiovascular parasitism in green sea turtles (Chelonia mydas)

Objective To investigate causes of ill health and mortality in juvenile wild green sea turtles ( Chelonia mydas ) found along the mid-north west coast of Western Australia between June and October of 1997.
Procedure Department of Conservation and Land Management rangers submitted four dead or dying green sea turtles from separate incidents for veterinary examination, necropsy, and bacteriological, parasitological and histopatho-logical examination.
Results Numerous different species of trematodes belonging to the families Pronocephalidae, Microscaphidiidae and Paramphistomidae were detected in the intestines of two turtles examined, and in all turtles there was severe spirorchid fluke infection including Haemoxenicon sp, Amphiorchis sp and Hapalotrema sp. Histopathological examination demonstrated severe multifocal to diffuse granulomatous vasculitis, aggregations of spirorchid fluke eggs and microabscesses throughout various tissues including intestines, kidney, liver, lung and brain. Cultures and or histopathological examination demonstrated disseminated Gram-negative bacterial infections including salmonella, E coli , Citrobacter freundii and Moraxella sp.
Conclusion Infections caused by salmonellae, E coli and other Gram-negative bacteria should be considered as causes of systemic illness and death in wild green sea turtles infected with spirorchid cardiovascular flukes and other internal parasites.     

Surgical repair of a depressed fracture in a green sea turtle, Chelonia mydas

Sea turtles are considered to be endangered species. A depressed fracture of a 35 kg green sea turtle was treated surgically. Isoflurane was used for induction and maintenance of anesthesia. Slow induction of and slow recovery from anesthesia was remarkable. After the operation, there was an improvement of general status, but head tilt and weakness of the left limbs persisted. As the turtle did not eat, force feeding using stomach tube was performed. The turtle died at about 6 months after the surgery.     

Intestinal volvulus and stricture associated with a leiomyoma in a green turtle (Chelonia mydas).

A previously stranded 30-kg female green turtle (Chelonia mydas) was referred to the Veterinary Medical Teaching Hospital at the University of Florida following a 2-mo history of anorexia, intermittent regurgitation, decreased fecal production, and positive buoyancy of the right side. Radiographs confirmed gaseous distension of bowel loops suggestive of intestinal obstruction. The coelom was surgically approached through a plastron osteotomy, and a 540 degrees volvulus of the small intestine was identified and derotated. An intestinal stricture was present at the site of the volvulus, and segments of bowel orad to the stricture were greatly distended. Following resection of abnormal bowel, the osteotomy was stabilized using self-tapping screws and figure-eight wire, and the defect was sealed with fiberglass cloth and fast-drying epoxy resin. A leiomyoma associated with the focal stricture was identified by histology. Appetite and defecation returned to normal. Six months after surgery, the turtle had regained normal buoyancy and showed no further clinical signs of gastrointestinal obstruction.     

Pharmacokinetics of tolfenamic acid in green sea turtles (Chelonia mydas) after intravenous and intramuscular administration

The present study aimed to evaluate the pharmacokinetic features of tolfenamic acid (TA) in green sea turtles, Chelonia mydas. Green sea turtles were administered single either intravenous (i.v.) or intramuscular (i.m.) injection of TA, at a dose of 4 mg/kg body weight (b.w.). Blood samples were collected at preassigned times up to 168 hr. The plasma concentrations of TA were measured using a validated liquid chromatography tandem mass spectrometry method. Tolfenamic acid plasma concentrations were quantifiable for up to 168 hr after i.v. and i.m. administration. The concentration of TA in the experimental green sea turtles with respect to time was pharmacokinetically analyzed using a noncompartment model. The C_max values of TA were 55.01 ± 8.34 µg/ml following i.m. administration. The elimination half-life values were 32.76 ± 4.68 hr and 53.69 ± 3.38 hr after i.v. and i.m. administration, respectively. The absolute i.m. bioavailability was 72.02 ± 10.23%, and the average binding percentage of TA to plasma protein was 19.43 ± 6.75%. Based on the pharmacokinetic data, the i.m. administration of TA at a dosage of 4 mg/kg b.w. might be sufficient to produce a long-lasting anti-inflammatory effect (7 days) for green sea turtles. However, further studies are needed to determine the clinical efficacy of TA for treatment of inflammatory disease after single and multiple dosages.     

全球海龟资源和保护概况

世界现存海龟仅2科6属7种,都属国际濒危物种。生物界很难找到像海龟—类受到众多世人强烈关注的动物了,国际上亦十分重视海龟的保护工作,不仅专门成立了海龟专家小组(MTSG),制定了许多国际公约,定期出版《海龟通讯》(MTN)等期刊,还成立了一些影响力广泛的国际组织,并经常召开一些与海龟保护相关的国际会议,掀起了全球范围的拯救海龟大行动。本研究概述了海龟资源现状、致危因素、国际保护措施及成果。近年来全球海龟资源总量呈直线下降趋势,海龟面临的主要生存威胁有:渔业副获、直接捕食、沿海经济发展、环境污染与疾病、全球变暖等。最后,简要介绍了海龟重要的经济、科研、观赏、文化和生态价值,并呼吁全人类共同保护海龟。     

Comparative pathology and pathogenesis of spontaneous and experimentally induced fibropapillomas of green turtles (Chelonia mydas)

Tumor biopsy samples from 25 Floridian and 15 Hawaiian green turtles (Chelonia mydas) with spontaneous green turtle fibropapillomatosis (GTFP) and from 27 captive-reared green turtles with experimentally induced GTFP were examined microscopically to differentiate the histologic features that result from GTFP pathogenesis and those that result from incidental factors that may vary according to geographic region. Common histologic features for spontaneous and experimentally induced tumors included fibroblast proliferation in the superficial dermis, epidermal acanthosis and hyperkeratosis, epidermal basal cell degeneration with dermal-epidermal cleft formation, spinous layer degeneration with intraepidermal vesicle and pustule formation, and ulceration. Visceral tumors, found in eight of 10 (80%) free-ranging turtles with cutaneous disease that were examined after death, had extensive interstitial fibrous proliferation. The presence of spirorchid trematode eggs and associated foreign body granulomas, common secondary findings within spontaneous tumors, varied by geographic location, and these findings were not observed in experimentally induced tumors. Eosinophilic intranuclear inclusions and intranuclear herpesvirus-associated antigen immunoreactivity were found in 18 of 38 (47%) experimentally induced cutaneous tumors and nine of 119 (7.5%) spontaneous tumors from Floridian but not Hawaiian turtles. The possible involvement of GTFP-associated herpesvirus in the pathogenesis of epidermal degenerative changes and GTFP pathogenesis is discussed.     

The use of echocardiography as a health assessment tool in green sea turtles (Chelonia mydas)

There are limited techniques available to assess the health of sea turtles as physical examination has little correlation to clinical findings, and blood reference intervals are broad and provide limited prognostic significance. Advances in the portability of ultrasound machines allow echocardiography to be increasingly used in the health assessments of wild animals. This study performed blood analysis and echocardiograms on 11 green sea turtles upon admission to a rehabilitation clinic and six animals before release. Significant differences were seen between groups, with admission animals having significantly smaller diameters of the cavum arteriosum at systole and diastole, smaller E‐waves and an increased fractional shortening. Pre‐release animals displayed significant increases in the maximum blood velocities of both the pulmonary artery and the left aorta. Significant negative correlations were seen between fractional shortening and uric acid and between the velocity time integral of the pulmonary artery and urea. The pulmonary artery velocity time integral was also significantly correlated to the E wave. Furthermore, there was asynchrony between the cavum arteriosum and the cavum pulmonale and the detection of a parasitic granuloma in the ventricular outflow tract of one animal. Overall, the results suggest that cardiac function in stranded green sea turtles is significantly impaired and that echocardiography has applications in the health assessments of green sea turtles.     

Spirorchiidiosis (Digenea: Spirorchiidae) and lesions associated with parasites in Caribbean green turtles (Chelonia mydas)

Forty-seven nesting green turtles (Chelonia mydas) were examined for parasitic pathogens. Four species of cardiovascular flukes (Digenea: Spirorchiidae), Learedius learedi, Hapalotrema postorchis, Monticellius indicum and Amphiorchis solus were collected from 39 of 40 of the turtles, and Neospirorchis species were identified in seven of the 47 by histological examination. The pathological changes associated with the spirorchiids and their eggs included aneurysms, arteritis of great vessels with dark nodular thickenings, endocarditis, haemorrhagic lesions, thrombi, vasculitis, and granulomatous reactions in all the turtles. Ozobranchus branchiatus (Hirudinea: Ozobranchidae) leeches were found on the skin of 27 of the 47 turtles and were associated with traumatic purulent and ulcerative dermatitis. Oesophagitis associated with Rameshwarotrema uterocrescens (Digenea: Pronocephalidae) was recorded in 11 of 43 of the turtles. Petechial haemorrhages and unspecific crater-like ulcerated lesions on the gastric mucosa associated with Charaxicephaloides species and Charaxicephalus robustus (Digenea: Pronocephalidae) were observed in four of 40 of the turtles. Cholycystitis and ectasia of mucosal glands associated with Rhytidodoides similis (Digenea: Rhytidodidae) were observed in one of the 47 turtles. Fibropapillomas were observed on the skin of one turtle and fibromas on the skin of two of them.     

Nasal Shedding of Equid Herpesvirus Type 1 and Type 4 in Hospitalized,Febrile Horses

The objective of this study was to determine the prevalence of shedding of equid herpesvirus 1 (EHV-1) or EHV-4 in nasal swab samples from any febrile, hospitalized horses during a 1-year period. It was hypothesized that some fevers in horses are associated with viral replication following recrudescence of latent virus or following a horizontal viral infection prior to or during admission to a referral hospital. During the observational period, nasal swab samples were collected from 64 febrile and 10 nonfebrile hospitalized horses. Routine DNA extraction was performed, and a validated quantitative polymerase chain reaction (qPCR) assay was used to detect and quantify genomic EHV-1 and -4 DNA. Genomic DNA of EHV-4 was detected in the nasal swab specimen of 1 of 64 febrile horses. EHV-1 DNA was not detected in any of the febrile horses. Samples from all nonfebrile horses were negative for both viruses. Considering the known association between fever and shedding of EHV-1 and EHV-4, we anticipated finding a higher percentage of PCR-positive samples from febrile patients. Fevers detected were likely a result of active disease processes for which the horses were hospitalized; concurrent other diseases appeared not to affect viral recrudescence. Further studies are warranted to examine frequency and factors of EHV latency and reactivation.     

Detection of Salmonella enterica Serovar Montevideo and Newport in Free‐ranging Sea Turtles and Beach Sand in the Caribbean and Persistence in Sand and Seawater Microcosms

Salmonellae are Gram‐negative zoonotic bacteria that are frequently part of the normal reptilian gastrointestinal flora. The main objective of this project was to estimate the prevalence of non‐typhoidal Salmonella enterica in the nesting and foraging populations of sea turtles on St. Kitts and in sand from known nesting beaches. Results suggest a higher prevalence of Salmonella in nesting leatherback sea turtles compared with foraging green and hawksbill sea turtles. Salmonella was cultured from 2/9 and identified by molecular diagnostic methods in 3/9 leatherback sea turtle samples. Salmonella DNA was detected in one hawksbill turtle, but viable isolates were not recovered from any hawksbill sea turtles. No Salmonella was detected in green sea turtles. In samples collected from nesting beaches, Salmonella was only recovered from a single dry sand sample. All recovered isolates were positive for the wzx gene, consistent with the O:7 serogroup. Further serotyping characterized serovars Montevideo and Newport present in cloacal and sand samples. Repetitive‐element palindromic PCR (rep‐PCR) fingerprint analysis and pulsed‐field gel electrophoresis of the 2014 isolates from turtles and sand as well as archived Salmonella isolates recovered from leatherback sea turtles in 2012 and 2013, identified two distinct genotypes and four different pulsotypes, respectively. The genotyping and serotyping were directly correlated. To determine the persistence of representative strains of each serotype/genotype in these environments, laboratory‐controlled microcosm studies were performed in water and sand (dry and wet) incubated at 25 or 35°C. Isolates persisted for at least 32 days in most microcosms, although there were significant decreases in culturable bacteria in several microcosms, with the greatest reduction in dry sand incubated at 35°C. This information provides a better understanding of the epizootiology of Salmonella in free‐ranging marine reptiles and the potential public health risks associated with human interactions with these animals in the Caribbean.     

Proliferative dermatitis in a loggerhead turtle, Caretta caretta, and a green turtle, Chelonia mydas, associated with novel papillomaviruses

A subadult loggerhead turtle, Caretta caretta, presented with generalized small, white, raised lesions over its neck, shoulders, and all four flippers. A juvenile green turtle, Chelonia mydas, recently treated for fibropapillomatosis, presented with four similar localized lesions on one flipper. To diagnose the conditions, biopsies of the lesions were taken for histopathology, electron microscopy, and molecular diagnostics. Histopathologic findings were similar in the two turtles and skin lesions were characterized by multifocal areas of epidermal hyperplasia accompanied by variation and abnormalities in the nuclear morphology of keratinocytes and a few intranuclear inclusions in some cells. Transmission electron microscopy revealed multiple epithelial cells with large intranuclear aggregates of virions consistent in morphology with papillomavirus. Papillomavirus was detected in samples from both turtles by polymerase chain reaction (PCR). Sequence analysis of the partial sequence of the papillomavirus E1 gene revealed two viruses (CcPV and CmPV) that were distinct from each other and from other species in Papillomaviridae, and likely represent two novel species and perhaps a new genus.