Efficacy of praziquantel in the treatment of green sea turtles with spontaneous infection of cardiovascular flukes

Objective To assess the efficacy of praziquantel as a treatment for cardiovascular flukes in turtles.
Procedure Six green sea turtles ( Chelonia mydas ) spontaneously infected with cardiovascular flukes (Digenea: Spirorchiidae) were treated orally with praziquantel, and necropsied 3 or 7 days later to look for flukes in the heart and major blood vessels. Six similar animals were maintained as untreated controls.
Results Absence of flukes in treated, but not control turtles, indicated that a one day course of treatment at a dose rate of 3 times 50 mg/kg body weight is effective.
Conclusion This result should be of value for preventing disease in wild caught green turtles introduced into farms or aquaria.     

Aerobic bacterial flora of nesting green turtles (Chelonia mydas) from Tortuguero National Park, Costa Rica.

Bacteriological examination of 70 nesting green turtles (Chelonia mydas) from Tortuguero National Park, Costa Rica was performed to investigate nasal and cloacal aerobic bacteria. A total of 325 bacterial isolates were obtained, including 10 Gram-negative and three Gram-positive genera. Two hundred thirty-nine were Gram-negative and 86 were Gram-positive isolates. Klebsiella pneumoniae was the most common microbe identified in turtle samples: 27/70 (38.5%) in cloacal, and 33/70 (47.1%) in nasal samples. The Enterobacteriaceae family, including Enterobacter agglomerans, E. cloacae, Escherichia coli, Klebsiella oxytoca, K. pneumoniae, and Serratia marcescens, was the largest Gram-negative group of bacteria recovered and comprised 127 of 239 (53.1%) of the Gram-negative isolates. Staphylococcus species was the largest Gram-positive bacteria group, including S. aureus, S. cromogenes, S. epidermis, and S. intermedius, and made up 63 of 86 (73.2%) of the Gram-positive isolates recovered. The results of this study demonstrate that the aerobic bacterial flora of nesting green turtles at Tortuguero National Park is composed of a very wide spectrum of bacteria, including several potential pathogens.     

Mycotic pneumonia in mariculture-reared green sea turtles

Microbiologic examination of 29 juvenile green sea turtles with a buoyancy abnormality revealed pulmonary infection with Sporotrichium sp, Cladosporium sp, and Paecilomyces sp. Histologic examination of the lungs revealed granulomatous lesions containing branching septate hyphae. The diagnosis was mycotic pneumonia.     

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.     

Two herpesviruses associated with disease in wild Atlantic loggerhead sea turtles (Caretta caretta)

Herpesviruses are associated with lung-eye-trachea disease and gray patch disease in maricultured green turtles (Chelonia mydas) and with fibropapillomatosis in wild sea turtles of several species. With the exception fibropapillomatosis, no other diseases of wild sea turtles of any species have been associated with herpesviral infection. In the present study, six necropsied Atlantic loggerhead sea turtles (Caretta caretta) had gross and histological evidence of viral infection, including oral, respiratory, cutaneous, and genital lesions characterized by necrosis, ulceration, syncytial cell formation, and intranuclear inclusion bodies. Nested polymerase chain reaction targeting a conserved region of the herpesvirus DNA-dependent-DNA polymerase gene yielded two unique herpesviral sequences referred to as loggerhead genital-respiratory herpesvirus and loggerhead orocutaneous herpesvirus. Phylogenetic analyses indicate that these viruses are related to and are monophyletic with other chelonian herpesviruses within the subfamily alpha-herpesvirinae. We propose the genus Chelonivirus for this monophyletic group of chelonian herpesviruses.     

Evaluation of hematology and serum biochemistry of cold-stunned green sea turtles (Chelonia mydas) in North Carolina, U.S.A

Hypothermia or cold-stunning is a condition in which the body temperature of an animal decreases below normal physiologic range and which has been linked to severe morbidity in sea turtles. Reports have focused on the physiologic changes caused by cold-stunning in Kemp's Ridley sea turtles (Lepidochelys kempii) and loggerhead sea turtles (Caretta caretta), but few have evaluated the green sea turtle (Chelonia mydas). This study evaluated hematologic and serum biochemical profiles of cold-stunned green sea turtles in North Carolina, USA. When compared with healthy, free-ranging juvenile green turtles from the same region, cold-stunned turtles exhibited hypoglycemia, hypocalcemia (both total and ionized calcium), hyponatremia, hypokalemia, hypoproteinemia, hypoalbuminemia, hyperphosphatemia, and elevations in uric acid and blood urea nitrogen. These findings contrast with some previously reported changes in cold-stunned Kemp's Ridley and loggerhead sea turtles. These results emphasize the importance of basing therapeutic regimens on biochemical analyses in cold-stunned sea turtles.     

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.     

Evaluation of magnetic resonance imaging for detection of internal tumors in green turtles with cutaneous fibropapillomatosis

OBJECTIVE: To describe the gross cross-sectional anatomy of green turtles (Chelonia mydas) and evaluate magnetic resonance imaging (MRI) for detection of internal tumors in green turtles with cutaneous fibropapillomatosis. DESIGN: Prospective study. ANIMALS: 3 dead green turtles, 1 healthy green turtle, and 8 green turtles with cutaneous fibropapillomatosis. PROCEDURES: Gross cross-sectional anatomy of a dead turtle was described. Each live turtle underwent a complete physical examination, and dorsoventral whole-body survey radiographic views were obtained. Magnetic resonance imaging was performed in dorsal and transverse planes. Radiographs and magnetic resonance images were examined for evidence of internal nodules. Results were compared with necropsy findings in 5 of 8 turtles. RESULTS: Nodules in the lungs of 2 turtles were detected via radiography, whereas pulmonary nodules were detected in 5 turtles via MRI. No other visceral nodules were detected via radiography; however, masses in the stomach and adjacent to the bladder and kidneys were detected in 1 turtle via MRI. Other extrapulmonary abnormalities observed at necropsy were not detected on MR images. CONCLUSIONS AND CLINICAL RELEVANCE: MRI may be valuable for detection of internal tumors in green turtles with cutaneous fibropapillomatosis. Nodules were more apparent in the lungs than in other organs. Results of MRI may serve as prognostic indicators for sea turtles undergoing assessment, treatment, and rehabilitation. Clinical application may be limited by cost and availability of MRI technology.     

Chronic shoulder osteoarthritis in a loggerhead turtle (Caretta caretta)

This report describes the lesions seen in an extremely weak and emaciated adult male loggerhead turtle (Caretta caretta) found stranded near Perth, Western Australia. Blood collected from a dorsal cervical sinus immediately prior to euthanasia demonstrated severe anaemia, markedly elevated plasma concentrations of creatinine kinase (2263 U/L), alkaline phosphatase (58 U/L), urea (18.1 mmol/L) and hypoglycaemia (glucose 0.7 mmol/L). Necropsy examination demonstrated a severe chronic osteoarthritis of the right shoulder joint with marked remodelling of the glenoid fossa, coracoid, scapula and humerus. There was marked synovial effusion and periarticular fibrosis. Cytological examination and culture of synovial fluid failed to demonstrate an infectious agent. Other findings were low numbers of trypanorhynch cysts present on the surface of the liver and numerous spirorchid eggs in the attached mesentery. Several large and small spirorchid flukes were present in the heart and there was a localised endarteritis in the left aortic arch associated with this infection. Numerous nematode eggs and at least three species of spirorchid eggs were seen microscopically in faeces collected from the terminal colon. The pathogenesis and significance of these lesions is discussed.     

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.     

Cryptosporidium sp.-associated enteritis without gastritis in rough green snakes (Opheodrys aestivus) and a common garter snake (Thamnophis sirtalis).

An epizootic of severe Cryptosporidium sp.-associated enteritis occurred in a group of 15 wild-caught juvenile rough green snakes (Opheodrys aestivus) at the Baltimore Zoo quarantine facility. All of the animals died with no premonitory signs. Histopathologic examination of the small and proximal large intestine of eight of the green snakes showed moderate to severe Cryptosporidium sp. infection and enteritis characterized by dense heterophilic and lymphocytic inflammatory infiltrates throughout the lamina propria with epithelial necrosis. Cryptosporidium sp. was also found in feces of an adult common garter snake (Thamnophis sirtalis) that was wild caught on zoo grounds and held in quarantine during the epizootic. After euthanasia, histologic examination of the garter snake showed a severe small intestinal Cryptosporidium sp. infection with only mild enteritis consisting of sparse heterophilic and lymphocytic infiltrates. There was no gross or histologic evidence of Cryptosporidium sp. gastritis in the nine snakes evaluated, and this is the first report of Cryptosporidium sp.-associated enteritis in snakes without gastric lesions.     

Conjunctivitis, tracheitis, and pneumonia associated with herpesvirus infection in green sea turtles

Fourteen juvenile (15- to 20-month-old) green sea turtles (Chelonia mydas), representative of a group of sea turtles with clinical signs of respiratory tract disease, were euthanatized and submitted for necropsy. Macroscopically, lesions included periglottal necrosis, tracheitis with intraluminal caseous and laminated necrotic debris, and severe pneumonia. Several turtles had caseous conjunctival exudate covering the eyes. Microscopically, the turtles had fibrinonecrotic inflammation around the glottal opening, tracheitis, and severe bronchopneumonia and interstitial pneumonia. In multifocal areas, periglottal and tracheal epithelial cells adjacent to areas of necrosis had hypertrophic nuclei with amphophilic intranuclear inclusions. A mixed population of primarily gram-negative microorganisms was isolated from the tracheal and glottal lesions. Attempts at viral isolation in cultures of green sea turtle kidney cells resulted in the development of cytopathic effects characterized by giant cell formation and development of intranuclear inclusions. Using electron microscopy, intranuclear viral particles (88 to 99 nm in diameter) were seen in inclusion-containing tracheal and glottal epithelial cells and infected green sea turtle kidney cells; particles were consistently seen enveloping from nuclear membranes, and mature particles (132 to 147 nm) were found in the cytoplasm. On the basis of size, conformation, location, and presence of an envelope, the particles most closely resembled those of herpes-viruses.     

Diseases of the respiratory tract of chelonians.

Diseases of the respiratory tract commonly occur in captive chelonians, and several diseases also have occurred in wild chelonians. Infectious causes include viruses, bacteria, fungi, and parasites. Herpesviruses have surfaced as important pathogens of the oral cavity and respiratory tract in Hermann's tortoise (Testudo hermanii), spur-thighed tortoise (Testudo graeca), and other tortoises in Europe and the United States. Herpesvirus-associated respiratory diseases also have been reported in the green turtle, Chelonia mydas, in mariculture in the Cayman Islands. Of diseases caused by bacteria, an upper respiratory tract disease caused by Mycoplasma sp has been reported in free-hanging and captive gopher tortoises in the southeastern United States and in desert tortoises in the Mojave Desert of the southwestern United States. Mycotic pulmonary disease is commonly reported in captive chelonians, especially in those maintained at suboptimal temperatures. An intranuclear coccidia has been seen in several species of captive tortoises in the United States, and, in one case, a severe proliferative pneumonia was associated with organisms in the lung. The most common noninfectious cause of respiratory disease in chelonians results from trauma to the carapace. Although pulmonary fibromas commonly occur in green turtles with fibropapillomatosis, for the most part, tumors of the respiratory tract are uncommon in chelonians.     

Digestive pathology of sea turtles stranded in the Canary Islands between 1993 and 2001

Digestive lesions were observed in 84 of 136 sea turtles (128 Caretta caretta, four Chelonia mydas and four Dermochelys coriacea) stranded in the Canary Islands between January 1993 and December 2001. In the oral cavity ulcerative and necropurulent stomatitis were the most frequently observed lesions, and in the oesophagus ulcerative and fibrinous oesophagitis, and traumatic oesophageal perforation were most frequently observed; all these lesions were mainly associated with the ingestion of fishing hooks. Different histological types of gastritis were observed in 35 of the turtles; necropurulent and fibrinous gastritis were associated with bacterial infections caused mainly by Proteus species, Vibrio alginolyticus, and Staphylococcus species, and larval nematodes of the genus Anisakis were responsible for a form of parasitic gastritis observed in 16 of the turtles. Different histological types of enteritis, including catarrhal, fibrinous, necropurulent and necrotising enteritis, affected 36 turtles; a wide range of gram-negative and gram-positive bacteria, including Bacillus species, Escherichia coli, Pasteurella species, Proteus species, Staphylococcus species, Streptococcus species and V. alginolyticus, were isolated from these lesions. All the cases of necrotising enteritis were associated with intestinal intussusception caused by the ingestion of monofilament fishing lines. Necrotising and/or multifocal granulomatous hepatitis were the lesions most commonly observed in the liver; they affected 29 of the turtles and were associated with Aeromonas hydrophila, Citrobacter species, E. coli, Proteus species, Staphylococcus species and V. alginolyticus infections. According to the stranding reports and the gross and histological lesions observed, 33 of the turtles had digestive lesions associated with the ingestion of hooks and monofilament lines, and two had lesions associated with the ingestion of crude oil.     

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

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

Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta)

A morphologic classification based on the cytochemical characteristics of blood cells of 35 juvenile loggerhead sea turtles (Caretta caretta) is described. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff, and toluidine blue. The morphologic characteristics of erythrocytes were similar to those reported in green turtles. Six types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes, monocytes and thrombocytes. Except for the basophils, the rest of the white blood cells from loggerhead turtles had different cytochemical characteristics compared to blood cells from other sea turtle species. The leukocyte differential count was different from that reported for other sea turtle species. Heterophils were the most numerous leukocytes from these loggerhead turtles, followed by lymphocytes, eosinophils, monocytes and basophils. This paper provides a morphologic classification of blood cells of loggerhead sea turtles that is useful for veterinary surgeons involved in sea turtle conservation.     

Altered in vitro immune responses in green turtles (Chelonia mydas) with fibropapillomatosis.

The immune competence of green sea turtles (Chelonia mydas) with fibropapillomatosis was assessed using in vitro techniques to measure lymphocyte proliferation in response to mitogens. In comparison with captive, healthy green sea turtles, those afflicted with fibropapillomas demonstrated diminished proliferation with Concanavalin A, phytohemagglutinin (T-cell mitogens), and lipopolysaccharide (B-cell mitogen). Also, markedly decreased proliferative responses to the lymphocyte polyclonal stimulator combination of ionomycin and phorbol myristate acetate were observed. Total circulating white blood cell counts were not statistically different between the two groups, although an overall decrease in lymphocyte number was observed in the papilloma group. The albumin/globulin ratio was decreased in the papilloma group because of decreased albumin and increased gamma globulins.     

Intestinal candidiasis in a loggerhead sea turtle (Caretta caretta): an immunohistochemical study

Post mortem examination of a juvenile loggerhead sea turtle (Caretta caretta) stranded in the Canary Islands revealed a fishing-line in the small intestine. Histologically, severe necrotic enteritis, multiple haemorrhages, and marked oedema of the intestinal submucosa were observed. Yeast cells and fungal hyphae were seen in the lamina propria of the intestinal mucosa and in the connective tissue of the submucosa. Because fungal cultures were not taken at the time of necropsy, an immunohistochemical study was performed in order to identify the fungus involved. Specific monoclonal and heterologously absorbed polyclonal antibodies served as the primary reagents for identification of aspergillosis, candidiasis, fusariosis, geotricosis, scedosporiosis, and zygomycosis, using an indirect immunofluorescence staining technique. The fungal elements were strongly stained only by a polyclonal antibody against Candida albicans and a monoclonal antibody against C. albicans. There are no known previous reports of Candida sp. causing skin disease or systemic mycotic infection in sea turtles.     

致肾脏、肠道病变大肠杆菌的分离及生物特性鉴定

利用常规培养基从病鸡的肾脏、肠道等组织器官中分离出了二株大肠杆菌，血清型为0152-160，该菌株能引起7／10的10日龄SPF鸡胚死亡，死亡鸡胚肾、肝肿大，全身出血。用分离物1^#、2^#大肠杆菌感染的20日龄雏鸡67％～88％死亡，从试验发病死亡鸡肾脏分离到的大肠杆菌仍能67％的感染鸡死亡。死亡鸡呈严重的肾肿大、尿酸盐沉积，肠壁出血、粘膜脱落，气囊炎和心包炎。该大肠杆菌对SPF鸡的致病死亡率低于普通健康鸡；试验证明用鸡毒支原体激发感染，可明显增强该大肠杆菌对SPF鸡的致死率和病变程度。另外试验证明此大肠杆菌对青、链霉素、庆大霉素等具有强耐药性。     

Effect of long-term tetracycline exposure (drinking water additive) on antibiotic-resistance of aerobic gram-negative intestinal flora of rats.

A study was undertaken to determine the effect of 2 years of intermittent administration of tetracycline in drinking water on antibiotic resistance in the aerobic gram-negative enterobacteria of rats in a closed colony. The bacterial isolates examined were resistant to tetracycline and streptomycin. Minimal inhibitory concentrations of tetracycline and streptomycin for intestinal organisms were similar in all of the animals, regardless of whether the animals were sampled while they were given drinking water with added tetracycline or at intervals of 3, 8, and 9 months after the antibiotic was no longer added to the drinking water. Biochemical examination of the isolates from each principal showed that Escherichia coli was the predominant enteric organism. In conjugation experiments, all E coli and Klebsiella pneumoniae isolated transferred tetracycline and streptomycin resistance to an E coli K-12 recipient. Four different strains of rats that had not been treated with tetracycline (controls) were examined for tetracycline resistance. Tetracycline-resistant Proteus mirabilis was isolated from the intestines of these animals. Plasmid-mediated resistance could not be demonstrated. The E coli and P vulgaris isolates from these control animals were susceptible to tetracycline.