Evaluation of five clinical chemistry analyzers for use in health assessment in sea turtles

OBJECTIVE: To compare blood biochemical values obtained from a handheld analyzer, 2 tabletop analyzers, and 2 diagnostic laboratories by use of replicate samples of sea turtle blood. DESIGN: Validation study. ANIMALS: 22 captive juvenile sea turtles. PROCEDURES: Sea turtles (18 loggerhead turtles [Caretta caretta], 3 green turtles [Chelonia mydas], and 1 Kemp's ridley turtle [Lepidochelys kempii]) were manually restrained, and a single blood sample was obtained from each turtle and divided for analysis by use of the 5 analyzers. Hematocrit and concentrations or activities of aspartate aminotransferase, creatine kinase, glucose, total protein, albumin, BUN, uric acid, P, Ca, K, Na, Cl, lactate dehydrogenase, and alkaline phosphatase were determined. Median values for each analyte were compared among the analyzers. RESULTS: Significant differences were found among the analyzers for most values; however, data obtained from the 2 diagnostic laboratories were similar for all analytes. The magnitude of difference between the diagnostic laboratories and in-house units was > or = 10% for 10 of the 15 analytes. CONCLUSIONS AND CLINICAL RELEVANCE: Variance in the results could be attributed in part to differences in analyzer methodology. It is important to identify the specific methodology used when reporting and interpreting biochemical data. Depending on the variable and specific case, this magnitude of difference could conceivably influence patient management.     

Steady-state plasma concentrations of itraconazole after oral administration in Kemp's ridley sea turtles, Lepidochelys kempi.

Pharmacokinetic studies of antifungal agents in reptiles are uncommon. Itraconazole, which has been used prophylactically in juvenile sea turtles suffering from hypothermia (cold stunning) on a regular basis, was evaluated for steady-state plasma concentrations. Five Kemp's ridley sea turtles (Lepidochelys kempi) receiving itraconazole at several dosages in a rehabilitation program had blood collected within 24 hr to estimate dosing frequency. Subsequently, serial blood samples of Kemp's ridley sea turtles that were given itraconazole at several dosages for 30 days to treat cold stunning were collected at various intervals to evaluate itraconazole plasma concentrations. Tissue samples were collected from one Kemp's ridley that died during rehabilitation. Plasma concentrations of itraconazole (and of hydroxyitraconazole [OH-ITRA], one of its major bioactive metabolites) were determined using a modified, validated reverse-phase high-performance liquid chromatography technique. Itraconazole concentrations in tissues were determined by bioassay to be far greater than the plasma concentrations measured in any of the turtles. At a 15-mg/kg dosage, the half-life (t1/2) was 75 hr for itraconazole and 55 hr for OH-ITRA. All dosages produced adequate concentrations in some turtles, but consistent therapeutic concentrations were produced only at 15 mg/kg q72hr and 5 mg/kg s.i.d., with the latter producing the highest plasma concentrations.     

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.     

Single-dose pharmacokinetics of ceftazidime and fluconazole during concurrent clinical use in cold-stunned Kemp's ridley turtles (Lepidochelys kempii)

Single-dose pharmacokinetics of intramuscularly administered ceftazidime (22 mg/kg) and subcutaneously administered fluconazole (21 mg/kg) were investigated during concurrent clinical use in naturally cold-stunned Kemp's ridley turtles (Lepidochelys kempii). Maximum mean concentration for ceftazidime was 61.31 μg/mL, and time of maximum concentration was 1.56 h postinjection. Maximum mean concentration for fluconazole was 26.16 μg/mL, and time of maximum concentration was 0.79 h postinjection. Results indicate that the ceftazidime dose and dosing interval used in this study are likely to be effective in treating susceptible strains of bacteria in Kemp's ridley turtles. However, the fluconazole dose and dosing interval are not likely to be effective against filamentous fungal pathogens that are often involved in marine turtle fungal infections.     

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.     

Ultrastructural characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta)

Ultrastructural characteristics of erythrocytes, heterophils, eosinophils, lymphocytes, monocytes and thrombocytes of the loggerhead sea turtle (Caretta caretta) were evaluated, using blood samples from 15 healthy juvenile animals. Except for the eosinophils, the rest of the white blood cells from loggerhead turtles had similar ultrastructural characteristics compared with blood cells from other sea turtle species. Eosinophils from loggerhead turtles were homogeneous in size, and no crystalline structures were observed within the granules. This paper provides an ultrastructural characterization of blood cells of loggerhead sea turtles, as a reference for future haematological studies of this species.     

Scintigraphic and radiographic evaluation of appendicular skeletal lesions in cold-stunned Kemp's ridley sea turtles

Osteolytic appendicular skeletal lesions in eight-stranded, cold-stunned Kemp's ridley sea turtles (Lepidochelys kempii) were evaluated using radiography and skeletal scintigraphy. Radiographic studies were performed monthly in most animals. Follow-up scintigraphy was performed 45-120 days after the initial exams in six turtles. Radiographically, lesions slowly progressed from an early osteolytic process contained to either the proximal or distal end of long bones, to a later stage characterized by thickening of the affected bone, sclerosis, and remodeling of the lesion borders. In seven turtles, the initial scintigrams were characterized by at least one focus of abnormal radiopharmaceutical uptake that correlated with a lytic site noted in radiographs. In five turtles, scintigraphic lesions were characterized by asymmetric radiopharmaceutical uptake rather than by increased intensity of uptake. Scintigraphic studies obtained more than 4 months after the appearance of clinical and radiographic signs had minimal, if any, abnormal radiopharmaceutical uptake, despite the persistence of abnormal radiographic findings. Skeletal scintigraphy is an effective method for more precisely determining if and when these animals can be returned to the wild. Animals were released if normal radiopharmaceutical uptake was seen during initial examination, or if decreased uptake was noted between serial examinations. In four of the turtles, resolution of abnormal scintigraphic findings permitted an objective decision to discontinue antibiotic and antifungal therapy. Seven of the eight turtles were released after correlation of the clinical signs with the imaging findings. Radiographs, however, are still needed to facilitate the correct identification of lesions with scintigraphy.     

Sectional anatomic and magnetic resonance imaging features of the head of juvenile loggerhead sea turtles (Caretta caretta)

Objective-To compare anatomic features of cross-sectional specimens with those of MRI images of the heads of loggerhead sea turtles (Caretta caretta). Animals-5 cadavers of juvenile female loggerhead sea turtles. Procedures-Spin-echo T1-weighted and T2-weighted MRI scans were obtained in sagittal, transverse, and dorsal planes with a 0.2-T magnet and head coil. Head specimens were grossly dissected and photographed. Anatomic features of the MRI images were compared with those of gross anatomic sections of the heads from 4 of these turtles. Results-In the MRI images, anatomic details of the turtles' heads were identified by the characteristics of signal intensity of various tissues. Relevant anatomic structures were identified and labeled on the MRI images and corresponding anatomic sections. Conclusions and Clinical Relevance-The MRI images obtained through this study provided valid information on anatomic characteristics of the head in juvenile loggerhead sea turtles and should be useful for guiding clinical evaluation of this anatomic region in this species.     

Comparative study of hematologic and plasma biochemical variables in Eastern Atlantic juvenile and adult nesting loggerhead sea turtles (Caretta caretta)

Background: Plasma biochemical and hematologic variables are important in the management of endangered sea turtles, such as loggerheads. However, studies on blood biochemistry and hematology of loggerheads are limited, and different concentrations according to variable criteria have been reported. Objective: The purpose of this study was to establish and compare baseline plasma chemistry and hematology values in Eastern Atlantic juvenile and adult nesting loggerhead sea turtles (Caretta caretta). Methods: Blood samples were collected from 69 healthy juvenile loggerhead sea turtles after their rehabilitation in captivity, and from 34 adult nesting loggerheads after oviposition. Fresh blood was used for leukocyte differential count and PCV determination. Heparinized blood was used for RBC and WBC counts. Plasma biochemical concentrations were measured using an automated biochemical analyzer. For the comparative study, nonparametric statistical analysis was done using the Mann–Whitney U‐test. Results: Minimum, maximum, and median concentrations were obtained for 14 hematologic and 15 plasma chemistry variables. Statistically significant differences between juvenile and adult turtles were found for PCV; RBC, WBC, and leukocyte differential counts; total protein, albumin, globulins, calcium, triglycerides, glucose, total cholesterol and urea concentrations; and lactate dehydrogenase activity. Conclusions: Age, size, and reproductive status cause important variations in the hematologic and plasma biochemical results of loggerheads. The reference values obtained in this study may be used as a standard profile, useful for veterinary surgeons involved in sea turtle conservation.     

Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity

A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.     

Surgical removal of an abscess associated with Fusarium solani from a Kemp's ridley sea turtle (Lepidochelys kempii)

A cold-stunned Kemp's ridley sea turtle, Lepidochelys kempii, developed an abscess associated with Fusarium solani, Vibrio alginolyticus, and a Shewenalla species after receiving a bite wound to the front flipper during rehabilitation. The lesion failed to respond to medical therapy and was treated successfully with surgery. Histopathology of the excised tissue demonstrated septic heterophilic inflammation with necrosis and granulation tissue, fungal elements, and bacteria, despite appropriate antimicrobial therapy. Variably thick bands of dense collagenous tissue partially surrounded affected areas which might have limited drug penetration into the tissue. Postoperative healing and eventual releases were uneventful. This is the first report of surgical treatment of cutaneous Fusarium infection in a sea turtle and supports surgery as an effective treatment for a fungal abscess in a reptile.     

Plasma concentrations of praziquantel after oral administration of single and multiple doses in loggerhead sea turtles (Caretta caretta)

OBJECTIVE: To determine the pharmacokinetics of praziquantel following single and multiple oral dosing in loggerhead sea turtles. ANIMALS: 12 healthy juvenile loggerhead sea turtles. PROCEDURE: Praziquantel was administered orally as a single dose (25 and 50 mg/kg) to 2 groups of turtles; a multiple-dose study was then performed in which 6 turtles received 3 doses of praziquantel (25 mg/kg, PO) at 3-hour intervals. Blood samples were collected from all turtles before and at intervals after drug administration for assessment of plasma praziquantel concentrations. Pharmacokinetic analyses included maximum observed plasma concentration (Cmax), time to maximum concentration (Tmax), area under the plasma praziquantel concentration-time curve, and mean residence time (MRTt). RESULTS: Large interanimal variability in plasma praziquantel concentrations was observed for all dosages. One turtle that received 50 mg of praziquantel/kg developed skin lesions within 48 hours of administration. After administration of 25 or 50 mg of praziquantel/kg, mean plasma concentrations were below the limit of quantification after 24 hours. In the multiple-dose group of turtles, mean plasma concentration was 90 ng/mL at the last sampling time-point (48 hours after the first of 3 doses). In the single-dose study, mean Cmax and Tmax with dose were not significantly different between doses. After administration of multiple doses of praziquantel, only MRTt was significantly increased, compared with values after administration of a single 25-mg dose. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of 25 mg of praziquantel/kg 3 times at 3-hour intervals may be appropriate for treatment of loggerhead sea turtles with spirorchidiasis.     

Endoscopic evaluation of the esophagus and stomach in three loggerhead sea turtles (Caretta caretta) and a Malaysian giant turtle (Orlitia borneensis).

Three loggerhead sea turtles (Caretta caretta) and a Malaysian giant turtle (Orlitia borneensis) were presented with suspected or confirmed esophageal foreign bodies. Esophagoscopy was performed on all turtles, and gastroscopy was performed on three turtles. In all cases, endoscopy was easy to perform, and allowed visualization of most upper gastrointestinal features. The papillated esophagus was easy to navigate, but mucosal papillae in the loggerhead sea turtles prevented examination of the underlying mucosa. The stomach was easily entered and examined in both species, but the working endoscope length (100 cm) prevented inspection of the pyloric antrum and the duodenum in all turtles. The turtles in this report may serve as references for future endoscopic examinations of these species.     

Evaluation of Doppler ultrasonography for the measurement of blood flow in young loggerhead sea turtles (Caretta caretta)

The aim of this study was to identify ultrasound accessible blood vessels in the loggerhead sea turtle (Caretta caretta) and describe their Doppler waveform patterns, peak systolic velocity, mean velocity, systolic/diastolic ratio as well as pulsatility and resistive indices. B-mode, colour and pulsed-wave Doppler examinations were performed on 10 turtles. Flow measurements were recorded for the left and right aorta, epigastric and internal iliac arteries, and right hepatic vein. Additionally, the large blood vessels of three dead turtles were injected with latex and dissected for anatomical support. A parabolic flow velocity profile was observed in all arteries. The waveforms of the right and left aortic arteries showed an unusual pattern when compared with mammals. The hepatic vein flow velocity waveform of the loggerhead sea turtle was found to be similar to that in the dog, although the flow velocity in the C-wave was higher than that in the A-wave. The low resistance flow pattern observed suggests that the loggerhead sea turtle's organs require a continuous blood supply.     

Pulmonary hyalohyphomycosis caused by Fusarium spp in a Kemp's ridley sea turtle (Lepidochelys kempi): an immunohistochemical study

AIM: To describe the microscopic and immunohistochemical findings in a case of pulmonary hyalohyphomycosis in a Kemp's ridley sea turtle (Lepidochelys kempi). METHODS: Samples of lung, liver and kidney from a stranded, dead Kemp's ridley sea turtle were routinely processed for histopathological studies. Two monoclonal antibodies that reacted specifically with antigens of Aspergillus spp and the Mucorales (Zygomycetes) group, and a panel of polyclonal antibodies raised against Aspergillus fumigatus, Candida albicans, Geotrichum candidum, Fusarium solani, and Scedosporium apiospermum were used for immunohistochemical or immunofluorescence staining. RESULTS: Histologically, a severe multifocal granulomatous pneumonia associated with fungal infection was diagnosed. All hyphae were identified as Fusarium spp because a strong and uniform reactivity was obtained only with a heterologously-absorbed polyclonal antibody raised against somatic antigens of Fusarium solani. CONCLUSIONS: Fusarium spp should be included in the differential diagnosis of mycotic pneumonia in Kemp's ridley sea turtles. CLINICAL RELEVANCE: This is the first report of a pulmonary mycotic infection in a sea turtle diagnosed using immunohistochemistry. Wildlife rehabilitators and pathologists should be aware of the availability of immunohistochemical techniques for identifying fungi in sea turtles.     

Sectional anatomic and magnetic resonance imaging features of coelomic structures of loggerhead sea turtles

OBJECTIVE: To compare cross-sectional anatomic specimens with images obtained via magnetic resonance imaging (MRI) of the coelomic structures of loggerhead sea turtles (Caretta caretta). ANIMALS: 5 clinically normal live turtles and 5 dead turtles. PROCEDURES: MRI was used to produce T1- and T2- weighted images of the turtles, which were compared with gross anatomic sections of 3 of the 5 dead turtles. The other 2 dead turtles received injection with latex and were dissected to provide additional cardiovascular anatomic data. RESULTS: The general view on the 3 oriented planes provided good understanding of cross-sectional anatomic features. Likewise, major anatomic structures such as the esophagus, stomach, lungs, intestine (duodenum and colon), liver, gallbladder, spleen, kidneys, urinary bladder, heart, bronchi, and vessels could be clearly imaged. It was not possible to recognize the ureters or reproductive tract. CONCLUSIONS AND CLINICAL RELEVANCE: By providing reference information for clinical use, MRI may be valuable for detailed assessment of the internal anatomic structures of loggerhead sea turtles. Drawbacks exist in association with anesthesia and the cost and availability of MRI, but the technique does provide excellent images of most internal organs. Information concerning structures such as the pancreas, ureters, intestinal segments (jejunum and ileum), and the reproductive tract is limited because of inconsistent visualization.     

Pathology of Fibropapillomatosis in Olive Ridley Turtles Lepidochelys olivacea Nesting in Costa Rica

Abstract

Fibropapillomatosis (FP) is a neoplastic disease that primarily affects green turtles Chelonia mydas in epidemic proportions worldwide. Although several infectious agents (herpesvirus, retrovirus, and papillomavirus) have been associated with the condition, the etiologic agent has not been isolated or characterized. Recently, FP has been reported in other sea turtle species including confirmed cases in loggerhead turtles Caretta caretta in Florida and field observations in olive ridley turtles Lepidochelys olivacea in the Pacific coasts of Mexico and Costa Rica. Skin and tumor specimens were collected from 72 olive ridley turtles nesting in Ostional Wildlife Refuge, Costa Rica, between July and September 1997. In all, 50 tumor biopsies were examined from 25 of the affected turtles. In addition, six biopsies were examined from five turtles that did not have visible masses and served as controls. Grossly, masses were 25 mm or less in diameter, white to gray, smooth to verruciform, raised tumors of the integument of the neck and flippers. Histologically, 42 of 50 were diagnosed as fibropapillomas and eight were classified as chronic active dermatitis and not tumors. Twenty of 42 fibropapillomas were in stages of regression and 9 of the remaining 22 tumors had histological changes that suggested early degeneration within the tumor. During field surveys based on gross lesions, prevalences of 1–10% have been reported in this nesting population. This is considered the first histopathologic confirmation of FP in olive ridley turtles.     

Relationship between barnacle epibiotic load and hematologic parameters in loggerhead sea turtles (Caretta caretta), a comparison between migratory and residential animals in Pamlico Sound, North Carolina.

Health status of a total of 57 loggerhead sea turtles (Caretta caretta; 42 migratory and 15 residential turtles) was analyzed using body condition and hematologic parameters. A subset of 18 juvenile migratory loggerhead sea turtles in the fall of 1997 and 15 residential turtles in the summer of 2000 were analyzed for barnacle epibiota. The migratory group had significantly higher red blood cell counts and percent heterophils and significantly lower percent lymphocyte and absolute eosinophil counts, as well as significantly lower plasma concentrations of calcium, sodium, chloride, potassium, glucose, alkaline phosphatase, and anion gap. Many of these variations may be because of physiology of migration. A positive association between turtle weight and hematocrit was detected and may be because of larger turtles diving for longer periods of time. There were no significant differences of epibiota load, health of the turtles, or condition index between turtles captured during the two events.     

Plasma concentrations of enrofloxacin after single-dose oral administration in loggerhead sea turtles (Caretta caretta).

Plasma concentrations and pharmacokinetics of enrofloxacin were determined in 12 loggerhead sea turtles (Caretta caretta) after oral administration. Six turtles in group 1 and group 2 received enrofloxacin at 10 mg/kg and 20 mg/kg of body weight, respectively. Blood was collected from the cervical sinus before administration and at timed intervals up to 168 hr following administration. Plasma concentrations of enrofloxacin were determined using a microbiologic assay. The mean peak plasma concentration (Cmax) was 4.07 microg/ml and 21.30 microg/ml for groups 1 and 2, respectively. Plasma levels were detectable at 168 hr postadministration, with mean values of 0.380 microg/ml for group I and 2.769 microg/ml for group 2. The mean elimination half-life for enrofloxacin was 37.80 hr for group I and 54.42 hr for group 2. These findings indicated that enrofloxacin is absorbed following oral administration in loggerhead sea turtles, and blood levels are maintained up to 168 hr following administration.     

Pharmacokinetics of florfenicol in loggerhead sea turtles (Caretta caretta) after single intravenous and intramuscular injections.

The pharmocodynamics of single injections of florfenicol in yearling loggerhead sea turtles (Caretta caretta) were determined. Eight juvenile loggerhead sea turtles weighing 1.25 (+/- 0.18) kg were divided into two groups. Four animals received 30 mg/kg of florfenicol i.v., and four received the same dose i.m. Plasma florfenicol concentrations were analyzed by reverse-phase high performance liquid chromatography. After the i.v. dose, there was a biphasic decline in plasma florfenicol concentration. The initial steep phase from 3 min to 1 hr had a half-life of 3 min, and there was a longer slow phase of elimination, with a half-life that ranged from 2 to 7.8 hr among turtles. The volume of distribution varied greatly and ranged from 10.46 to -60 L/kg. Clearance after the i.v. dose was 3.6-6.3 L/kg/hr. After the i.m. injection, there was a peak within 30 min of 1.4-5.6 microg/ml, and florfenicol was thereafter eliminated with a half-life of 3.2-4.3 hr. With either route, florfenicol plasma concentrations were below the minimum inhibitory concentrations for sensitive bacteria within 1 hr. Florfenicol does not appear to be a practical antibiotic in sea turtles when administered at these doses.