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.     

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.     

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.     

Pharmacokinetics of marbofloxacin after a single oral dose to loggerhead sea turtles (Caretta caretta)

The single-dose disposition kinetics of marbofloxacin (MBX) were determined in clinically healthy loggerhead sea turtles (n = 5) after oral (PO) administration of 2 mg kg⁻¹ bodyweight. Marbofloxacin plasma concentrations were determined by DAD–HPLC (LOD/LOQ 0.015/0.05 μg ml⁻¹). Data were subjected to non-compartmental analysis. Following PO administration, marbofloxacin achieved maximum plasma concentrations of 11.66 ± 2.53 mg L⁻¹ at 15.00 ± 3.00 h. The absence of general adverse reactions in the turtles of the study, and the favourable pharmacokinetic properties (long half-life and high maximum plasma concentration) of MBX administered PO at the single-dose of 2 mg kg⁻¹ suggest the possibility of its safe and effective clinical use in loggerhead sea turtles.     

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.     

Computed tomographic anatomy of the head of the loggerhead sea turtle (Caretta caretta)

The heads of three loggerhead sea turtles were disarticulated and imaged immediately to minimize postmortem changes and then frozen and sectioned. For computed tomography (CT) imaging, the heads were positioned in ventral recumbency. Transverse CT images with soft-tissue window were obtained from the olfactory sac region to the temporomandibular joint region. After CT imaging, the heads were sectioned and the gross sections were compared to CT images, to assist in the accurate identification of the anatomic structures. Different clinically relevant anatomic structures were identified and labelled in two series of photographs (CT images and anatomic cross-sections). CT images provided good differentiation between the bones and the soft tissues of the head. The information presented in this paper should serve as an initial reference to evaluate CT images of the head of the loggerhead sea turtle and to assist in the interpretation of lesions of this region.     

Ultrasonographic imaging of loggerhead sea turtles (Caretta caretta)

Twenty live and five dead juvenile and subadult loggerhead sea turtles were examined ultrasonographically. Ten soft tissue areas of the integument were used as acoustic windows: cervical-dorsal and cervical-ventral, left and right cervicobrachial, left and right axillary, left and right prefemoral and left and right postfemoral windows. Anatomical cross-sections were performed on the dead turtles to provide reference data. The fourth and fifth cervical vertebrae, the spinal cord, and the venous sinuses of the external jugular vein were clearly visible through the cervical-dorsal acoustic window, and the oesophagus and the heart were imaged through the cervical-ventral acoustic window. The stomach was more frequently visible through the left axillary acoustic window. The liver could be imaged through both sides, but the right axillary acoustic window was better for visualising the gall bladder. The large and small intestines and the kidneys were visible through the right and left prefemoral acoustic windows; the kidneys were easily identified by their intense vasculature.     

Radiographic features of the limbs of juvenile and subadult loggerhead sea turtles (Caretta caretta)

This study aimed to provide the normal radiographic anatomic appearance of the limbs of the loggerhead sea turtle, Caretta caretta. Dorsopalmar and dorsoplantar radiographs were taken of the forelimbs and hindlimbs of 15 juvenile and 15 subadult loggerhead sea turtles, 17 alive and 13 dead. For comparison, computed tomographic, gross anatomic, osteologic, and histologic studies were performed on the limbs of 5 of the sea turtles. Bones from the distal part of the fore and hind flippers were seen in detail with a mammographic film-screen combination. The pectoral and pelvic girdles, superimposed by the carapace, were better seen on standard radiographs with the use of rare-earth intensifying screens. Mammographic radiographs of the manus of 5 small juvenile turtles showed active growth zones. Visualization of bone contours in the distal part of the limbs was clearer than in mammals owing to the very few superimpositions. The presence of a substantial amount of cartilage in the epiphyses produced better visibility of limb ends. We conclude that use of a mammography film-screen combination is the best way to evaluate the bony and joint structures of the limbs of sea turtles. Radiography provides reliable images for clinical purposes. Considering the low cost and logistics of this technique, it is a practical ancillary test for marine animal rehabilitation centers to use.     

Intraocular pressure of juvenile loggerhead sea turtles (Caretta caretta) held in different positions.

The intraocular pressure of 12 apparently healthy juvenile loggerhead sea turtles (Caretta caretta) was determined by applanation tonometry while the turtles were held in dorsoventral, ventrodorsal, and head-down suspended positions. The median intraocular pressures were 5 mmHg (range 4 to 9 mmHg) in the dorsoventral position, 7 mmHg (range 5 to 12 mmHg) in the ventrodorsal position, and 23 mmHg (range 17 to 33 mmHg) in the suspended position.     

Ingesta passage and gastric emptying times in loggerhead sea turtles (Caretta caretta)

Ingesta passage times of soft flat foam dishes and gastric emptying time of barium-impregnated polyethylene spheres (BIPS) were measured in 22 and 8 loggerhead sea turtles (Caretta caretta), respectively. Transit time (T(1)) was considered as the time between ingestion and first elimination, and retention time (T(50)) and total transit time (T(85)) the expulsion time of 50% and 85% of the markers, respectively. The experiments were carried out at different times of the year and water temperature was recorded. A set of dorso-ventral radiographs was taken to locate the BIPS, and the gastrointestinal anatomy of 5 dead turtles was studied to help with interpretation of the radiographs. No significant correlation was observed between T(1), T(50), T(85) and minimum straight carapace length (SCLmin) or body mass and no statistical difference was found in ingesta passage transit times between juvenile (n = 6) and sub-adult turtles (n = 16). Mean passage times of the dishes (in days) were: T(1) = 9.05, T(50) = 12.00 and T(85) = 13.19. Gastric emptying time using BIPS was 24-48 h. The transit time (T(1)) for the BIPS was longer (13.25 +/- 4.86 days) than the foam markers (8.5 +/- 2.73 days) in 8 turtles studied simultaneously. Although the total transit time tended to be faster in turtles submitted to water temperatures between 20 degrees C and 23.6 degrees C no significant correlation was observed between T(1), T(50) and T(85) and the temperature.     

Ketoprofen pharmacokinetics of R‐ and S‐isomers in juvenile loggerhead sea turtles (Caretta caretta) after single intravenous and single‐ and multidose intramuscular administration

Ketoprofen is a nonsteroidal anti‐inflammatory and analgesic agent that nonselectively inhibits cyclooxygenase, with both COX‐1 and COX‐2 inhibition. Recent studies on COX receptor expression in reptiles suggest that nonselective COX inhibitors may be more appropriate than more selective inhibitors in some reptiles, but few pharmacokinetic studies are available. The goal of this study was to determine single‐ and multidose (three consecutive days) pharmacokinetics of racemic ketoprofen administered intravenously and intramuscularly at 2 mg/kg in healthy juvenile loggerhead turtles (Caretta caretta). The S‐isomer is the predominant isomer in loggerhead sea turtles, similar to most mammals, despite administration of a 50:50 racemic mixture. Multidose ketoprofen administration demonstrated no bioaccumulation; therefore, once‐daily dosing will not require dose adjustment over time. S‐isomer pharmacokinetic parameters determined in this study were C_max of 10.1 μg/ml by IM injection, C₀ of 13.4 μg/ml by IV injection, AUC of 44.7 or 69.4 μg*hr/ml by IM or IV injection, respectively, and T½ of 2.8 or 3.6 hr by IM or IV injection, respectively. Total ketoprofen plasma concentrations were maintained for at least 12 hr above concentrations determined to be effective for rats and humans. A dose of 2 mg/kg either IM or IV every 24 hr is likely appropriate for loggerhead turtles.     

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.     

Effects of anticoagulant and autoanalyzer on blood biochemical values of loggerhead sea turtles (Caretta caretta).

We evaluated the effect of anticoagulant (lithium heparin, sodium heparin, or none) and type of autoanalyzer on selected blood biochemical values of the loggerhead sea turtle (Caretta caretta). More differences were observed between the analytes in serum and those in the 2 types of plasma than were observed between the 2 types of plasma. Differences in electrolyte concentrations were not significant when plasma from sodium-heparinized blood was compared with plasma from lithium-heparinized blood. Serum is not recommended for reptilian studies because clot formation is unpredictable and because the time required for clotting may allow substantial changes in the chemical composition of the sample. For most determinants, values varied more between the 2 types of autoanalyzers than among the 3 anticoagulant treatments. These sources of variation must be considered when performing comparative studies.     

Successive changes of hematologic characteristics and plasma chemistry values of juvenile loggerhead turtles (Caretta caretta).

Hematologic characteristics and plasma chemistry values of juvenile loggerhead turtles (Caretta caretta) from the ages of 1 mo to 3 yr were obtained to establish baseline values. Five clinically normal loggerhead turtles were selected from the same clutch and raised in an indoor artificial nesting beach. Blood samples were successively collected and examined for various blood characteristics for a maximum total of 15 times. Hematologic characteristics, including packed cell volume, white blood cell counts, and white blood cell differentials; and plasma chemistry values, including total bilirubin, total protein, albumin, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, gamma-glutamic transpeptidase, creatinine, blood urea nitrogen, uric acid, alkaline phosphatase, amylase, triglyceride, total cholesterol, ionized sodium, ionized potassium and ionized chlorine, were measured. These results were used to establish a hematology and blood chemistry baseline for captive juvenile loggerhead turtles and will aid in their medical management.     

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

The pharmacokinetics of oxytetracycline in 2-yr-old loggerhead sea turtles (Caretta caretta) after single i.v. and i.m. injections were studied for biologic marking and therapeutic applications. Twenty juvenile turtles were divided into two treatment groups. Ten animals received 25 mg/kg of oxytetracycline i.v. and 10 received the same dosage i.m. Plasma oxytetracycline concentrations were analyzed by reverse-phase high-performance liquid chromatography. Data from the i.v. route best fit a three-compartment model, whereas noncompartmental analysis was used to compare data from both the i.v. and i.m routes. For the i.v. route, means for maximum plasma concentration, terminal phase half-life, systemic clearance, and apparent volume of distribution at steady state were 6.6 microg/ml, 66.1 hr, 290.7 ml/hr/kg, and 18.4 L, respectively. For the i.m. route, means for systemic availability, maximum plasma concentration, and elimination half-life were 91.8%, 1.6 microg/ml, and 61.9 hr, respectively. The remarkably high apparent volume of distribution may possibly be associated with a deep compartment of drug disposition such as bone deposition associated with the large skeletal mass of turtles and the fact that these were well-nourished, growing juveniles. Although maximum plasma concentration by i.m. administration was lower than for the i.v. route, the long elimination time indicates that an infrequent dosing interval may be effective for sensitive bacteria.     

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.     

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

The pharmacokinetics of ceftazidime in yearling loggerhead sea turtles (Caretta caretta) following single i.m and i.v. injections were studied. Eight juvenile 1.25+/-0.18 kg turtles were divided into two groups. Four animals received 20 mg/kg of ceftazidime i.v. and four received the same dose i.m. Plasma ceftazidime concentrations were analyzed by reverse-phase high-performance liquid chromatography. The i.v. and i.m. administration half-lives were 20.59+/-3.24 hr and 19.08+/-0.77 hr, respectively. The volume of distribution was 0.42+/-0.07 L/kg, and the systemic clearance was 0.217+/-0.005 ml/min/kg. Ceftazidime was detected in all blood samples and its concentration exceeded the minimum inhibitory concentration for Pseudomonas for 60 hr after i.m. and i.v. injections.     

Relationship between separation time of plasma from heparinized whole blood on plasma biochemical analytes of loggerhead sea turtles (Caretta caretta)

Concentrations and activities of selected biochemicals of loggerhead sea turtles (Caretta caretta) were determined for plasma that was separated from whole blood samples that were kept up to 96 hr post collection (PC) in a refrigerator. Blood samples collected from seven juvenile captive loggerhead sea turtles were added to tubes containing lithium heparin and were placed on ice. Equal amounts of anticoagulated whole blood from the lithium heparin tubes were then aliquoted into plastic tubes and stored as whole blood under refrigeration until they were centrifuged at 0, 4, 24, 48, and 96 hr PC. Plasma was removed and the analytes that were measured were alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), creatine kinase (CK), sodium, chloride, potassium, magnesium, calcium, phosphorus, cholesterol, glucose, urea nitrogen, uric acid, total protein, albumin, and globulin. Compared with values at 0 time, the only analyte to be significantly different at 24 hr PC was GGT (activity decreased by 25%). Compared with values at 0 time, significant differences at 96 hr PC were only seen in AST (2% increase), GGT (25% decrease), glucose (7% decrease), and uric acid (25% increase). Although a statistically significant difference was found in concentrations of phosphorus and cholesterol over time by repeated measures analysis of variance (ANOVA), the follow-up multiple comparison procedure could not define the specific time points at which significant differences occurred. For all other analytes, significant differences over the time course of the study were not found. In these instances, the power of the ANOVA was sufficient (> or = 0.80) to detect any arithmetic differences of a clinically relevant magnitude. Although plasma should be separated from the cellular component of blood as soon as possible PC, in a field situation in which a centrifuge is unavailable, samples can be stored in a portable cooler up to 24 hr without appreciable change in select biochemical analytes.     

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.