Relationships between Spatial Biology and Physiological Ecology in the Gopher Tortoise, Gopherus polyphemus.

AbstractThe overlap between spatial and physiological ecology is generally understudied, yet both fields are fundamentally related in assessing how individuals balance limited resources. Herein, we quantified the relationships between spatial ecology using two parameters of home range (annual home range area and number of burrows used in 1 yr) and four measures of physiology that integrate stress and immunity (baseline plasma corticosterone [CORT] concentration, plasma lactate concentration, heterophil-to-lymphocyte [H∶L] ratio, and bactericidal ability [BA]) in a wild free-ranging population of the gopher tortoise (Gopherus polyphemus) to test the hypothesis that space usage is correlated with physiological state. We also used structural equation models (SEMs) to test for causative relationships between the spatial and physiological parameters. We predicted that larger home ranges would be negatively correlated with traditional biomarkers of stress and positively correlated with immunity, consistent with our hypothesis that home ranges are determined based on individual condition. Males had larger home ranges, used more burrows, and had higher baseline CORT than females. We found significant negative correlations between lactate and home range (r=-0.456, df=21, P=0.029). CORT was negatively correlated with the number of burrows used in both sexes (F=7.322, df=2,20, P=0.003, adjusted R2=0.383). No correlations were observed between space use and BA or, notably, H∶L ratio. SEMs suggested that variation in the number of burrows used was a result of variation in baseline CORT. The lack of a relationship between H∶L ratio and home range suggests that home range differences are not associated with differences in chronic stress, despite the pattern between baseline CORT and number of burrows used. Instead, this study indicates that animals balance trade-offs in energetics, likely by way of baseline corticosteroid, in such a way as to maintain function across continuously variable home range strategies.

AEROBIC BLOOD CULTURES AND COMPARISON TO CLINICAL FINDINGS OF FREE-RANGING GREEN TURTLES (CHELONIA MYDAS) IN EAST CENTRAL FLORIDA.

Positive blood cultures have been identified in debilitated, stranded, and deceased green turtles (Chelonia mydas), suggestive of septicemia. Interpretation of these results is often difficult because multiple studies have previously identified bacteremia in clinically healthy reptiles. In this study, paired blood cultures and skin cultures obtained after aseptic preparation of the venipuncture site were collected from 50 immature free-ranging green turtles from Port Canaveral, Florida. Blood culture results were compared with health status (apparently healthy versus unhealthy, based on physical examination findings and appropriate body condition), date of collection, presence of external fibropapillomatosis, healed or unhealed injuries, and presence of barnacles. Weight, body condition score, body condition index, morphometric measures, volume of blood collected, and body temperature were compared between blood culture-positive and blood culture-negative turtles. Positive blood cultures were identified in 14% (7 of 50) of all turtles, including 15.6% (5 of 32) of apparently healthy turtles. Vibrio spp., Bacillus megaterium, Cellulomonas sp., and Staphylococcus pasteuri were isolated in blood culture from apparently healthy individuals. There was a significant association (P = 0.048) between positive skin cultures and positive blood cultures, but isolates obtained were consistently different between paired results. There was no significant association (P > 0.05) between blood culture results and health status, evidence of healed or unhealed injuries, external fibropapillomatosis, or presence of barnacles. Based on the results of this study, positive blood cultures suggestive of nonclinical bacteremia may be present in apparently healthy green turtles. The results of this study will aid the attending clinician in interpretation of blood culture results of apparently healthy or presumed septicemic captive and rehabilitating green turtles as part of the conservation and population recovery of this threatened species.

LACK OF AGREEMENT BETWEEN A POINT-OF-CARE BLOOD URIC ACID METER AND A BENCHTOP CHEMISTRY ANALYZER IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Uric acid (UA) is excreted as an end product of protein metabolism in many reptiles, including some chelonians. Elevated plasma UA concentrations can occur due to many physiologic and pathologic changes, and determining plasma UA concentrations is part of a complete general health assessment in this taxon. UA concentrations are typically measured using benchtop chemistry analyzers, but point-of-care (POC) UA meters have also been developed for human use. However, these POC UA meters have not been investigated for use in any reptile species. The purpose of this study was to assess agreement between UA measurements produced by a standard benchtop chemistry analyzer and a POC UA meter in free-living eastern box turtles (Terrapene carolina carolina). UA concentrations were measured with a POC meter using fresh whole blood and frozen-thawed plasma and with a standard benchtop chemistry analyzer using frozen-thawed plasma. Poor-to-moderate agreement was present between each of the three methods as evidenced by mixed models, Passing-Bablok regression, Bland-Altman plots, and Cohen's κ. Differences between methods fell outside of clinically acceptable limits, indicating that the POC UA meter should not be used in eastern box turtles.

PROTEIN ELECTROPHORESIS AND REFERENCE INTERVALS OF SERUM PROTEINS OF A CAPTIVE POPULATION OF ROTI ISLAND SNAKE-NECKED TURTLES (CHELODINA MCCORDI).

Protein electrophoresis (PEP) is an important tool in mammals to characterize specific dysproteinemias and detect acute and chronic inflammatory responses. In reptiles, PEP is the gold standard method for globulin fraction determination and albumin measurement. In this study, preliminary reference intervals were established for serum PEP in 22 clinically healthy adult Roti Island snake-necked turtles (Chelodina mccordi), a critically endangered species, kept in captivity and sampled over two monsoon seasons. The species has a prominent prealbumin fraction and ß-globulins were the dominant globulin fraction. Significant differences between females and males were found in prealbumin (P < 0.01), albumin (P = 0.02), α1-globulin (P = 0.05) and γ-globulin (P = 0.01). Gravid females had significantly lower total protein (P < 0.01), prealbumin (P < 0.01), albumin (P < 0.01) and albumin:globulin ratio (P = 0.01). These preliminary reference intervals should aid in clinical investigation in this species as well as further research studies seeking to understand the application of PEP in reptilian species.

Subcutaneous amikacin administration in red-eared sliders (Trachemys scripta elegans) produces prolonged detectable plasma concentrations without biochemistry evidence of impaired renal function.

OBJECTIVE: To establish pilot data on the plasma concentrations of SC amikacin at 2 doses in red-eared sliders and evaluate concurrent plasma biochemistry parameters. ANIMALS: 8 adult red-eared sliders (Trachemys scripta elegans). METHODS: Amikacin was administered SC at target doses of 5 and 10 mg/kg with a 3-week washout period. Blood samples were collected at 0, 24, 48, 72, and 96 hours postadministration. Plasma amikacin concentrations were quantified using liquid chromatography tandem mass spectrometry. Plasma biochemistry analyses were performed before amikacin administration, 1 week post 5-mg/kg administration, and 1 week post 10-mg/kg administration. RESULTS: Mean maximum amikacin plasma concentrations were recorded 24 hours after 5-mg/kg and 10-mg/kg dosing and were 17.5 ± 2.32 µg/mL and 23.6 ± 2.92 µg/mL, respectively. Mean plasma concentrations after 5-mg/kg dosing steadily decreased to 9.1 ± 0.92 µg/mL by 96 hours postadministration. Amikacin remained detectable in all plasma samples 3 weeks post 5-mg/kg dosing with a mean plasma concentration of 1.04 ± 0.22 µg/mL. Mean plasma concentrations after 10-mg/kg dosing did not decrease over the 96-hour study period. There were no clinically relevant changes in biochemistry parameters. CLINICAL RELEVANCE: Amikacin persists at detectable plasma levels for at least 3 weeks after SC administration of a 5-mg/kg dose in red-eared sliders, which has not previously been reported in any species. No biochemistry changes consistent with renal toxicity occurred after either dose. Use caution with repeated amikacin dosing in this species until further studies can better characterize cumulative amikacin pharmacokinetics and toxic threshold.

No Ranaviral DNA Found in Australian Freshwater Turtles, 2014-19, Despite Previous Serologic Evidence.

Ranaviruses are pathogens of ectothermic vertebrates (fish, amphibians, and reptiles). Turtles are the most common group of reptiles reported with ranaviral infections. However, there have been no surveys for wild ranaviral infection in any turtles from the suborder Pleurodira, despite ranaviral distributions and experimentally susceptible pleurodiran turtle populations overlapping in several areas, including Australia. We assayed 397 pooled blood samples from six Australian freshwater turtle species collected from five different sites in northern Australia between 2014 and 2019. Historical serologic surveys in the area had found antiranaviral antibodies; however, we did not detect any ranaviral DNA in our samples. Discrepancies between historical serologic and our molecular results may be explained by low viral prevalence during the years that these samples were collected, survivorship bias, or possibly an age class bias in sampling.

Comparison of the blood biochemical values of foraging and nesting Olive Ridley turtles (Lepidochelys olivacea) from Sinaloa, Mexico.

Our study aimed to establish reference values for nesting females and compare them with those previously reported to understand olive ridley turtles' health status and contribute to long-term health assessment and monitoring in foraging and nesting areas from the state of Sinaloa, Mexico. In August and September 2018, morphometric data and biochemical profiles were collected from 33 nesting olive ridley turtles from Ceuta Beach Sanctuary (CBS) and 14 foraging female turtles captured at the foraging site, Navachiste Marine Area (NMA). Nesting turtles sampled had greater CCL (65.86 ± 1.70 cm) than those from the foraging area (61.54 ± 1.22) (p < 0.05). Regarding biochemical profiles, post-nesting turtles had higher packed cell volume (PCV), albumin, blood urea nitrogen (BUN), cholesterol, triglycerides, and calcium than turtles from the foraging area (p < 0.05). Phosphorus levels were higher in foraging turtles than in nesting turtles (p = 0.001), while the remaining parameters showed no significant differences. The present study describes for the first time the blood biochemical values of nesting turtles from the Ceuta Beach Sanctuary in southern Sinaloa, Mexico, similar to those of foraging turtles from the north of the state. The significant differences observed between the two analysis groups may be due to the energy reserves and reproductive and nesting activity of the nesting turtles, so the blood biochemistry values described in this study can be used as a standard reference blood value for the olive ridley turtle population of Sinaloa, Mexico.

Pharmacokinetic characteristics of florfenicol in green sea turtles (Chelonia mydas) and hawksbill sea turtles (Eretmochelys imbricata) after intramuscular administration.

The pharmacokinetics of florfenicol (FFC) in green sea and hawksbill sea turtles were evaluated following intramuscular (i.m.) administration at two different dosages of 20 or 30 mg/kg body weight (b.w.). This study (longitudinal design) used 5 green sea and 5 hawksbill sea turtles for the two dosages. Blood samples were collected at assigned times up to 168 h. FFC plasma samples were analyzed using validated high-performance liquid chromatography equipped with diode array detection. The pharmacokinetic analysis was performed using a non-compartment approach. The FFC plasma concentrations increased with the dosage. The elimination half-life was similar between the treatment groups (range 19-25 h), as well as the plasma protein binding (range 18.59%-20.65%). According to the surrogate PK/PD parameter (T > MIC, 2 µg/mL), the 20 and 30 mg/kg dosing rates should be effective doses for susceptible bacterial infections in green sea and hawksbill sea turtles.

Hemoparasite Analysis and Hematological Parameters of Four Freshwater Turtle Species (Testudines: Podocnemididae) from the Brazilian Amazon.

METHODS: Sampling was performed at three sites in the Tapajós River mouth, Pará State, Brazil, and an aliquot of blood was collected from each turtle by post-occipital dorsal vein puncture. The aliquots were used for the determination of hematological parameters and for the study of hemoparasites in the blood samples. RESULTS: A total of 37 turtles were sampled: 21 Podocnemis expansa, three P. sextuberculata, nine P. unifilis, and four P. erythrocephala. Parasitological analyses showed inclusions in erythrocytes compatible with Haemogregarina sp., representing an overall prevalence of parasitized animals of 64.8% with an average of 94,789 parasites/ml of blood. In this study, the evolutionary forms of Haemogregarina spp. from trophozoites, pre-meronts, meronts, and mature and immature gamonts were observed in three species of freshwater turtle species, and one morphotype of the parasite with high similarity to Haemogregarina podocnemis has been observed. CONCLUSION: Despite recent research on freshwater turtles, this study provides new information on the intensity of blood parasite infection and hematological parameters of free-living animals, highlighting the need for studies on infected and uninfected animals from the same population.

Development of reference intervals for serum biochemistry and haematology of juvenile green sea turtles (Chelonia mydas) in a Thai rehabilitation centre.

No reference intervals for serum biochemistry and haematology of sea turtles in Thailand exists to assist veterinarians who are responsible for sea turtle health management and treatment. This study determined serum biochemistry and basic haematology of healthy juvenile green sea turtles (n = 92) in captivity in Thailand following the American Society for Veterinary Clinical Pathology (ASVCP), Quality Assurance and Laboratory Standards Committee (QALS) guidelines for the determination of reference intervals in veterinary species. Biochemistry tests, including blood urea nitrogen, creatinine, uric acid, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were analysed using an IDEXX VetTest Chemistry Analyzer. Haematology parameters were measured manually using a microhaematocrit for packed cell volume (PCV), Neubauer counting chamber for red blood cell count and cyanmethemoglobin method for haemoglobin concentration. mean corpuscular volume and mean corpuscular haemoglobin concentration were calculated using the PCV, red blood cell count and haemoglobin. Turtles in this study were found to have higher mean values for PCV (28.70%), haemoglobin (92.13 g/L), mean corpuscular haemoglobin concentration (327.03 g/L), uric acid (247.15 µmol/L), alanine aminotransferase (16.53 IU/L), aspartate aminotransferase (209.44 IU/L), and alkaline phosphatase (245.08 IU/L) compared to sea turtles in Brazil. The reference intervals established using high numbers of healthy turtles in this study will assist veterinarians with diagnostic and treatment decisions when evaluating laboratory results for juvenile green sea turtles.

Husbandry Protocols for Juvenile Loggerhead Sea Turtles (Caretta caretta) Based on Stress Response to Stocking Density and Dry-Dock Time.

When necessary, sea turtles are held captive for veterinarian care and research purposes. Protocols and basic guidelines have been described for husbandry of sea turtles with veterinarian needs but not considering physiological indicators of animal welfare. Because all sea turtle are imperiled species, monitoring their welfare is important. The aim of this study was to standardize husbandry protocols for loggerhead (Caretta caretta) juveniles held under seminatural conditions, based on circulating concentration of plasma corticosterone (Cort) and behavior. Two experiments were performed to analyze physiological and behavioral responses of the animals facing changes in stocking density and different dry-docking times. Cort analyses suggested that the number of animals per tank can be modified occasionally, without affecting their health and welfare. However, dry-docking time should be < 30 min, as indicated by the significant elevation of circulating Cort at ≥ 30 min, rising from 1.51- ng/ml to 5.28-ng/ml. Protocols tested did not affect behavioral responses, except for the breaths per move, which increased while Cort increased, despite differences exhibited by experimental animals in behavioral responses according to daily times (morning vs afternoon) and the sex of the animals.

An 18-µm microaggregate blood filter does not cause hemolysis during in vitro whole blood transfusions in sea turtles.

OBJECTIVE: Determine the hemolytic effect of an 18-µm microaggregate blood filter during in vitro sea turtle whole blood transfusions as well as describe the average diameter of leatherback (Dermochelys coriacea) and Kemp's ridley sea turtle (Lepidochelys kempii) RBCs. ANIMALS: 5 green (Chelonia mydas), 5 loggerhead (Caretta caretta), and 5 Kemp's ridley sea turtles (total n = 15). METHODS: Heparinized sea turtle blood was infused at 60 mL/h through a microbore extension set without and then with a postsyringe, inline 18-µm microaggregate blood filter. Pre- and postfiltration PCV, Hct, total solids, sodium, chloride, potassium, glucose, and free plasma hemoglobin concentrations were measured. With the use of light microscopy and archived blood smears, the maximum and minimum diameter of 20 RBCs from each of the 5 leatherback and 5 Kemp's ridley sea turtles were measured with a calibrated ocular micrometer using 400X magnification. RESULTS: There were no significant differences between pre- and postfiltration samples for Hct, total solids, sodium, chloride, potassium, glucose, and free plasma hemoglobin concentrations; however, there was a significant median postfiltration decrease in PCV of approximately 4%, representing a 13% decrease of the total RBCs transfused. Average maximum diameters for leatherback and Kemp's ridley sea turtle RBCs were 19.7 and 16.1 µm, respectively. CLINICAL RELEVANCE: Although the 18-µm microaggregate blood filter does not hemolyze transfused sea turtle RBCs and is likely safe for in vivo blood transfusions, the filter's pores may retain a small proportion of infused RBCs given their diameter.

Subcutaneous administration of ceftazidime at 20 and 40 mg/kg produces theoretically therapeutic plasma concentrations for at least 120 hours in red-eared sliders (Trachemys scripta elegans).

OBJECTIVE: To evaluate and compare the pharmacokinetic parameters of SC ceftazidime administered at 20 and 40 mg/kg to red-eared sliders. ANIMALS: 8 adult red-eared sliders (Trachemys scripta elegans). METHODS: In a sequential, 2-period study with a 3-week washout period between treatments, ceftazidime was administered SC to turtles at 20 and 40 mg/kg. Blood samples were collected from the subcarapacial sinus at 0, 24, 48, 72, 96, and 120 hours after ceftazidime administration. Plasma ceftazidime concentrations were quantified using reversed-phase HPLC. RESULTS: Mean plasma half-life after 20- and 40-mg/kg dosing was 39.75 ± 8.0 hours and 33.03 ± 6.56 hours, respectively. Mean maximum plasma concentration after 20- and 40-mg/kg dosing was 71.0 ± 15.93 µg/mL and 120.0 ± 30.62 µg/mL, respectively. Mean plasma ceftazidime concentrations remained ≥ 8 µg/mL, the theoretical MIC for various reptile pathogens for all time points. CLINICAL RELEVANCE: Results indicate that ceftazidime dosed at either 20 or 40 mg/kg produces plasma concentrations exceeding the theoretical MIC of various reptile pathogens for at least 120 hours. An ideal dosing interval could not be determined, as all plasma concentrations remained above the threshold of interest for all time points. Follow-up studies should focus on establishing a dosing interval and more rigorous monitoring for potential adverse effects.

Analysis of risk factors associated with gas embolism and evaluation of predictors of mortality in 482 loggerhead sea turtles.

Sea turtles that are entrapped in static and towed nets may develop gas embolism which can lead to severe organ injury and death. Trawling characteristics, physical and physiologic factors associated with gas-embolism and predictors of mortality were analysed from 482 bycaught loggerheads. We found 204 turtles affected by gas-embolism and significant positive correlations between the presence of gas-embolism and duration, depth, ascent rate of trawl, turtle size and temperature, and between mortality and ascent time, neurological deficits, significant acidosis and involvement of > 12 cardiovascular sites and the left atrium and sinus venosus-right atrium. About 90% turtles with GE alive upon arrival at Sea Turtle Clinic recovered from the disease without any supportive drug therapy. Results of this study may be useful in clinical evaluation, prognostication, and management for turtles affected by gas-embolism, but bycatch reduction must become a priority for major international organizations. According to the results of the present study the measures to be considered to reduce the catches or mortality of sea turtles for trawling are to be found in the modification of fishing nets or fishing operations and in greater awareness and education of fishermen.

Plasma electrophoresis profiles of Blanding's turtles (Emydoidea blandingii) and influences of month, age, sex, health status, and location.

Baseline plasma electrophoresis profiles (EPH) are important components of overall health and may aid in the conservation and captive management of species. The aim of this study was to establish plasma protein fractions for free-ranging Blanding's turtles (Emydoidea blandingii) and evaluate differences due to age class (adult vs. sub-adult vs. juvenile), sex (male, female, or unknown), year (2018 vs. 2019), month (May vs. June vs. July), health status, and geographical location (managed vs. unmanaged sites). Blood samples were obtained from 156 Blanding's turtles in the summer of 2018 and 129 in 2019 at two adjacent sites in Illinois. Results of the multivariate analysis demonstrated that age class, sex, year, month, health status, and geographical location all contributed to the variation observed in free-ranging populations. Adult females had the highest concentration of many protein fractions, likely associated with reproductive activity. Juveniles had lower protein concentrations. Temperature and rainfall differences between years impacted concentrations between 2018 and 2019, while May and June of both years saw higher levels in some protein fractions likely due to peak breeding and nesting season. Individuals with evidence of trauma or disease also showed increased plasma protein fractions when compared to those that were considered healthy. The two sites showed a wide/large variation over the two years. All of these factors emphasize the importance of considering multiple demographic or environmental factors when interpreting the EPH fractions. Establishing ranges for these analytes will allow investigation into disease prevalence and other environmental factors impacting this endangered species.

Evaluation of a dexmedetomidine-midazolam-ketamine combination administered intramuscularly in captive ornate box turtles (Terrapene ornata ornata).

OBJECTIVE: To characterize the effects of a combination protocol of dexmedetomidine-midazolam-ketamine (DMK) administered intramuscularly (IM) in ornate box turtles (Terrapene ornata ornata). STUDY DESIGN: Prospective experimental trial. ANIMALS: A total of 16 apparently clinically healthy adult ornate box turtles (eight male, eight female). METHODS: Each turtle was treated with dexmedetomidine (0.1 mg kg-1), midazolam (1 mg kg-1) and ketamine (10 mg kg-1) administered IM. Time to first response, time to maximal effect, the plateau phase and time to recovery from reversal administration were recorded. Physiologic variables, muscle tone, reflexes and the ability to perform endotracheal intubation were recorded at 5 minute intervals. Movement in response to an IM injection of 0.1 mL sterile 0.9% NaCl administered in the left pelvic limb, using a 25 gauge needle to a depth of just past the bevel of the needle, was assessed every 15 minutes. Atipamezole (0.5 mg kg-1) IM and flumazenil (0.05 mg kg-1) SC were administered 60 minutes after the initial DMK injections. RESULTS: The mean time to first response, time to maximal effect, the plateau phase and time to recovery were 2.1, 14.9, 38.7 and 7.8 minutes, respectively. A respiratory rate was not observed in most turtles. The body temperature significantly increased over time. The palpebral reflex was persistent in 43% of turtles and the tail pinch reflex remained intact in 13% of turtles. All turtles recovered with no observed adverse effects. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, this DMK protocol administered to ornate box turtles resulted in a rapid-onset, light anesthesia lasting approximately 40 minutes and a smooth recovery with no adverse effects noted.

Circulating miRNome of Trachemys scripta after elective gonadectomy under general anesthesia.

Post-surgical management is an important issue in veterinary medicine, requiring biomarkers with high sensitivity and specificity for timely and effective treatment. Emerging evidence suggests that miRNAs are promising stress- and pain-related markers. The aims were to profile the circulating miRNA signature in plasma of turtles (Trachemys scripta) and point out potential candidate biomarkers to assess the status of the animal. The plasma of female turtles underwent surgical gonadectomy were collected 24 h pre-surgery, and 2.5 h and 36 h post-surgery. The expression of miRNAs was profiled by Next Generation Sequencing and the dysregulated miRNAs were validated using RT-qPCR. The diagnostic value of miRNAs was calculated by ROC curves. The results showed that 14 miRNAs were differentially expressed over time. RT-qPCR validation highlighted that 2-miR-499-3p and miR-203-5p-out of 8 miRNAs tested were effectively modulated. The Area Under the Curve (AUC) of miR-203-5p was fair (AUC 0.7934) in discriminating pre- and 36 h post-surgery samples and poor for other time points; the AUC of miR-499-3p was excellent (AUC 0.944) in discriminating pre-surgery and 2.5 h post-surgery samples, and fair in discriminating pre-surgery and 36 h post-surgery (AUC 0.7292) and 2.5 h and 36 h post-surgery (AUC 0.7569) samples. In conclusion, we demonstrated for the first time that miRNAs profile changes in plasma of turtles underwent surgical oophorectomy and identified miR-203-5p and miR-499-3p as potential candidate biomarkers to assess animals' status. Further studies are necessary to confirm their diagnostic value and to investigate functional and mechanistic networks to improve our understanding of the biological processes.

Hematologic variables of the Arrau turtle (Podocnemis expansa) under the effects of different anticoagulants and cytologic stains.

BACKGROUND: The hematology of turtles of the Amazon, such as the Arrau turtle (Podocnemis expansa), has not been well described in the literature. Referencing how to collect and analyze blood samples is fundamental for the medical practice of these exotic animals. OBJECTIVES: The aim of this study was to evaluate the hematologic parameters of Arrau turtles of the Amazon to determine the best anticoagulant (EDTA, citrate, or sodium heparin) and cytologic stain (Giemsa, Quick Panoptic, or Rosenfeld) for use with this species. METHODS: Blood samples from eight turtles were collected. Three blood smears were made using blood without anticoagulant, and the rest of the sample was distributed in three tubes, containing EDTA, citrate, and sodium heparin, for erythrogram and leukogram analyses. RESULTS: All blood samples anticoagulated with EDTA showed marked hemolysis. Blood collected with citrate showed minimal hemolysis, and blood collected with sodium heparin did not have any hemolysis. Some analyses were not performed on blood containing EDTA because of the intense hemolysis. The erythrocyte variables were obtained and showed better results for blood samples in heparin. Blood smears without anticoagulant were appropriate for differential leukocyte counts with the three stain types, while blood with any of the anticoagulants made it difficult to differentiate leukocytes. CONCLUSIONS: Sodium heparin showed the best results and should be the anticoagulant of choice for hematologic studies in P expansa. All cytologic stains were efficient, allowing adequate identification of distinct cell groups. Leukocyte differentiation was assured and had better contrast using blood smears made from blood without an anticoagulant.

TERBINAFINE PHARMACOKINETICS FOLLOWING SINGLE-DOSE ORAL ADMINISTRATION IN RED-EARED SLIDER TURTLES (TRACHEMYS SCRIPTA ELEGANS): A PILOT STUDY.

In this pilot study, the pharmacokinetics of terbinafine were determined in six apparently healthy red-eared slider turtles (Trachemys scripta elegans) after a single PO administration. Terbinafine suspension (15 mg/kg, once) was administered via gavage tube to all turtles. Blood samples were collected immediately before (time 0) and at 1, 2, 4, 8, 24, and 48 h after drug administration. Plasma terbinafine concentrations were quantified by ultra-performance liquid chromatography-mass spectrometry, and noncompartmental pharmacokinetic analysis was performed. None of the animals showed any adverse responses following terbinafine administration. Mean area under the curve from time 0 to 24 h was 1,213 h × ng/ml (range 319-7,309), mean peak plasma concentration was 201.5 ng/ml (range 45.8-585.3), mean time to maximum plasma concentration was 1.26 h (range 1-4), mean residence time was 7.71 h (range 3.85-14.8), and mean terminal half-life was 5.35 h (range 2.67-9.83). The administration of terbinafine (15 mg/kg, PO) may be appropriate for treatment of select fungal organisms with low minimum inhibitory concentrations in red-eared slider turtles but may require q12h administration even for organisms with low minimum inhibitory concentrations. Multiple-dose studies as well as clinical studies are needed to determine ideal dosages and efficacy.

PLASMA VORICONAZOLE CONCENTRATIONS FOLLOWING SINGLE- AND MULTIPLE-DOSE SUBCUTANEOUS INJECTIONS IN WESTERN POND TURTLES (ACTINEMYS MARMORATA).

A recently characterized fungal pathogen, Emydomyces testavorans, has been associated with ulcerative shell disease and significant morbidity in Western pond turtles. Voriconazole is a second-generation triazole antifungal medication that prevents fungal growth through disruption of ergosterol synthesis, causing abnormalities in the fungal cell membrane. Preliminary reports of minimum inhibitory concentrations (MIC) indicate that voriconazole is effective in vitro against E. testavorans. Ultraperformance liquid chromatography was used to measure voriconazole plasma concentrations in blood samples from healthy Western pond turtles after administration of a single SC injection of 10 mg/kg and after multiple doses (10 mg/kg SC q48h for seven doses). The data were analyzed using a naïve pooled approach. Mean (SE) observed time to maximum concentration was 2 (0.18) h for a single dose and 50 (2.2) h for multiple doses; the multiple-dose trial observed mean (SE) maximum concentration was 12.4 (2.2) µg/ml, and observed mean (SE) trough concentration was 1.7 (0.7) µg/ml. Multifocal skin sloughing following the single-dose trial was observed in one turtle and there was a significant increase in polychromatophilic cells amongst the study turtles after the multiple-dose voriconazole trial. No other adverse effects were observed. When voriconazole was administered at 10 mg/kg SC q48h, observed trough plasma concentrations were consistently higher than reported E. testavorans MIC concentrations. Further study is needed to determine the clinical safety and in vivo efficacy of this dose in Western pond turtles.