Evaluation of cyclooxygenase protein expression in traumatized versus normal tissues from eastern box turtles (Terrapene carolina carolina)

This pilot study was designed to determine whether cyclooxygenase (COX)-1, COX-2, or both are expressed in normal turtle tissues and whether level of expression changes when tissue becomes inflamed. Five eastern box turtles, Terrapene carolina carolina, that either died or were euthanatized due to disease or injuries were used for this work. Tissues were obtained from the five turtles. Western blot analysis was used to evaluate tissues for COX-1 and COX-2 proteins. Densiometric analysis was used to compare Western blot bands within each turtle. COX-1 and COX-2 were found in the liver, kidney, grossly normal muscle, and grossly traumatized (inflamed) muscle of all study turtles. In all cases, COX-1 and COX-2 proteins were increased in traumatized muscle over grossly normal nontraumatized muscle. The highest levels of COX-1 and COX-2 proteins were found in kidney and liver. There was no statistical difference between the amount of COX-1 protein in liver and kidney, but traumatized muscle compared with grossly normal muscle had significantly greater COX-1 but not COX 2 protein concentrations. There was no statistical difference between the amount of COX-2 protein in liver and kidney. Traumatized muscle expressed nonstatistically significant greater amounts of COX-2 compared with grossly normal muscle. COX-1 and COX-2 proteins are expressed in turtle tissues, and both isoforms are upregulated during inflammation of muscle tissue. Traditional nonsteroidal anti-inflammatory drugs (NSAIDs) that block both COX isoforms might be more efficacious than COX-2-selective drugs. This work suggests that NSAIDs should be evaluated for potential liver and kidney toxicity in turtles.     

Pharmacokinetics of ketorolac in wild Eastern box turtles (Terrapene carolina carolina) after single intramuscular administration

Ketorolac is a nonsteroidal anti‐inflammatory drug that possesses potent analgesic activity comparable to morphine. The opioid shortage in the United States has led to an unreliable supply of opioids for use in rehabilitation facilities, thus underscoring the need for research on the safe and effective use of nonopioid alternatives. The goal of this study was to determine the pharmacokinetics of ketorolac after a single 0.25 mg/kg intramuscular injection administered to injured Eastern box turtles (Terrapene carolina carolina). A sparse blood sampling protocol was used to collect samples from 32 wild turtles that presented to the Turtle Rescue Team at North Carolina State University for traumatic injuries. Blood was collected from 0 to 24 hr after injection and analyzed via high‐pressure liquid chromatography (HPLC). A nonlinear mixed‐effects (NLME) model was fitted to the data to obtain typical values for population parameters. Using this approach, we identified a long half‐life (T_1/2) of 9.78 hr and a volume of distribution (V_ss) of 0.26 L/kg. We have concluded that this long T_1/2 for a dose of 0.25 mg/kg ketorolac‐injected IM provides plasma levels above a previously published target level for 24‐hour analgesia to allow for once daily dosing.     

Radiographic anatomy and barium sulfate contrast study of the gastrointestinal tract of eastern box turtles (Terrapene carolina carolina)

Gastrointestinal disorders are an important cause of morbidity in box turtles (Terrapene carolina Carolina), however published information is currently lacking on the normal radiographic anatomy, transit, and emptying times of the gastrointestinal tract. A total of 15 healthy box turtles were recruited for this prospective, anatomic, reference interval study. Three‐view radiographic series (vertical beam dorsoventral, horizontal beam latero‐lateral, and horizontal beam rostrocaudal views) were acquired prior to contrast administration, and following contrast administration at 0, 20, 40, 60, and 90 min, 2, 4, 8, 12, and 24 h post administration, and every 24 h thereafter until all contrast was eliminated (15 mL/kg barium sulfate diluted to 30% weight per volume was administered via orogastric gavage). Vertical beam dorsoventral and horizontal beam latero‐lateral views were of excellent quality to identify gastrointestinal structures. The horizontal beam rostrocaudal view immediately postcontrast administration provided gastric and pyloric identification but had lesser diagnostic use at later time points due to anatomical superimposition. The gastrointestinal tract was composed of a tubular stomach, a pyloric sphincter near midline, a duodenum with a cranial flexure in the right cranial coelomic cavity, small intestines within the right coelom, a small cecal bulb, and a transverse and descending colon. Contrast media entered the large intestine by 24 h in all turtles, and a pyloro‐colic indentation was noted at the proximal descending colon. The large intestinal emptying was highly variable due to the interindividual variability of contrast sequestration within the cecal bulb. Findings from the current study serve as a reference on the gastrointestinal anatomy, transit, and emptying times in healthy eastern box turtles; and introduce a novel, horizontal beam, rostrocaudal view for gastrointestinal contrast studies in chelonians.     

Quantifying visual acuity of Terrapene carolina triunguis utilizing optokinetic response

Objective

Quantify visual acuity of a semi-aquatic turtle species, Terrapene carolina triunguis (three-toed box turtles), utilizing optokinetic response/reflex/nystagmus.

Methods

Ten turtles were placed on a custom platform and an image of white and black gratings of varying sized widths was projected onto a white screen in front of the turtles. Optokinetic response (OKR) was observed through a web camera as well as physically by an observer. The grating sizes were decreased until OKR could not be elicited. The size of grating was recorded and was used to calculate visual acuity.

Results

The average visual acuity was 0.26 cycle per degree (cpd) with the highest recorded value being 0.43 cpd and lowest being 0.15 cpd. Neither sex nor weight were significantly associated with visual acuity (p = .24 and .98, respectively).

Conclusions

Visual acuity of these semi-aquatic turtles was much lower than previously reported visual acuities of aquatic turtles. This could be due to differences in refractive indices between water and air, behavioral adaptations for the different species and/or method of obtaining visual acuity.     

Identification of a novel herpesvirus in captive Eastern box turtles (Terrapene carolina carolina)

Herpesviruses are significant pathogens of chelonians which most commonly cause upper respiratory tract disease and necrotizing stomatitis. Herpesvirus infection was identified in two populations of captive Eastern box turtles (Terrapene carolina carolina) using histopathology and polymerase chain reaction (PCR) with DNA sequencing. Necrotizing lesions with eosinophilic to amphophilic intranuclear inclusion bodies were identified in the tissues of one hatch-year individual in January 2013, which was herpesvirus positive by PCR. A separate captive group of adults had an observed herpesvirus prevalence of 58% using PCR in July 2011. In these cases, a novel herpesvirus, Terrapene herpesvirus 1 (TerHV1), was identified and serves as the first herpesvirus sequenced in the genus Terrapene. Similar to the other herpesviruses of the Order Testudines, TerHV1 clusters with the genus Scutavirus of the subfamily Alphaherpesvirinae.     

Myiasis in two box turtles.

Two eastern box turtles (Terrapene carolina carolina) were treated for myiasis caused by Sarcophaga cistudinis. The tortoises were examined because of swellings of the proximal cervical regions. Both fully recovered following surgical removal of multiple larvae.     

Evaluation of lidocaine for brachial plexus blockade in eastern box turtles (Terrapene carolina carolina)

ObjectiveTo evaluate latency and duration of a brachial plexus block technique in eastern box turtles performed with 2% lidocaine at three dose rates.Study designProspective, randomized, blinded crossover study.AnimalsAdult eastern box turtles, two for drug dose evaluation and a group of six (three male, three female) weighing 432 ± 40 g (mean ± standard deviation) for the main study.MethodsAnimals were randomly assigned to four brachial plexus blocks with lidocaine at 5, 10 and 20 mg kg^–1 or 0.9% saline (treatments LID5, LID10, LID20 and CON, respectively), separated by 1 week. Treatment side was randomized and blocks were performed unilaterally. Baseline observations of mentation, heart rate (HR), respiratory rate (f_R), skin temperature and limb response to manipulation or toe pinch were evaluated. Assessments were made every 10 minutes until 1 hour of normal sensory and motor function to the treated thoracic limb, or for a total of 2 hours if no block was evident.ResultsMotor and sensory blockade was achieved in treatments LID10 and LID20 in one turtle, with a latency of 10 minutes and duration of 50 minutes for both doses. Raising of the ipsilateral lower palpebra occurred with both blocks. Turtles administered lidocaine experienced higher HR compared with CON, and HR decreased over time for all individuals. Mentation and f_R were not changed with any lidocaine dose.ConclusionsThe technique was unreliable in producing brachial plexus motor and sensory blockade at the lidocaine doses evaluated in this study. HR was higher in lidocaine-administered turtles but remained within normal limits for the species. No change in mentation or f_R was observed among treatments.Clinical relevanceGeneral anesthesia with systemic analgesia is recommended for surgical procedures involving the chelonian thoracic limb. Further studies are needed to optimize a brachial plexus block in this species.     

Prevalence of Salmonella spp in cloacal, fecal, and gastrointestinal mucosal samples from wild North American turtles

OBJECTIVE: To determine prevalence of Salmonella spp in samples collected from wild North American turtles. ANIMALS: 94 wild North American turtles of 6 species in 2 genera. DESIGN: Prospective microbiologic study. PROCEDURES: A convenience sample of wild North Carolina turtles admitted to a veterinary college was evaluated for Salmonella spp by use of standard techniques via microbiologic culture of cloacal swab and fecal samples. Gastrointestinal mucosa samples were also collected at necropsy from turtles that died or were euthanized. Cloacal swab samples were also collected from wild pond turtles for bacteriologic culture. Controls were established by use of wild-type Salmonella Typhimurium LT2. RESULTS: 94 turtles were tested for Salmonella spp; Salmonella spp were not detected in any sample. By use of a pathogen-prevalence and sample-size table, the true prevalence of Salmonella spp was estimated as < 5%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that wild turtles in central North Carolina may not be active shedders or carriers of Salmonella spp. Despite this 0% prevalence of infection, proper hygiene practices should be followed when handling wild turtles.     

Hematology of the keeled box turtle (Cuora mouhotii)

A healthy adult, intact female keeled box turtle (Cuora mouhotii) was found to have a marked heterophilic leukocytosis using normal hematologic parameters established for the eastern box turtle (Terrapene carolina carolina), a related chelonian species. This animal was monitored with serial complete blood counts (CBCs) over the next 15 years despite remaining asymptomatic for an infectious condition. Retrospective CBC data were compiled from 38 presumably healthy keeled box turtles to establish hematologic values for comparison in this species. Using this species-specific data, over the 15-year period, the female keeled box turtle had two times where the white blood cell (WBC) count was greater than 2 standard deviations (SD) above the mean, six times where the WBC count was greater than 1 SD above the mean, six times where the PCV was greater than 2 SD above the mean, and eight times where the PCV was greater than 1 SD above the mean. Infection and inflammation are the most common causes of leukocytosis in reptiles; however, given the clinical presentation of this patient, it was postulated that these clinicopathologic changes could be secondary to a stress response. Establishing reference intervals and understanding how stress impacts CBC parameters are important for evaluating the health status of keeled box turtles kept in captivity and for assessing the effects of environmental changes on the health status of wild populations of this endangered chelonian species.     

Herpesvirus-like infection in map turtles

Two map turtles housed in an aquarium with a group of other map turtles became lethargic and anorectic, and subcutaneous edema developed prior to their deaths. Gross postmortem findings included subcutaneous edema and swollen, diffusely pale livers. Histologic examination of the livers revealed multifocal to diffuse areas of necrosis, with intranuclear eosinophilic inclusions in hepatocytes. Intranuclear and intracytoplasmic aggregates of particles morphologically resembling herpesvirus were identified in degenerating hepatocytes by electron microscopy.     

Avipoxvirus infection in peregrine falcons (Falco peregrinus) from a reintroduction programme in Germany

Poxvirus infections are common in domestic birds in Germany, but they are rare in birds of prey. Only species of falconidae imported from Arabian or Asian countries have so far tested positive for poxvirus, and, among these, only raptors kept for falconry. As part of a reintroduction programme in the northern county of Mecklenburg-Western Pomerania, which is adjacent to the Baltic Sea, 21 young peregrine falcons were released into the wild; six of them died and one was examined postmortem, its tissues being examined by light and electron microscopy. In addition, an ELISA for fowlpox, pigeonpox and canarypox was applied. No virus could be isolated and propagation in culture failed, but virus particles were detected by electron microscopy in lesions from its skin and tongue.     

Renal myxozoanosis in crowned river turtles Hardella thurjii: description of the putative agent Myxidium hardella n. sp. by histopathology, electron microscopy, and DNA sequencing

Chelonian myxozoanosis is rarely reported and has previously not been documented to cause disease. This report describes myxozoanosis associated with significant renal disease in two Crowned River turtles (Hardella thurjii). One turtle presented with emaciation and died. The cage mate presented with emaciation and was euthanized. Histologically, renal intratubular myxozoan spores were associated with renal tubular necrosis, tubular mineralization, and chronic interstitial nephritis, with membranoproliferative and mes-angioproliferative glomerulopathy. Both turtles also had disseminated metastatic mineralization. On the basis of these findings, chronic renal insufficiency from myxozoanosis and subsequent metastatic mineralization were considered the primary problems. By light and electron microscopy, the myxozoan spores had features of the genus Myxidium. Maximum parsimony analysis of small-subunit rDNA sequences placed the turtle myxozoan basal to a clade containing Myxidium truttae and a Myxidium sp. with strong bootstrap support. This myxozoan agent appears to be a significant pathogen in H. thurjii on the basis of morphologic changes in the kidneys of in the infected turtles.     

Generalized amyloidosis in an Eastern Bongo (Tragelaphus eurycerus isaaci)

Amyloidosis has been described in a wide range of domestic and wild species and man. A 10-yr-old male Eastern (Mountain) Bongo (Tragelaphus eurycerus isaaci) was submitted for postmortem examination after a period of 24-hr malaise. Gross examination found evidence of biventricular cardiac hypertrophy, congestive heart failure, and focal pulmonary abscessation. Histologic changes in the heart were consistent with hypertrophic change. Amyloid deposits were found within the liver, kidney medulla, heart, adrenal cortex, and pituitary gland and were confirmed as reactive systemic amyloid (AA) by immunohistochemistry. The pulmonary abscessation was thought to be the stimulus for excessive serum amyloid associated protein production leading to the reactive systemic amyloidosis. Colloidal goiter was also identified as an incidental finding.     

Detection of a novel herpesvirus associated with squamous cell carcinoma in a free-ranging Blanding’s turtle

The spread of both infectious and noninfectious diseases through wildlife populations is of increasing concern. Neoplastic diseases are rarely associated with population-level impacts in wildlife; however, impacts on individual health can be severe and might reflect deteriorating environmental conditions. An adult male free-ranging Blanding’s turtle (Emydoidea blandingii) originally captured in 2005 and deemed healthy, was recaptured in 2018 with a 1 × 1.5 cm intra-oral broad-based right mandibular mass. An excisional biopsy was performed, and histopathology revealed squamous cell carcinoma (SCC). Consensus herpesvirus PCR identified a novel herpesvirus (proposed name Emydoidea herpesvirus 2 [EBHV-2]) within the tumor. EBHV-2 shares 85% sequence homology with Terrapene herpesvirus 2 (TerHV-2), a herpesvirus linked to fibropapillomas in eastern box turtles (Terrapene carolina carolina). Virus-associated fibropapillomas have been identified in multiple marine turtle species and have had debilitating effects on their populations, but to date, virus-associated SCCs are rarely reported.     

Reptiles with dermatological lesions: a retrospective study of 301 cases at two university veterinary teaching hospitals (1992-2008)

This retrospective study reviews the medical records of 301 reptiles with dermatological lesions that were examined at the Veterinary Medical Teaching Hospital, University of California at Davis (VMTH-UCD) and the Unité de Dermatologie-Parasitologie-Mycologie, Ecole Nationale Vétérinaire de Nantes (UDPM-ENVN) from 1 January 1992 to 1 July 2008. The most common reptile groups differed between the two hospitals, with lizards being the most common at the VMTH-UCD and chelonians at the UDPM-ENVN. At the VMTH-UCD, boa constrictors (Boa constrictor), ball pythons (Python regius) and other Python species were over-represented, and box turtles (Terrapene carolina) were under-represented in the dermatological lesion caseload. When institutional data were combined, 47% of all reptiles at both institutions with confirmed or suspected cases of sepsis had petechiae, with the highest association seen in chelonians at 82%. Dependent on institution and reptile group, from 29% to 64% of the cases had underlying husbandry issues. Sixty-two per cent of all cases were alive at final status. Veterinarians treating reptiles with skin disease should be aware of the following: (i) that boa constrictors and Python species may be predisposed to dermatological lesions; (ii) that client education is important for proper husbandry; and (iii) that there is a possible association between petechiae and sepsis, especially in chelonians. The conjectural association between certain skin lesions and sepsis remains to be confirmed by systematically derived data that demonstrate a causal relationship between the two.     

First confirmed canine case of Ehrlichia canis infection in Japan

An 11-year-old castrated Pekinese dog that had been moved from Indonesia to Japan eight years previously was diagnosed with an Ehrlichia canis infection by haematological characteristics (normocytic anaemia, mild thrombocytopenia and hypergammaglobulinaemia) and serological findings (antibody titre to E canis 1:3,200 or more). The dog did not respond to treatment with tetracycline and died from renal failure. The diagnosis was confirmed postmortem by pathological evaluation and polymerase chain reaction (PCR) followed by sequencing of the 16S rRNA gene. Typical morulae of Ehrlichia were detected in the cytoplasm of macrophages in spleen tissue by immunohistological staining. Ehrlichia-like organisms were also detected in the spleen by electron microscopy. E canis-specific PCR analysis of DNA extracted from the spleen gave a positive signal, and sequence analysis of the fragment revealed that it was identical to part of the 16s rRNA gene of E canis. The dog was the first confirmed clinical case of E canis infection in Japan.