A mystery revealed: an update on eosinophil and other blood cell morphology of the Argentine black and white tegu (Salvator merianae).

Reptile white blood cell (WBC) morphological features are strikingly variable across species. In the Argentine black and white tegu (Salvator merianae), red tegu (Salvator rufescens), and Savannah monitor (Varanus exanthematicus), previous reports described a WBC type with a single distinct, clear, linear- to ovoid- to crescent-shaped inclusion of presumptive monocytic origin. The objective of this study was to further investigate the origin of this unique WBC type with crescent-shaped inclusions. Blood samples from two Argentine black and white tegus, tegu 1, a 4-year-old female, and tegu 2, a 2-year-old presumed male, were submitted for routine hematological evaluation. Additional blood films were prepared and stained with these cytochemical stains: alkaline phosphatase (ALP; naphthol AS-MX phosphate substrate), alpha-naphthyl butyrate esterase, alpha-chloroacetate esterase, myeloperoxidase, Periodic acid-Schiff, and Sudan black B. Blood films from tegu 1 were also stained with a second ALP stain (5-bromo-4-chloro-3-indoxyl-phosphate and nitroblue tetrazolium substrate), Luna, luxol fast blue, and toluidine blue. The blood from tegu 1 was cytocentrifuged to isolate and fix the buffy coat in glutaraldehyde 2.5% aqueous solution for transmission electron microscopy. Six morphologically distinct WBC types were identified from tegu 1, including heterophils, basophils, monocytes, azurophils, lymphocytes, and the unique WBC type, which were identified as eosinophils with inclusions. WBC types in tegu 2 were similar; however, eosinophils lacked a discernable inclusion. Proper WBC identification will be useful in obtaining accurate hemogram data for this species.

Use of Blood Lactate in Assessment of Manual Capture Techniques of Zoo-Housed Crocodilians.

Species-specific welfare indicators are important in promoting positive welfare for zoo animals. Reptiles are a notoriously understudied group in regards to behavior, welfare needs, and husbandry requirements. Using opportunistically obtained samples, we evaluated how blood lactate is affected by variation in manual capture and restraint in crocodilians. Lactate is an indicator of anerobic metabolism in reptiles. It offers a relatively simple and indirect way to assess physiological stress. Determining the best way to reduce struggling during capture and associated stress is of great importance to these species' overall welfare. Blood samples (N = 53) were collected from 10 different species of crocodilians. It was found that age class was a significant predictor variable of lactate levels after capture, and longer handling time seemed to cause an increase in lactate. Finally, draining enclosure pools for a small number of the captures was associated with higher lactate levels compared to other capture factors that were recorded. This study showed that welfare of captive crocodilians could be improved by shortening the duration of physical restraint events when possible. Developing objective measures of welfare and establishing baseline recommendations for care and handling of crocodilians will ultimately promote and improve their wellbeing, along with that of other understudied reptiles in zoos.

AUSTWICKIA CHELONAE IN A WILD GOPHER TORTOISE (GOPHERUS POLYPHEMUS) AND EVIDENCE OF POSITIVE SELECTION ON THE DIPHTHERIA-LIKE TOXIN GENE.

Austwickia (Dermatophilus) chelonae is a filamentous, Gram-positive Actinobacteria in the Dermatophilaceae family. It has caused fatal granulomatous disease in diverse captive reptile species on three continents, but its presence in wild or free-ranging populations was unknown. An adult female gopher tortoise (Gopherus polyphemus) was presented euhydrated, but cachectic and infested with ticks, with two firm, encapsulated masses over the cranioventral neck and right stifle. The tortoise had moderate nonregenerative anemia and evidence of inflammation; plasma biochemistry data was within normal limits. Fine needle aspirate of the neck lesion revealed abundant necrosis and aggregates of cocci. Computed tomography delineated the masses and revealed an additional mass adjacent to the left zygomatic bone. After surgical excision, histology identified chronic granulomas with intralesional filamentous bacteria. Pan-bacterial 16S rRNA PCR and sequencing of the masses identified A. chelonae. Despite treatment with oxytetracycline and ceftazidime, the tortoise deteriorated and was euthanatized. An esophageal lesion consistent with A. chelonae was seen on postmortem examination, although it was determined that the tortoise ultimately succumbed to fungal pneumonia caused by Metarhizium robertsii, an entomopathogenic biotoxin sprayed as insect control. This case reveals A. chelonae is present in free-ranging chelonians in North America. This organism produces a toxin gene similar to diphtheria toxin, one of the most potent known biotoxins, which has not been previously identified outside the genus Corynebacterium. Novel PCR primers were designed for the toxin and rpoB genes, which were amplified and sequenced from two cases and compared with two available genomes. Selection analysis revealed that the toxin gene is under positive selection, which implies it interacts significantly with the immune system, making it a good candidate for immunodiagnostic test development.

Sedation and Anesthesia of Galapagos (Chelonoidis nigra), Aldabra (Aldabrachelys gigantea), and African Spurred Tortoises (Centrochelys sulcata): A Retrospective Review (2009-2019).

Tortoises belong to the taxonomic family Testudinidae, which is considered one of the most imperiled families of the order Testudines. Anesthesia is often required for the medical and surgical management of large tortoises. The objectives of this retrospective study were to review drug regimens used to successfully anesthetize Galapagos (Chelonoidis nigra), Aldabra (Aldabrachelys gigantea) and African spurred (Centrochelys sulcata) tortoises, and to compare the times to effect and to extubation in tortoises administered different premedication protocols. Anesthetic records of giant tortoises admitted to the University of Florida College of Veterinary Medicine between January 2009 and December 2019 were reviewed. A total of 34 tortoises (six Aldabra, 23 Galapagos, and five African spurred) were included, resulting in 64 anesthetic events. Frequently used premedication protocols included an α2-adrenergic agonist and ketamine combined with either midazolam (group α2-adrenergic agonist, midazolam, ketamine, AMK; n = 34), a µ-opioid receptor agonist (group α2-adrenergic agonist, µ-opioid receptor agonist, ketamine, AOK; n = 13), or a µ-opioid receptor agonist and midazolam (group α2-adrenergic agonist, midazolam, µ-opioid receptor agonist, ketamine, AMOK; n = 10). Inhalant anesthetics (isoflurane, n = 21; sevoflurane, n = 23) were frequently used for maintenance of anesthesia following premedication. Out of the 34 total tortoises, 22 had only one anesthetic event, five had two anesthetic events, three had three anesthetic events, and four had four or more anesthetic events. Few adverse effects were observed and there was no mortality reported during the peri-anesthetic period. Sedation and general anesthesia of giant tortoises can be successfully performed with a combination of an α2-adrenergic agonist and ketamine in combination with midazolam and/or a µ-opioid receptor agonist.

Identification of a novel mortality-associated Helicobacter species in gopher tortoises (Gopherus polyphemus), qPCR test development and validation, and correlation with mortality in a wildlife rehabilitation population.

The genus Helicobacter includes spiral-shaped bacteria in the phylum Proteobacteria, class Epsilonproteobacteria, order Campylobacteriales, that have been associated with disease in animals, including reptiles. Three wild gopher tortoise (Gopherus polyphemus) index cases presented between 2012 and 2019 with nasal discharge, lethargy, and weight loss. Cytological examination of nasal discharge from all 3 tortoises identified marked heterophilic and mild histiocytic rhinitis with abundant extracellular and phagocytized spiral shaped bacteria that stained positive with Warthin-Starry stain. Polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene revealed this to be a novel Helicobacter species. Two tortoises died despite treatment attempts, and the third was moribund and was euthanized. Histological examination of the nasal mucosa (n = 3) showed granulocytic to lymphocytic rhinitis with variable mucosal hyperplasia, erosion, and ulceration; Warthin-Starry staining highlighted the presence of spiral bacteria in the untreated tortoise. Genus-specific primers were designed, and the gyrA and groEL genes were amplified by PCR and sequenced. Phylogenetic analysis shows that this organism and other previously characterized Helicobacter from tortoises form a clade. Development and cross-validation of two qPCR diagnostic assays for the gyrA and groEL genes showed significant correlation of the results of two assays (P < 0.0001). These assays were used to survey nasal wash samples from 31 rehabilitating gopher tortoises. Mortality of tortoises significantly correlated with higher Helicobacter loads detected by qPCR (P = 0.028). Appropriate quarantine protocols for tortoises during rehabilitation should consider this organism. Upper respiratory disease in tortoises may involve complex microbial ecology; factors beyond Mycoplasmopsis (Mycoplasma) agassizii should be taken into account.

GRANULOMATOUS ENCEPHALOMYELITIS IN A FALSE GHARIAL (TOMISTOMA SCHLEGELII) ASSOCIATED WITH A NOVEL CHLAMYDIA SPECIES.

A 5-yr-old, captive, hatched, female false gharial (Tomistoma schlegelii) presented with a 1-mo history of cervical spinal curvature. Antemortem diagnostics, including blood work, electromyography, muscle biopsies, and advanced imaging tests, were either within reference ranges or did not identify any specific etiology. Necropsy revealed extensive, marked, chronic granulomatous encephalomyelitis along with neuronal necrosis, rarefaction, gliosis, and astrocytosis of the white and gray matter of the cerebrum, cerebellum, brainstem, and spinal cord. Pan-chlamydiae polymerase chain reaction protocols for the 16S ribosomal RNA and ompA genes were performed on samples of spinal cord and brain, and both resulted in amplicons. Sequencing of the products revealed that they were positive for a novel Chlamydia species. Infections by members of the phylum Chlamydiae have been reported in a diverse range of vertebrate hosts, including crocodilians. Chlamydia spp. infections are likely underdiagnosed because of a paucity of diagnostic techniques specific for detection. This is the first case report of a novel Chlamydia species associated with severe granulomatous encephalomyelitis in a false gharial.

Gastric Intussusceptions in a Red Corn Snake (Pantherophis guttatus) Associated with Cryptosporidiosis.

A 3-year-old female red corn snake (Pantherophis guttatus) was presented for a three-week history of anorexia and decreased defecations. On physical examination, a soft midbody intracoelomic swelling was palpated. Transcutaneous coelomic ultrasound revealed a target-like mass on a transverse section of the stomach, suggesting the presence of a gastrointestinal intussusception. On exploratory coeliotomy, a double compounded esophagogastric and gastroduodenal intussusception was diagnosed and reduced surgically. A gastropexy was also performed to prevent recurrence. On histopathology, the gastric glandular mucosa showed moderate to marked proliferation. Diffusely lining the luminal surface of glandular epithelium and free within the lumen were a myriad of protozoa consistent with Cryptosporidium sp. A diagnosis of chronic proliferative gastritis due to Cryptosporidium sp. was made based on these findings. Intussusceptions are rare in reptiles and are infrequently reported in snakes. This is the first report of a double compounded intussusception in a nonmammalian species and the first report of an intussusception involving the stomach in a snake with gastritis due to Cryptosporidium sp.

BLASTOCYSTIS SP. AND BLASTOCYSTIS RATTI IN A BRAZILIAN PORCUPINE (COENDOU PREHENSILIS) WITH DIARRHEA.

A hand-raised, 5-mo-old, intact male Brazilian porcupine (Coendou prehensilis) was evaluated for chronic diarrhea, failure to thrive, and anorexia. On presentation the porcupette was dull, dehydrated, and passing yellow, malodourous, watery diarrhea. Cytologic examination of feces revealed a large number of organisms, morphologically consistent with Blastocystis. Blastocystis polymerase chain reaction (PCR) performed on feces was positive. Direct sequencing on two sequential samples confirmed the presence of Blastocystis ratti and a novel Blastocystis sequence. The porcupette was treated supportively, which included a 4-wk metronidazole course. Diarrhea resolved within 2 wk of treatment, and the animal's growth rate dramatically improved. Recheck PCR was negative for Blastocystis. Although an important and controversial cause of diarrhea in immunocompromised humans, this organism is not well recognized as a potential pathogen and zoonosis in zoo animals. Clinicians should be aware of the potential for disease associated with this organism, especially in immunocompromised animals.

MANAGEMENT OF A PATENT URACHUS AND YOLK COELOMITIS IN A PREHENSILE-TAILED SKINK (CORUCIA ZEBRATA).

An 11-day-old, captive-born, male prehensile-tailed skink (Corucia zebrata) was evaluated for a chronically swollen umbilicus. On presentation, the skink appeared dehydrated and weak. The umbilical stump was sensitive, edematous, and erythematous. Yellow fluid was readily expressed during palpation of the surrounding area, suggestive of urine. Following several days of supportive care, a positive contrast cloacagram supported the diagnosis of a patent urachus. An exploratory celiotomy was performed, confirming yolk coelomitis and a patent urachus. Both were corrected surgically, and the skink improved steadily thereafter. This report confirms the presence of a urinary bladder in C. zebrata and is the first report of a patent urachus in a reptile. Surgical intervention and medical management of concurrent infectious coelomitis were curative in this case.

Successful management of hypertrophic cardiomyopathy in a Matschie's tree kangaroo (Dendrolagus matschiei).

A 3-yr-old, intact male Matschie's tree kangaroo (Dendrolagus matschiei) was examined for a 1-wk history of intermittent lethargy and tachypnea. An echocardiogram revealed concentric hypertrophy of the left ventricular free wall and interventricular septum. These findings were compared to measurements from healthy Matschie's tree kangaroos, supporting a diagnosis of hypertrophic cardiomyopathy. At the time of publication, the patient has been managed for over 11.5 yr, using a combination of enalapril, furosemide, diltiazem, and diet modifications. Hypertrophic cardiomyopathy should be considered as a differential diagnosis in tree kangaroos exhibiting signs of cardiovascular or respiratory distress. This case represents the first report of antemortem diagnosis and successful management of hypertrophic cardiomyopathy in a Matschie's tree kangaroo.

Non-lethal sampling of liver tissue for toxicologic evaluation of Florida cottonmouths snakes, Agkistrodon piscivorus conanti.

Due to their longevity, strong site tenure, poikilothermic metabolism, and low-energy specializations, reptiles might serve as excellent environmental sentinels. Cottonmouth snakes are generalist predators and scavengers, and as such, may have higher exposure to persistent environmental contaminants as a result of bioaccumulation. Traditionally, assessment and monitoring of contaminant exposure in reptiles have involved lethal sampling techniques. In this paper, we describe a non-destructive technique for sampling liver tissue in live anesthetized Florida cottonmouths. Wild-caught snakes (n = 21) were anesthetized with propofol, and a liver wedge biopsy was obtained by clamping the edge of the organ with two small hemostatic mosquito forceps via right-sided coeliotomy incision. A minimum required tissue sample weighing >100 mg was harvested from all except one of the animals. No mortalities occurred during the procedures or recovery from anesthesia, and all snakes were released back into the field after the animal had consumed prey and defecated, usually within 2 weeks following surgery. Hemorrhage was a minor complication in most snakes, especially those with friable discolored livers. The procedure appeared to have no short-term deleterious effects, and two biopsied individuals were captured after being released into the field and appeared to be normal and healthy. However, follow-up studies and recapture of more snakes are needed to assess long-term survivability. Our non-destructive liver sampling technique might be implemented in toxicological studies of other squamates and could help to minimize the lethal sampling of threatened species.

Molecular detection of hematozoa infections in tundra swans relative to migration patterns and ecological conditions at breeding grounds.

Tundra swans (Cygnus columbianus) are broadly distributed in North America, use a wide variety of habitats, and exhibit diverse migration strategies. We investigated patterns of hematozoa infection in three populations of tundra swans that breed in Alaska using satellite tracking to infer host movement and molecular techniques to assess the prevalence and genetic diversity of parasites. We evaluated whether migratory patterns and environmental conditions at breeding areas explain the prevalence of blood parasites in migratory birds by contrasting the fit of competing models formulated in an occupancy modeling framework and calculating the detection probability of the top model using Akaike Information Criterion (AIC). We described genetic diversity of blood parasites in each population of swans by calculating the number of unique parasite haplotypes observed. Blood parasite infection was significantly different between populations of Alaska tundra swans, with the highest estimated prevalence occurring among birds occupying breeding areas with lower mean daily wind speeds and higher daily summer temperatures. Models including covariates of wind speed and temperature during summer months at breeding grounds better predicted hematozoa prevalence than those that included annual migration distance or duration. Genetic diversity of blood parasites in populations of tundra swans appeared to be relative to hematozoa prevalence. Our results suggest ecological conditions at breeding grounds may explain differences of hematozoa infection among populations of tundra swans that breed in Alaska.

Comparison of intravenous medetomidine and medetomidine/ketamine for immobilization of free-ranging variable flying foxes (Pteropus hypomelanus).

Medetomidine (0.03 mg/kg) and medetomidine/ketamine (0.05/5.0 and 0.025/2.5 mg/kg), administered by intravenous injection, were evaluated for short-term immobilization of wild-caught variable flying foxes (Pteropus hypomelanus). Medetomidine alone produced incomplete chemical restraint and a stressful, prolonged induction. Both ketamine/medetomidine doses produced a smooth induction and complete immobilization. The combined medetomidine/ketamine dose of 0.025/2.5 mg/kg produced a rapid induction (232±224 sec) with minimal struggling and vocalization, a complete and effective immobilization period, and tended to lead to a faster and better quality recovery than medetomidine alone or a higher dose of medetomidine and ketamine (0.05/5.0 mg/kg), thus reducing holding time and permitting an earlier release of the bat back into the wild.

Detection and phylogenetic characterization of a novel herpesvirus from the trachea of two stranded Common Loons (Gavia immer).

Two wild adult Common Loons (Gavia immer) were evaluated after being found stranded in mainland north-central Florida on separate occasions. On the basis of upper airway endoscopic and cytologic findings, we diagnosed severe ulcerative tracheitis antemortem in one of the birds while more subtle lesions were observed in the other. A novel herpesvirus was detected in antemortem tracheal samples using nested consensus PCR amplification of the polymerase gene and sequencing. Despite prolonged intensive medical care, the bird with severe lesions failed to improve and was euthanized 9 days after endoscopy. No viral inclusions were evident histologically in the lesions. However, an undulating tracheal mucosa in a "mountain ridge" pattern, resulting from epithelial regeneration and hyperplasia, was present, as is seen in the late stages of infectious laryngotracheitis in chickens. The second bird recovered and was released. The genetic distance between this and other characterized herpesviruses supports placement of this virus as a novel species, referred to as Gaviid herpesvirus 1 (GavHV1). Phylogenetically, GavHV1 clusters within the genus Iltovirus. The relationship between the observed lesions and the virus remains to be demonstrated.

Evaluation of parenteral drugs for anesthesia in the blue crab (Callinectes sapidus).

The objective of this study was to evaluate the efficacy and safety of several parenteral anesthetics in blue crabs (Callinectes sapidus). Thirty-one animals were administered one or more of the following drugs by injection into the hemolymph (i.v.) through an arthrodial membrane: etomidate, ketamine, lidocaine, pentobarbital, propofol, tiletamine-zolazepam, xylazine, and ketamine-xylazine. A subset of crabs received intracardiac ketamine. Etomidate had no effect. Lidocaine effects were ultrashort (<3 min). Pentobarbital had prolonged inductions (2 min) and often caused violent excitement and poor anesthesia. Propofol induced light anesthesia accompanied by distress and limb autotomy. Inductions with ketamine, xylazine, tiletamine-zolazepam, and ketamine-xylazine were usually fast (<60 sec). Their anesthetic effects were generally very short (5-10 min) but predictable, smooth, and with good muscle relaxation. The latter two protocols induced a deep plane of anesthesia more consistently but also more significant bradycardia. Intracardiac ketamine injection was fatal in four of five crabs. In conclusion, the anesthetic protocols were considered unsuitable for prolonged anesthesia. However, if very short-term anesthesia is desired, then tiletamine-zolazepam or ketamine-xylazine is recommended. Further studies are indicated to identify a safe anesthetic protocol of longer duration in C. sapidus as well as in other crab species.

Application of the laryngeal mask airway for anesthesia in three chimpanzees and one gibbon.

Three pediatric chimpanzees and one pediatric gibbon were anesthetized for routine physical examination. Anesthesia was maintained with inhalant delivered via a laryngeal mask airway (LMA). The LMA was easy to insert, provided adequate control of the airway for ventilation, and caused no tracheal stimulation. No complications were observed. As compared with a face mask, the LMA has the advantage of a more secure airway; the ability to effectively ventilate the patient; less dead space, which leads to lower rebreathing of carbon dioxide; and less exposure of personnel to waste gases. As compared with an endotracheal tube, the LMA causes less airway trauma, is easier to place, and is less stimulating to the patient. The LMA should be considered for use in fasted non-human primates presented for procedures lasting less than 60 min where high peak inspiratory pressures are not needed.

Venom extraction from anesthetized Florida cottonmouths, Agkistrodon piscivorus conanti, using a portable nerve stimulator.

Florida cottonmouth snakes (Agkistrodon piscivorus conanti) were anesthetized with the injectable anesthetic propofol, and venom expulsion was induced with a commercially available human nerve stimulator. We observed rapid anesthetic induction with strong correlation between animal mass and both propofol dose and induction time. We also found a positive correlation between venom yield and animal mass. The method we describe produced consistent venom extraction, maximized yield by completely emptying the glands, potentially reduced animal stress by reducing time of conscious physical restraint, and decreased the likelihood of human envenomation. This technique could also be used in remote field locations.

Education of global veterinarians.

In 2003, the University of Florida (UF) College of Veterinary Medicine (CVM) created an Office of International Programs (OIP) in response to one of ten initiatives of the UF Strategic Plan: internationalization of the curriculum. The OIP has developed coursework that provides students with an opportunity for international exposure during the veterinary curriculum at three levels. In Level 1 (on campus) students can participate in a seminar series in global health: www.ufglobalhealth.org. This is an elective course offered to professional students at the UF Health Science Center (Dentistry, Medicine, Pharmacy, Public Health, and Veterinary Medicine). In Level 2 (abroad), students can participate in structured study abroad programs under the supervision of UF faculty and international scholars from collaborative institutions abroad. In Level 3 (on campus and abroad), students can participate in a certificate program in international veterinary medicine. This is a 15-credit program, parallel to the veterinary curriculum. By offering courses on campus and abroad, we want to empower the curriculum with a global perspective of the veterinary profession, as well as with a humanist education that can help students recognize the importance of respect for cultural differences and the reasons for different degrees of development and growth in the world. In addition, this paper presents the need for veterinary medicine and other disciplines in the health sciences to communicate with other disciplines in the social sciences and natural sciences to create development practitioners equipped with cross-disciplinary knowledge and skills needed to formulate, implement and evaluate solutions aimed at breaking the cycle of poverty and disease in low income societies. Finally, this paper makes a call to the American Veterinary Medical Association Council on Education to assess the need to recognize the importance of internationalization of the veterinary curriculum as a key standard for accreditation of colleges or schools of veterinary medicine.