RETROSPECTIVE ANALYSIS OF BLOOD CULTURES AND THEIR ASSOCIATION WITH CLINICAL FINDINGS AND OUTCOME IN GREEN SEA TURTLES (CHELONIA MYDAS) AT A FLORIDA SEA TURTLE REHABILITATION FACILITY, 2017-2020.

Septicemia is commonly suspected of sea turtles entering rehabilitation. However, blood culture results of green sea turtles (Chelonia mydas) are infrequently reported in the literature. Aerobic blood cultures were performed for intake examinations of 167 green sea turtles undergoing rehabilitation at Brevard Zoo's Sea Turtle Healing Center, Melbourne, Florida, USA from 2017 to 2020. The incidence of positive cultures during intake examinations was 24% (40/167). The most common bacterial isolates identified were Vibrio alginolyticus, Shewanella algae, Achromobacter xylosoxidans, Photobacterium damselae, Sphingomonas paucimobilis, and Vibrio parahaemolyticus. There was a statistically significant association (P < 0.05) between culture status and evidence of external injury. There was no significant association between culture status and Caryospora-like coccidia infection, or fibropapillomatosis. Culture-positive turtles had significantly lower (P < 0.05) total white blood cell, lymphocyte, monocyte, total protein, albumin, and calculated globulin values compared to turtles with negative blood cultures. Significantly more culture-positive turtles died in rehabilitation compared to culture-negative (P = 0.042). Positive blood cultures suggestive of septicemia are commonly found during intake examinations at a Florida sea turtle rehabilitation facility.

Aquatic and Terrestrial Invertebrate Welfare.

Invertebrates are a diverse group of animals that make up the majority of the animal kingdom and encompass a wide array of species with varying adaptations and characteristics. Invertebrates are found in nearly all of the world's habitats, including aquatic, marine, and terrestrial environments. There are many misconceptions about invertebrate sentience, welfare requirements, the need for environmental enrichment, and overall care and husbandry for this amazing group of animals. This review addresses these topics and more for a select group of invertebrates with biomedical, economical, display, and human companionship importance.

The Utility of Bile Acids for the Diagnosis of Liver Disease in Exotic Animals.

The synthesis of bile acids occurs during the degradation of cholesterol in hepatocytes. Thus, this analyte is expected to be a sensitive indicator of hepatocellular dysfunction or alterations in portal circulation. Bile acids can be quantified via an enzymatic reaction to a highly conserved moiety across species. The evidence for the clinical utility of bile acids for the diagnosis of liver disease is strongest in birds and ferrets with equivocal evidence in rodents, rabbits, and reptiles. Current limitations to the interpretation of bile acids in exotic animal species include a paucity of species-specific reference intervals and incomplete understanding of bile acid metabolism in nonmammalian species and the diversity of bile acids synthesized by vertebrates.

A Retrospective Study of Pathology in Bats Submitted to an Exotic and Zoo Animal Diagnostic Service in Georgia, USA (2008-2019).

Pathology records of bats submitted to the University of Georgia from managed care settings were reviewed to identify naturally occurring diseases. Fifty-nine cases were evaluated during an 11-year period (2008-2019), including representatives from four families: Pteropodidae (Yinpterochiroptera), Phyllostomidae, Vespertilionidae and Molossidae (Yangochiroptera). Pathology reports were reviewed to determine the primary pathological process resulting in death or the decision to euthanize. Cases were categorized as non-infectious (34/59; 58%), infectious/inflammatory (17/59; 29%) or undetermined due to advanced autolysis (8/59; 14%). Musculoskeletal diseases and reproductive losses were the most frequent pathological processes. Among the infectious processes identified, bacterial infections of the reproductive and haemolymphatic systems were most frequently observed. The first two reports of neoplasia in small flying foxes (Pteropus hypomelanus) are described. Bats under managed care present with a wide range of histopathological lesions. In this cohort, non-infectious disease processes were common.

PLASMA SEPARATOR TUBES DO NOT HAVE ANY OVERT EFFECTS ON ROUTINE PLASMA CHEMISTRY DATA OF GREEN SEA TURTLES (CHELONIA MYDAS).

Plasma separator tubes (PSTs) are a variant of lithium heparin blood tube containing a polymer gel, which, when centrifuged, creates a physical barrier between plasma and blood cells. Their use is common in laboratory procedures of reptilian species. This study aimed to determine whether the use of plasma separator tubes impacts plasma biochemistry data in green sea turtles (Chelonia mydas) at time of collection and after 24 hr of contact time with the separator gel after centrifugation at refrigerator temperature. A single blood sample was collected from 42 rehabilitating green sea turtles at the Sea Turtle Healing Center, Brevard Zoo, Melbourne, Florida, USA and divided into one lithium heparin tube [LHT (0 hr)] and two PSTs. After immediate centrifugation of all three tubes, plasma was transferred from the LHT (0 hr) and one PST (0 hr) into tubes without additive. The plasma was left in contact with the separator gel in the second PST (24 hr). After 24 hr of refrigeration, all three plasma aliquots were analyzed for the following 23 analytes: sodium, potassium, chloride, carbon dioxide, calcium, phosphorus, magnesium, iron, total protein, albumin, globulin (calculated), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltransferase, creatine kinase, glucose, urea nitrogen, creatinine, uric acid, triglyceride, and cholesterol. No statistically significant differences were found for any biochemical analytes between LHT (0 hr), PST (0 hr), and PST (24 hr). The use of PST does not appear to impact routine plasma biochemical analytes in green sea turtles and analytes appear stable in refrigerated plasma for up to 24 hr after centrifugation when using PSTs.

Tumor re-growth, case outcome, and tumor scoring systems in rehabilitated green turtles with fibropapillomatosis.

Fibropapillomatosis (FP) is an infectious, neoplastic disease of major concern in sea turtle rehabilitation facilities. Rehabilitating sea turtles that undergo tumor removal surgery often have tumor regrowth and may experience mortality. We evaluated tumor score, removal, and regrowth in rehabilitating green sea turtles with FP in 4 rehabilitation facilities in the southeastern USA during 2009-2017. Of 756 cases, 312 (41%) underwent tumor removal surgery, 155 (50%) of those had tumor regrowth within an average of 46 ± 45 d, and 85 (27%) had multiple (>1) regrowth events. Of 756 turtles with FP, 563 (75%) did not survive after admission into a rehabilitation facility, including 283 (37%) that were euthanized and 280 that died without euthanasia (37%), and 193 survived, including 186 (25%) released and 7 (1%) placed in permanent captive care. Tumor removal surgery increased the odds of tumor regrowth but also enhanced survivorship, whereas tumor regrowth was not a significant predictor of case outcome. Three FP tumor scoring systems were used to assign tumor scores to 449 cases, and differing results emphasize that tumor scoring systems should be applied to the situations and/or location(s) for which they were intended. FP tumor score was not a significant predictor for the event or extent of FP tumor regrowth after surgical excision. Under current rehabilitation regimes, outcomes of rehabilitation for tumored turtles have a low probability of success. The results of this study may be used to help guide clinical decision-making and determine prognoses for rehabilitating sea turtles with FP.

Caryospora-Like Coccidia Infecting Green Turtles (Chelonia mydas): An Emerging Disease With Evidence of Interoceanic Dissemination.

Protozoa morphologically consistent with Caryospora sp. are one of the few pathogens associated with episodic mass mortality events involving free-ranging sea turtles. Parasitism of green turtles (Chelonia mydas) by these coccidia and associated mortality was first reported in maricultured turtles in the Caribbean during the 1970s. Years later, epizootics affecting wild green turtles in Australia occurred in 1991 and 2014. The first clinical cases of Caryospora-like infections reported elsewhere in free-ranging turtles were from the southeastern US in 2012. Following these initial individual cases in this region, we documented an epizootic and mass mortality of green turtles along the Atlantic coast of southern Florida from November 2014 through April 2015 and continued to detect additional, sporadic cases in the southeastern US in subsequent years. No cases of coccidial disease were recorded in the southeastern US prior to 2012 despite clinical evaluation and necropsy of stranded sea turtles in this region since the 1980s, suggesting that the frequency of clinical coccidiosis has increased here. Moreover, we also recorded the first stranding associated with infection by a Caryospora-like organism in Hawai'i in 2018. To further characterize the coccidia, we sequenced part of the 18S ribosomal and mitochondrial cytochrome oxidase I genes of coccidia collected from 62 green turtles found in the southeastern US and from one green turtle found in Hawai'i. We also sequenced the ribosomal internal transcribed spacer regions from selected cases and compared all results with those obtained from Caryospora-like coccidia collected from green turtles found in Australia. Eight distinct genotypes were represented in green turtles from the southeastern US. One genotype predominated and was identical to that of coccidia collected from the green turtle found in Hawai'i. We also found a coccidian genotype in green turtles from Florida and Australia with identical 18S and mitochondrial sequences, and only slight inter-regional differences in the internal transcribed spacer 2. We found no evidence of geographical structuring based on phylogenetic analysis. Low genetic variability among the coccidia found in green turtle populations with minimal natural connectivity suggests recent interoceanic dissemination of these parasites, which could pose a risk to sea turtle populations.

Host sex, size, and hemoparasite infection influence the effects of ectoparasitic burdens on free-ranging iguanas.

Investigations focusing on host-ectoparasite interactions in animals have revealed asymptomatic to severe health and fitness consequences suggesting that species mobilize different interspecific response mechanisms. Fewer studies, however, have examined intraspecific responses to ectoparasitic burdens. In this study, we analyzed host health and fitness responses to increasing ectoparasite burdens along with the presence/absence of hemoparasites of free-ranging insular rock iguanas (Cyclura cychlura) in The Bahamas. Using hematology, plasma biochemistry, as well as body condition and growth rate comparisons, we failed to find significant associations of tick burdens with annual growth rate, corticosterone, packed cell volume, total white blood cell, and heterophil, monocyte, eosinophil or hemoglobin measures. We did, however, find mixed and significant associations of tick burdens with lymphocyte and basophil counts, heterophil-to-lymphocyte ratios, and body condition indices. These associations varied by sex, size, and hemoparasite infection status suggesting that different life stages of iguanas may invest differently in immune responses, and impacts may be modulated based on size and sex of hosts, and coinfection status.

T-cell Thymic Lymphoma With Proventricular Metastasis in a Florida Scrub Jay ( Aphelocoma coerulescens).

An adult, wild-caught, female Florida scrub jay ( Aphelocoma coerulescens) was evaluated because of an observable mass on the ventral neck. Initial physical examination and diagnostic tests were performed, which revealed a subcutaneous mass. Surgical removal of the mass was attempted, but the bird died during surgery. Results of necropsy and histopathologic evaluation identified the mass as thymic lymphoma with proventricular metastasis. Immunohistochemical staining revealed strong cytoplasmic immunoreactivity for CD3 in the thymic mass and within the predominant lymphoid population in the serosal proventricular masses, which confirmed metastasis of T-cell lymphoma. To our knowledge, this is the first report of T-cell thymic lymphoma in a wild Florida scrub jay.

Evidence for henipavirus spillover into human populations in Africa.

Zoonotic transmission of lethal henipaviruses (HNVs) from their natural fruit bat reservoirs to humans has only been reported in Australia and South/Southeast Asia. However, a recent study discovered numerous HNV clades in African bat samples. To determine the potential for HNV spillover events among humans in Africa, here we examine well-curated sets of bat (Eidolon helvum, n = 44) and human (n = 497) serum samples from Cameroon for Nipah virus (NiV) cross-neutralizing antibodies (NiV-X-Nabs). Using a vesicular stomatitis virus (VSV)-based pseudoparticle seroneutralization assay, we detect NiV-X-Nabs in 48% and 3-4% of the bat and human samples, respectively. Seropositive human samples are found almost exclusively in individuals who reported butchering bats for bushmeat. Seropositive human sera also neutralize Hendra virus and Gh-M74a (an African HNV) pseudoparticles, as well as live NiV. Butchering bat meat and living in areas undergoing deforestation are the most significant risk factors associated with seropositivity. Evidence for HNV spillover events warrants increased surveillance efforts.

Effects of sevoflurane anesthesia on righting reflex and hemolymph gas analysis variables for Chilean rose tarantulas (Grammostola rosea).

OBJECTIVE: To determine the safety, efficacy, and effects on hemolymph gas analysis variables of sevoflurane anesthesia in Chilean rose tarantulas (Grammostola rosea). ANIMALS: 12 subadult Chilean rose tarantulas of unknown sex. PROCEDURES: Spiders were anesthetized in a custom chamber with sevoflurane (5% in oxygen [1.0 L/min]), then allowed to recover in 100% oxygen. Righting reflex was evaluated every 3 minutes during anesthesia to determine time to anesthetic induction and recovery. Hemolymph samples were collected from an intracardiac location prior to and after induction of anesthesia and evaluated to determine various gas analysis variables. RESULTS: Mean ± SD induction and recovery times were 16 ± 5.91 minutes and 29 ± 21.34 minutes, respectively. Significant differences were detected for Po2, base excess, and glucose and ionized magnesium concentrations between hemolymph samples obtained before anesthesia and those obtained after induction of anesthesia. CONCLUSIONS AND CLINICAL RELEVANCE-Results of this study suggested that the use of sevoflurane as an anesthetic agent for Chilean rose tarantulas was safe and effective. Various hemolymph sample gas analysis values changed during anesthesia.

Physiological effects of tourism and associated food provisioning in an endangered iguana.

Deliberately feeding wildlife is an increasingly popular tourism-related activity despite a limited understanding of long-term impacts on the species being fed. As a result, tourist behaviours that may have adverse impacts on imperiled species have often been encouraged without the necessary evaluation or oversight. Here, we report the responses of Northern Bahamian Rock Iguanas (Cyclura cychlura) to human-visitation pressure and associated food provisioning. We compared a variety of blood chemistry parameters of iguanas subjected to supplemental feeding at popular tourist destinations with iguanas occurring on islands where supplemental feeding does not take place. We demonstrate that male and female iguanas inhabiting tourist-visited islands where supplemental feeding occurs do not differ in body condition or baseline stress and stress response (determined by corticosterone levels) compared with iguanas from non-visited islands. Both males and females from tourist-visited sites experienced a greater incidence of endoparasitic infection and atypical loose faeces. Indicators of dietary nutrition, including glucose, potassium, and uric acid values, also differed for both sexes from tourist-visited and unvisited islands. Male iguanas from visited islands differed significantly from those on non-visited islands in calcium, cholesterol, cobalt, copper, magnesium, packed cell volume, selenium, and triglyceride concentrations, whereas female iguanas from visited islands differed significantly in ionized calcium. Although the interpretation of these differences is challenging, chronic biochemical stressors could compromise individual health over time or decrease survivorship during periods of environmental stress. We suggest protocols that can be adopted throughout the region to ensure that supplemental feeding has fewer impacts on these long-lived iguanas.

Evaluation of critical care blood analytes assessed with a point-of-care portable blood analyzer in wild and aquarium-housed elasmobranchs and the influence of phlebotomy site on results.

OBJECTIVE: To establish reference ranges for critical care blood values measured in wild and aquarium-housed elasmobranchs by use of a point-of-care (POC) blood analyzer and to compare values on the basis of species category (pelagic, benthic, or intermediate) and phlebotomy site. DESIGN: Cross-sectional study. ANIMALS: 66 wild and 89 aquarium-housed elasmobranchs (sharks and rays). PROCEDURES: Aquarium-housed elasmobranchs were anesthetized for sample collection; wild elasmobranchs were caught via hook and line fishing, manually restrained for sample collection, and released. Blood was collected from 2 sites/fish (dorsal sinus region and tail vasculature) and analyzed with the POC analyzer. Reference values of critical care blood analytes were calculated for species most represented in each population. Values were compared on the basis of species categorization (pelagic, intermediate, or benthic) and collection site. RESULTS: Oxygen saturation and circulating concentrations of lactate and glucose were significantly different among aquarium-housed pelagic, intermediate, and benthic species. Lactate concentration was significantly different among these categories in wild elasmobranchs. Significant differences were detected between samples from the 2 collection sites for all blood analytes. In both study populations, pH and lactate values were infrequently < 7.2 or > 5 mmol/L, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Brevity of handling or chemical restraint may have reduced secondary stress responses in fish because extreme variations in blood analyte values were infrequent. Sample collection site, species categorization, acclimation to handling, and restraint technique should be considered when assessing values obtained with the POC analyzer used in this study for blood analytes and immediate metabolic status in elasmobranchs.

Ophthalmic examination findings in a captive colony of western gray kangaroos (Macropus fuliginosus).

Complete ophthalmic examination of a mob of western gray kangaroos (Macropus fuliginosus) was performed under chemical restraint. Examination included intraocular pressure (IOP) measurement by rebound and applanation tonometry, fluorescein staining, corneal diameter measurement, slit-lamp biomicroscopy and indirect funduscopy. The corneal diameters had a mean of 19.52 mm, SD 2.16 mm, 95% confidence interval (CI) 18.71-20.32 mm. Ocular abnormalities were noted in 4/8 (50%) of examined kangaroos. Intraocular pressure as estimated by rebound tonometry was 9.00 mm Hg with a 25-75% quartile range of 6.5-10.75 mm Hg and a minimum-maximum range of 5.00-23.00 mm Hg. Intraocular pressure as estimated by applanation tonometry was 11.50 mm Hg with a 25-75% quartile range of 10.00-17.00 mm Hg and a minimum-maximum range of 9.00-20.00 mm Hg. This is the first report of ocular examination findings in a mob of captive western gray kangaroos and provides ranges and values for tonometry and corneal diameter.

Orthoreovirus infection and concurrent cryptosporidiosis in rough green snakes (Opheodrys aestivus): pathology and identification of a novel orthoreovirus strain via polymerase chain reaction and sequencing.

Reoviruses are nonenveloped, segmented, double-stranded RNA viruses capable of infecting a wide range of invertebrate, vertebrate, fungus, and plant hosts. Though sporadic infection has been reported in a variety of reptilian species, infection of rough green snakes (Opheodrys aestivus) has not been previously described. Five wild-caught, adult rough green snakes were obtained by a zoological institution. Clinical deterioration was first noted in all snakes after 3 weeks in quarantine. Despite treatment, clinical decline progressed, and all 5 snakes died or were euthanized by 48 days post-arrival. Moderate, multifocal, acute, necrotizing hepatitis with hepatocellular syncytia was diagnosed in 1 snake. Two additional snakes had severe, diffuse, subacute to chronic pancreatitis. All 5 snakes had gastroenteric cryptosporidiosis. Electron microscopic examination of liver from the snake with hepatic lesions revealed scattered hepatocytes containing 1 or more intranuclear clusters of approximately 90 nm in diameter viral particles arranged in loose arrays. Polymerase chain reaction (PCR) amplification of a segment of the reovirus RNA-dependent RNA polymerase gene was performed on RNA extracted from tissues of all 5 snakes. PCR amplification of samples extracted from the snake with hepatic lesions resulted in a 109-base pair (bp) product. Phylogenetic analyses indicated the virus was a novel strain distinct from other reoviruses at a level consistent with species difference. The source of infection was unknown. PCR amplification of samples extracted from the other 4 snakes was negative.

Vitamin D3 in the hemolymph of goliath birdeater spiders (Theraphosa blondi).

Vitamin D3 is an important vitamin in vertebrates. This fat-soluble vitamin is associated with the regulation of many physiologic processes, most importantly calcium metabolism. The presence or importance of vitamin D3 has been determined in only a handful of invertebrate species. In this study, hemolymph was collected from six wild-caught, subadult goliath birdeater spiders (Theraphosa blondi) and analyzed for the presence of 25(OH)-vitamin D3, the precursor to the active form of vitamin D3. The metabolite 25(OH)-vitamin D3 was detected in all of the spiders (mean: 5.7 nmol/L, SD: 1.5 nmol/L, range: 3-7 nmol/L). The method by which spiders acquire vitamin D3 is unknown. It is possible, though unlikely, that they synthesize it via exposure to ultraviolet radiation. Many of the invertebrate species upon which theraphosid spiders prey are not known to have high circulating levels of vitamin D3 or its precursors. However, dietary intake is a possible means of vitamin D3 acquisition in this study.

Isoflurane anesthesia of wild-caught goliath birdeater spiders (Theraphosa blondi) and Chilean rose spiders (Grammostola rosea).

Anesthesia is used in theraphosid spiders to facilitate medical procedures (e.g., physical examination, sample collection, surgery); however, most information on this subject is anecdotal. This study was conducted to systematically determine the anesthetic parameters of wild-caught, subadult goliath birdeater spiders (Theraphosa blondi) (n = 11) and Chilean rose spiders (Grammostola rosea) (n = 12). Each spider was placed in a 3-L gas anesthetic chamber and subjected to an induction of 5% isoflurane at a rate of 1 L/min oxygen. Anesthetic depth was monitored by evaluating the righting reflex every 5 min. Animals were recovered in 100% oxygen. Induction, recovery, and overall anesthetic times were determined. After an 8-wk washout period, the procedure was repeated. For both species, median induction time was 10 min. Median recovery time was 30 min for T. blondi and 12.5 min for G. rosea.

Effects of ultraviolet radiation on plasma 25-hydroxyvitamin D3 concentrations in corn snakes (Elaphe guttata).

OBJECTIVE: To determine whether corn snakes exposed to UVB radiation have increased plasma 25-hydroxyvitamin D3 concentrations, compared with control snakes. ANIMALS: 12 corn snakes (Elaphe guttata). PROCEDURES: After an acclimation period in individual enclosures, a blood sample was collected from each snake for assessment of plasma 25-hydroxyvitamin D3 concentration. Six snakes were provided with no supplemental lighting, and 6 snakes were exposed to light from 2 full-spectrum coil bulbs. By use of a radiometer-photometer, the UVA and UVB radiation generated by the bulbs were measured in each light-treated enclosure at 3 positions at the basking surface and at 2.54 cm (1 inch) below each bulb surface; the arithmetic mean values for the 3 positions at the basking surface and each individual bulb surface were calculated immediately after the start of the study and at weekly intervals thereafter. At the end of the study (day 28), another blood sample was collected from each snake to determine plasma 25-hydroxyvitamin D3 concentration. RESULTS: Mean +/- SD plasma concentration of 25-hydroxyvitamin D3 in snakes that were provided with supplemental lighting (196 +/- 16.73 nmol/L) differed significantly from the value in control snakes (57.17 +/- 15.28 nmol/L). Mean exposure to UVA or UVB did not alter during the 4-week study period, although the amount of UVA recorded near the bulb surfaces did change significantly. CLINICAL RELEVANCE: These findings have provided important insight into the appropriate UV radiation requirements for corn snakes. Further investigation will be needed before exact husbandry requirements can be determined.