INVESTIGATION OF A POINT-OF-CARE VISCOELASTIC COAGULATION MONITOR AND ITS COMPARISON TO THROMBOELASTOGRAPHY IN CLINICALLY HEALTHY AFRICAN ELEPHANTS (LOXODONTA AFRICANA).

Elephant endotheliotropic herpesvirus (EEHV) can induce fatal hemorrhagic disease (HD) in African elephants (Loxodonta africana). Once clinical signs develop, progression is rapid, even with aggressive treatment. There is a critical need to develop point-of-care diagnostic tests to aid in identification of EEHV-HD prior to the onset of overt clinical signs. Study objectives were to investigate a novel, point-of-care viscoelastic coagulation monitor (VCM Vet), compare the results to thromboelastography (TEG), and report traditional hemostatic analytes in adult African elephants. Whole blood was collected from seven clinically healthy elephants (four females and three males, 18-47 yr) and analyzed in duplicate via VCM Vet and kaolin-activated TEG 1-3 and 30 min following collection, respectively. Separated plasma was frozen for ancillary coagulation testing. Both analyses generated quantifiable clotting reactions with variables (median [range]) describing clot formation rate (VCM Vet, clot time = 682 s [530-987 s], clot formation time = 244 s [186-744 s], Alpha = 40° [14-47°]; TEG, reaction time = 6.2 min [3.7-11.8 min], kinetic time = 1.3 min [0.9-2.6 min], Alpha = 70° [57-77°]), clot strength (VCM Vet, maximum clot formation = 34 units [20-45 units]; TEG, maximum amplitude = 75 mm [69-80 mm], shear elastic modulus strength = 14.7 Kdynes/s [11.3-19.5 Kdynes/s]), and clot lysis (VCM Vet, lysis index at 30 min = 100% [100-99%], lysis index at 45 min = 98% [95-100%]; TEG, lysis index at 30 min = 0% [0-0.4%], lysis index at 60 min = 1.4% [0-2.6%]) recorded. Additional testing (median [range]) included D-dimer concentration (33 ng/ml [28-94 ng/ml]), prothrombin time (12.4 s [12.2-13.2 s]), activated partial thromboplastin time (17.2 s [14.2-18.8 s]), and fibrinogen concentration (297 [282-383] mg/dL). Tracings generated by VCM Vet and TEG were clinically similar, and there was visual agreement and minimal difference between quantitative variables for duplicate tests. VCM Vet is a promising, user-friendly tool for use in identification and management of coagulopathies in African elephants.

Retrospective review of eyelid coloboma in snow leopards (Panthera uncia) housed under managed care in North America: 49 cases (2000-2020).

OBJECTIVE: To determine incidence, trends in presentation, associated ocular lesions and other diseases, treatment modalities, and case outcomes of eyelid coloboma cases in snow leopards. ANIMALS: 49 snow leopards with eyelid coloboma living under managed care in North America. METHODS: Medical records were retrospectively searched to identify snow leopards in which eyelid coloboma was diagnosed between January 1, 2000, and December 31, 2020. Data recorded from each animal included signalment, ophthalmic examination information, clinical signs, concurrent health conditions, medical and/or surgical interventions, time to resolution of signs, recurrence of clinical signs, and direct relatives with a history of eyelid coloboma. RESULTS: Ocular clinical signs were present at diagnosis in most cases but not seen in all cases. Corrective procedures were undertaken in 39 cases. Clinical signs were resolved by the total combination of interventions in 84.6% of individuals; however, signs resolved in only 33.3% of cases after a single surgical reconstruction or cryoablation procedure per eye. CLINICAL RELEVANCE: Eyelid coloboma is widespread in the North American snow leopard population. A high percentage (73.5%) have an affected sibling, parent, or grandparent, suggesting a heritable component. Surgical correction resolves or improves clinical signs in most cases, but there is a high rate of postprocedural complications with all procedure types employed. Most complications are minor and manageable, but these can also impact case outcomes. Animals require long-term monitoring, as clinical signs may recur (in some cases, years after initial signs are reported to be resolved), and some animals may require long-term care to manage signs.

A novel herpesvirus from a wild-caught Madagascar spider tortoise shows evidence of host-viral coevolution with a duplication event in Durocryptodira.

Herpesviruses can be significant reptile pathogens. Herpesviral infection in a wild-caught, male spider tortoise (Pyxis arachnoides) under human care was detected during a routine wellness examination prior to transition between zoologic organizations. The tortoise had no clinical signs of illness. Oral swabs obtained during a physical examination as part of pre-shipment risk mitigation for infectious disease were submitted for consensus herpesvirus PCR assay and sequencing. Based on comparative sequence analysis, the novel herpesvirus identified is a member of the subfamily Alphaherpesvirinae. Studies of herpesviral phylogeny in chelonian species support branching patterns of turtle herpesviruses that closely mirror those of their hosts. The symmetry of these patterns is suggestive of close codivergence of turtle herpesviruses with their host species. The distribution of these viruses in both tortoises and emydids suggests a phylogenetic duplication event in the herpesviruses after host divergence of the Pleurodira and basal to the divergence of Americhelydia. Herpesviral infections have been documented to cause higher morbidity when introduced to aberrant host species, and significant consideration must be given to the presence of herpesviruses in the management of tortoise collections, particularly collections that include various species of testudines.

RETROSPECTIVE REVIEW OF UROLITHIASIS-RELATED MORBIDITY AND MORTALITY IN ASIAN COLOBINE MONKEYS.

Obstructive and incidental urolithiasis cases were evaluated in 5 Asian colobine monkey species (n= 21 individuals) that included 12 silvery langurs (Trachypithecus cristatus), 6 spectacled langurs (Trachypithecus obscurus), 1 Javan langur (Trachypithecus auratus auratus), 1 François' langur (Trachypithecus francoisi), and 1 red-shanked douc langur (Pygathrix nemaeus) from eight zoologic institutions. All institutions that responded were Association of Zoos and Aquariums, European Association of Zoos and Aquaria, or World Association of Zoos and Aquariums accredited. Males were more commonly represented in the total number of cases (86%), and all cases of obstructive urolithiasis occurred in males. The most common clinical signs observed in obstructive cases included stranguria (58.8%), lethargy (41.2%), anorexia (29.4%), depression (17.6%), and penile manipulation (11.8%). Clinicopathologic abnormalities revealed azotemia (76.5%), anemia (35.3%), and hyperkalemia (23.5%). Eleven of the 21 cases included urinalysis results, and crystalluria was reported in all 11. Obstructive cases were more commonly managed surgically, with medical management following. Instances of individual obstruction ranged from 0 (incidental finding) to 18, with a median of 8 (mean of 4.3) recurrent obstructions. A total of 39 urolith analyses were available from 17 of 21 cases, with calcium carbonate being the most common type isolated (37 of 39, 94.9%). Calcium oxalate was observed in the remaining 5.1% of cases (2 of 39). No cases exhibited a consistent match of crystalluria and urolith type. Death or euthanasia secondary to obstructive urolithiasis occurred in 52.4% of cases. Urinary obstruction secondary to urolithiasis appears to be a relevant cause of morbidity and mortality in Asian colobine species, and further study into etiology and preventive medicine should be undertaken.

MORTALITY REVIEW FOR THE NORTH AMERICAN SNOW LEOPARD (PANTHERA UNCIA) ZOO POPULATION FROM JANUARY 1999 TO DECEMBER 2019.

The objective of this 20-yr retrospective study was to review and summarize causes of mortality in the North American (NA) snow leopard population to inform and enhance animal health and husbandry practices. Pathology reports were requested from all NA zoological institutions housing snow leopards that died between 01 January 1999 and 31 December 2019. Data were reviewed and cause of death (COD) and concurrent diseases were summarized and compared by age group, organ system, and disease process. The 241 snow leopards in this report include 109 males, 130 females, and two of undetermined sex. Among them were 116 geriatric snow leopards (>15 yr), 72 adults (15-3 yr), 16 juveniles (3 yr to 2 mo), 32 neonates (2 mo to 0 days), and five fetuses (<0 days). Overall, noninfectious diseases were the most common COD across all age groups (73%). In adult and geriatric snow leopards, chronic renal disease (CRD) (38.8%) and malignant neoplasia (19.7%), including oral squamous cell carcinoma (6.4%), were a common COD. In juveniles and neonates, perinatal death and congenital diseases, including ocular coloboma (15.6%), were a common COD. Individuals with CRD were 13.5 and 4.36 times more likely to have veno-occlusive disease and cardiac fibrosis, respectively. Snow leopards with urolithiasis were 5.27 times more likely to have CRD. Infectious (14.1%) and inflammatory diseases (8.7%) for which no specific etiology was identified were less common overall and more common in juveniles and neonates (25% and 21%, respectively). Neoplasms not previously reported in snow leopards or that are generally uncommon in the veterinary literature included transitional cell carcinoma of the urinary bladder (n = 7) and mesothelioma (n = 1).

REVIEW OF CANINE DISTEMPER VACCINATION USE AND SAFETY IN NORTH AMERICAN CAPTIVE LARGE FELIDS (PANTHERA SPP.) FROM 2000 TO 2017.

Data on canine distemper virus (CDV) vaccination were collected on 812 large felids (351 tigers, Panthera tigris; 220 lions, Panthera leo; 143 snow leopards, Panthera uncia; 50 leopards, Panthera pardus; and 48 jaguars, Panthera onca) from 48 institutions to assess vaccine use and safety. The documented individual vaccination events with multiple products numbered 2,846. Canarypox-vectored CDV vaccines were the most commonly used vaccines (96.3% of all vaccinations) and the Purevax® Ferret Distemper (PFD) vaccine was the most commonly used canarypox-vectored vaccine (91.0% of all vaccinations). Modified live virus (MLV) CDV vaccines were used for 3.7% of all vaccinations, and only in tigers, lions, and snow leopards. Adverse effects were reported after 0.5% (13 of 2,740) of the canarypox-vectored vaccinations and after 2.9% (3 of 104) of the MLV CDV vaccinations. This low complication rate suggests large felids may not be as sensitive to adverse effects of MLV CDV vaccines as other exotic carnivores. Serological data were available from 159 individuals (69 tigers, 31 lions, 31 snow leopards, 22 jaguars, and 6 Amur leopards, Panthera pardus orientalis) vaccinated with the PFD vaccine, and 66.0% of vaccinates seroconverted (defined as acquiring a titer ≥1: 24) at some point postvaccination: 24.3% after one vaccination, 55.8% after two vaccinations, 54.3% after three vaccinations, and 79.2% after four or more vaccinations. Among animals exhibiting seroconversion after the initial PFD vaccinations, 88.9% still had titers ≥12 mo and ≥24 mo after the last vaccination, and 87.5% had titers ≥1: 24 at ≥36 mo after the last vaccination. The study was unable to assess fully the safety of vaccination with either canarypox-vectored or MLV CDV vaccines during gestation because of the small number of animals vaccinated while pregnant (n = 6, all vaccinated with PFD).

RED PANDAS' (AILURUS FULGENS) SEROLOGICAL RESPONSE TO CANARYPOX-VECTORED CANINE DISTEMPER VACCINES.

Red pandas (Ailurus fulgens) are susceptible to canine distemper, with a number of reported vaccine-induced canine distemper cases. Canarypox-vectored recombinant canine distemper vaccines (PureVax Ferret Distemper [PFD] and Recombitek CDV [rCDV]) provide protection without inoculating a live distemper virus, but there are currently no published data regarding these vaccines' safety and efficacy in red pandas. One hundred twenty-two serum samples were collected from 50 captive red pandas and analyzed for antibodies to canine distemper. All naïve red pandas (n = 20) had negative titers. Naïve pandas receiving two PFD vaccinations had either negative or intermediate titers (n = 4). In contrast, naïve pandas receiving a series of two or three rCDV vaccinations (n = 14) had greater antibody responses. Red pandas vaccinated with PFD >12 mo since their last vaccination and a rCDV booster vaccination showed the highest titers observed. We recommend red pandas be administered a series of at least three recombinant vaccine (PDF or rDCV) vaccinations, followed by annual booster vaccinations.

THE PASSAGE AND DURATION OF ANTIBODIES TO WEST NILE VIRUS IN HUMBOLDT PENGUINS (SPHENISCUS HUMBOLDTI).

West Nile virus (genus Flavivirus) outbreaks and mortality events have been documented in both wild and captive avian species, including penguins. Serologic response to vaccination in avian species has varied and appears to be largely species dependent; however, Humboldt penguins ( Spheniscus humboldti ) previously showed excellent rates of seroconversion. The goal of this study was to determine virus neutralization titers of 17 Humboldt penguin hens and their subsequent eggs, chicks, or both following vaccination with a killed West Nile vaccine. Chicks were also vaccinated at 56, 70, and 84 days old. Titers were measured from 10-346 days prior to lay as well as serially in seven chicks. Data collected showed positive rank correlation between maternal titers and yolk titers (ρ = 0.90, P < 0.0001, n = 14) but no association between booster vaccination and yolk titers. All seven chicks had detectable antibody on days 14 and 28, and antibody levels had increased (relative to day 56) in 3 of 6 chicks (50%; 95% confidence interval 14-86%) by day 112. Further information is provided on a suggested vaccination schedule for Humboldt penguin chicks based on a time-dependent decline in maternal antibody titers. Cell-mediated immunity and experimental challenge following vaccination have not yet been investigated in this species.

VORICONAZOLE TOXICITY IN MULTIPLE PENGUIN SPECIES.

Aspergillosis is a common respiratory fungal disease in penguins managed under human care. Triazole antifungal drugs, including itraconazole, are most commonly used for treatment; however, itraconazole treatment failures from drug resistance are becoming more common, requiring newer treatment options. Voriconazole, a newer triazole, is being used more often. Until recently, no voriconazole pharmacokinetic studies had been performed in penguins, leading to empiric dosing based on other avian studies. This has led to increased anecdotal reporting of apparent voriconazole toxicity in penguins. This report describes 18 probable and 6 suspected cases of voriconazole toxicity in six penguin species from nine institutions: 12 African penguins (Spheniscus demersus), 5 Humboldt penguins (Spheniscus humboldti), 3 Magellanic penguins (Spheniscus magellanicus), 2 gentoo penguins (Pygoscelis papua papua), 1 macaroni penguin (Eudyptes chrysolophus), and 1 emperor penguin (Aptenodytes forsteri). Observed clinical signs of toxicity included anorexia, lethargy, weakness, ataxia, paresis, apparent vision changes, seizure-like activity, and generalized seizures. Similar signs of toxicity have also been reported in humans, in whom voriconazole therapeutic plasma concentration for Aspergillus spp. infections is 2-6 µg/ml. Plasma voriconazole concentrations were measured in 18 samples from penguins showing clinical signs suggestive of voriconazole toxicity. The concentrations ranged from 8.12 to 64.17 µg/ml, with penguins having plasma concentrations above 30 µg/ml exhibiting moderate to severe neurologic signs, including ataxia, paresis, and seizures. These concentrations were well above those known to result in central nervous system toxicity, including encephalopathy, in humans. This case series highlights the importance of species-specific dosing of voriconazole in penguins and plasma therapeutic drug monitoring. Further investigation, including pharmacokinetic studies, is warranted. The authors recommend caution in determining voriconazole dosages for use in penguin species.

Ophidiomyces ophiodiicola dermatitis in eight free-ranging timber rattlesnakes (Crotalus horridus) from Massachusetts.

Eight free-ranging timber rattlesnakes (Crotalus horridus) from two geographically isolated Massachusetts populations were observed with skin lesions located primarily on the head but occasionally also on the lateral and ventral surfaces of the body. The snakes underwent health assessments that included physical examination, clinical pathology, full body radiographs, and full thickness biopsies of skin lesions. Each snake had fungal elements present histologically in tissue sections from skin lesions. Ophidiomyces ophiodiicola was identified from skin lesions using polymerase chain reaction in all eight snakes.

Efficacy of treatment and long-term follow-up of Batrachochytrium dendrobatidis PCR-positive anurans following itraconazole bath treatment.

All anuran specimens in the Wildlife Conservation Society's collections testing positive for Batrachochytrium dendrobatidis (Bd) were treated with itraconazole and then studied after treatment to assess the long-term effects of itraconazole and the drug's effectiveness in eliminating Bd carriers. Twenty-four individuals and eight colonies of 11 different species (75 total specimens) tested positive for Bd via polymerase chain reaction (PCR) on multicollection survey. All positive individuals and colonies were treated with a 0.01% itraconazole bath solution and retested for Bd via one of two PCR methodologies within 14 days of treatment completion, and all were negative for Bd. A total of 64 animals received secondary follow-up PCR testing at the time of death, 6-8 mo, or 12-15 mo post-treatment. Fourteen animals (14/64, 21.9%) were PCR positive for Bd on second follow-up. The highest percentage positive at second recheck were green-and-black poison dart frogs (Dendrobates auratus; 5/5 specimens, 100%), followed by red-eyed tree frogs (Agalychnis callidryas; 4/11, 36.4%), grey tree frogs (Hyla versicolor; 1/3, 33.3%), and green tree frogs (Hyla cinera; 3/11, 27.3%). Re-testing by PCR performed on 26/28 individuals that died during the study indicated 11/26 (42.3%) were positive (all via DNA extracted from formalin-fixed paraffin-embedded skin sections). However, there was no histologic evidence of chytridiomycosis in any of 27/28 individuals. The small number of deceased animals and effects of postmortem autolysis limited the ability to determine statistical trends in the pathology data, but none of the necropsied specimens showed evidence of itraconazole toxicity. Problems with itraconazole may be species dependent, and this report expands the list of species that can tolerate treatment. Although itraconazole is effective for clearance of most individuals infected with Bd, results of the study suggest that repeat itraconazole treatment and follow-up diagnostics may be required to ensure that subclinical infections are eliminated in amphibian collections.

Evaluation of medetomidine-ketamine-butorphanol anesthesia with atipamezole-naltrexone antagonism in captive male guanacos (Lama guanicoe).

Seven captive adult male guanacos (Lama guanicoe) weighing 112.0 +/- 10.9 kg (mean +/- standard deviation) were anesthetized with a combination of medetomidine (90.0 +/- 8.8 microg/kg), ketamine (2.7 +/- 0.3 mg/kg), and butorphanol (0.3 +/- 0.03 mg/kg) administered intramuscularly to evaluate its anesthetic and cardiopulmonary effects. Inductions were smooth and rapid, with a mean time to initial effect of 3 +/- 1.5 min and a mean time to recumbency of 5.1 +/- 3.1 min. Anesthesia was predictable, smooth, and characterized by excellent muscle relaxation. Spontaneous ventilation was maintained throughout anesthesia in all animals. Marked bradycardia ranging from 24 to 52 beats/min was noted across all time points for all individuals. Median heart rates decreased during the procedures, but median heart rate, temperature, respiratory rate, and end-tidal carbon dioxide values over the 20 min monitoring period were not significantly different. Mean arterial partial pressure of oxygen (PaO2) on initial sampling was 65.9 +/- 14.8 mm Hg, with six of seven animals exhibiting hypoxemia (PaO2 <80 mm Hg). After oxygen supplementation for 20 min, mean PaO2 values showed statistically significant increases to a mean value of 127.7 +/- 32.4 mm Hg (P = 0.0014). Mean arterial partial pressure of carbon dioxide (PaCO2) showed a significant increase over the monitoring period (P = 0.0004), and mild hypoventilation (PaCO2 >45 mm Hg) was noted in four animals. Mean total duration of procedure time was 23.3 +/- 3.3 min. All guanacos received 0.45 +/- 0.04 mg/kg atipamezole and 2.7 +/- 0.25 mg/kg naltrexone administered intramuscularly for anesthetic antagonism. Recoveries were smooth and uncomplicated. Mean time to sternal recumbency after antagonist administration was 7.7 +/- 4.5 min, and time to successful standing was 12.9 +/- 5.0 min, with all animals standing on first attempt.

Use of a Mycobacterium tuberculosis rapid lateral flow test for assessment of non-specific tuberculin responses in silvered langurs (Trachypithecus cristatus ultima).

Nonspecific responses to intradermal tuberculin skin testing (TST) have been frequently documented in silvered langurs (Trachypithecus cristatus ultima) at the Wildlife Conservation Society's Bronx Zoo. A commercial rapid lateral flow immunoassay (RT) for the identification of Mycobacterium tuberculosis and M. bovis antibodies was employed to assess this test's correlation with TST results and tuberculosis status. Twenty-two silvered langurs were tested with both TST and RT. TST results were characterized into three groups as nonspecific tuberculin responses (n=9), responses to avian tuberculin only (n=6), or negative (n=7). Rapid lateral flow test results were negative in 20 of 22 individuals with equivocal results in two avian tuberculin responders. There was no difference in RT results between TST groups (P = 0.178). Rapid lateral flow test results were not in agreement with mammalian old tuberculin TST results (kappa = 0.08) but showed raw agreement with tuberculosis status (proportion of overall agreement = 0.909). Results suggest the RT assay may useful in the confirmation of nonspecific TST responders in silvered langurs.

Retrospective evaluation of idiopathic hematuria and associated pathology in Grant's gazelles (Gazella granti): 10 cases.

Ten cases of hematuria in Grant's gazelle (Gazella granti) (two male and eight female) from five institutions were examined and the clinicopathologic data summarized. Five gazelles died spontaneously and five were euthanized. All gazelles had marked hematuria without pyuria. Mean age at the onset of clinical signs and time of euthanasia or death was 5.0 +/- 1.4 yr and 8.2 +/- 3.7 yr, respectively. The severity of clinical signs with hematuria ranged from episodes of chronic intermittent hematuria to marked dysuria, with urinary bladder rupture secondary to obstructive blood clots in one case. Submandibular edema was the most common associated clinical sign (five of 10 cases). Serum chemistries from eight gazelles obtained during hematuria episodes revealed hypocalcemia (8/8), hypoproteinemia (7/8), hypoalbuminemia (7/8), and hyperphosphatemia (6/8). Fifty percent of the gazelles (4/8) developed anemia over the course of hematuria episodes. Prothrombin times and partial thromboplastin times were presumed increased in two of four animals evaluated. The predominant histologic lesions in seven of 10 gazelles reviewed were vascular necrosis, vasculitis, and perivasculitis in the urinary tract. Lesions in necropsied gazelles were identified in the urinary bladder (7/10 gazelles), kidney (3/10), and ureter (3/10). Additional urinary tract lesions included tubulointerstitial nephritis (5/10 gazelles), hemorrhagic cystitis (4/10), renal tubular necrosis (4/10), and subacute renal infarcts (2/10). Polymerase chain reaction testing on paraffin-embedded urinary tract tissue for alcelaphine herpesvirus-1 and -2, ovine herpesvirus-2, bluetongue virus, and epizootic hemorrhagic disease virus was negative for the six cases tested. One gazelle that had been vaccinated for Leptospira interrogans had a titer to serovar icterohaemorrhagiae, but serum from the six other gazelles tested was negative for all L. interrogans serovars. No exposure to any toxic agent was identified. An underlying cause for vascular lesions associated with episodic hematuria in Grant's gazelles remains to be determined.

Seroprevalence of retrovirus in North American captive macropodidae.

Laboratory records of serology results from captive macropodidae sampled between 1997 and 2005 were reviewed to assess the seroprevalence of retrovirus exposure. Serum samples from 269 individuals (136 males, 133 females) representing 10 species of macropods housed in 31 North American captive collections were analyzed for retrovirus antibody using an indirect immunofluorescent assay. The prevalence of positive antibody titers comparing male versus female, between species, between age groups, and among animals with identified parentage was examined by nonparametric statistical analyses. Median age of animals at time of sample collection was 36 mo (range 2-201 mo). Total percentage seropositive was 20.4%. Serum antibody was detected in 31 of 47 (66.0%) tammar wallaby (Macropus eugenii), nine of 24 (37.5%) yellow-footed rock wallaby (Petrogale xanthopus), four of 11 (36.4%) swamp wallaby (Wallabia bicolor), 10 of 80 (12.5%) red-necked wallaby (Macropus rufogriseus), and one of 54 (1.9%) parma wallaby (Macropus parma). No individuals of western gray kangaroo (n=3) (Macropus fuliginosus), eastern gray kangaroo (n=19) (Macropus giganteus), common wallaroo (n=6) (Macropus robustus), red kangaroo (n=11) (Macropus rufus), or Matschie's tree kangaroo (n=14) (Dendrolagus matschiei) were positive for retrovirus antibody. These results demonstrate that five species of captive macropods have a history of exposure to retrovirus, with the highest percentage seropositive and highest statistical correlation in M. eugenii (pair-wise Fisher's exact test, alpha = 0.05). Additionally, one wild-caught M. eugenii was confirmed seropositive during quarantine period, indicating that retrovirus exposure may exist in wild populations.