Itraconazole is an antifungal drug used to treat chytridiomycosis, one of the leading causes of global amphibian decline in species such as the critically endangered Panamanian golden frog (Atelopus zeteki). Despite its widespread use for prophylaxis and treatment in both assurance colonies and free-ranging amphibians, there is minimal pharmacologic information to guide dosing. In experiment A, frogs were exposed to 0.01% itraconazole for 10 min and tissue samples were analyzed at various time points from 1 to 84 h. In experiment B, frogs were divided into six groups and exposed to itraconazole in different combinations of concentration (0.01% or 0.001%) and time (5, 10, or 15 min) over 10 days of treatment. Tissue concentrations were quantified via high-performance liquid chromatography. In experiment A, following a one-time dose, itraconazole concentrations remained high until the end of the experiment at 84 h. In experiment B, at 0.01% itraconazole daily for 10 days, skin and liver concentrations were high and increased substantially over the 10-day treatment course. Frogs exposed at the lower concentration (0.001%) had tissue concentrations that appeared to remain steady. At the reported doses over 10 days, there was no histologic evidence of hepatic toxicity, although one frog was found dead in the low-dose bath at 84 h and could not be further analyzed. This experiment is an excellent example of assurance colonies providing evidence-based information for the improved care and welfare of amphibians in order to prepare for future free-ranging populations.

Penguins under managed care are widely considered to have high susceptibility to infection by Aspergillus spp. Antemortem laboratory diagnostic options vary in sensitivity and specificity, and antibody detection has been problematic in penguin species given elevated levels of reactivity observed in clinically normal patients using traditional whole antigen enzyme-linked immunosorbent assays (ELISA). In the present study, an alternative assay was implemented to detect reactivity to Afmp1p, an Aspergillus cell wall antigen, in samples obtained from several different penguin species. With confirmed infection, abnormal protein electrophoretograms were consistently observed, and gliotoxin was detected in the majority of cases. An increase in reactivity to Afmp1p was observed in penguins with confirmed (n = 18, p < 0.0001) and probable (n = 13, p = 0.08) aspergillosis versus normal adult penguins (n = 33). Interestingly, increased reactivity to Afmp1p (p < 0.0001) was noted in normal adult penguins (n = 33) versus juvenile penguins (n = 22, p < 0.0001). Overall, the area under the curve for this assay was 0.890, with a sensitivity of 94.4% and a specificity of 57.6%, with an antibody assay cutoff of 1.0. Increasing reactivity resulted in an increase in specificity. These data support the use of Afmp1p antibody quantitation as part of a diagnostic workup in penguins with suspected aspergillosis.

Aspergillosis is a major cause of morbidity and mortality in penguins, with triazole antifungal drugs being commonly used for prophylaxis and treatment. This report describes 15 cases of fatal hemolysis associated with liquid itraconazole and voriconazole formulations administered to African penguins (Spheniscus demersus) from four institutions. All penguins underwent stressful events (e.g. relocation, induced molt) and were administered commercial liquid itraconazole formulations or compounded voriconazole liquid suspension. Observed clinical signs in affected penguins prior to death included hyporexia, weight loss, lethargy, dyspnea, red-tinged droppings, and obtunded mentation. Intra- and extravascular hemolysis and hemoglobinuric nephrosis were the primary pathologic manifestations on postmortem examination. The concentration-dependent hemolytic potentials of itraconazole, voriconazole, and commercial and compounded vehicle suspensions were evaluated in vitro by exposing chicken whole blood as a surrogate for penguin blood. Hemoglobin content in blood plasma was then measured by spectrophotometry. Neither itraconazole nor voriconazole alone induced hemolysis in vitro. The vehicle ingredients sorbitol and hydromellose induced hemolysis, but not at predicted plasma levels in chicken erythrocytes, suggesting neither the azole antifungals nor their major vehicles alone were likely to contribute to hemolysis in vivo in these penguins. Potential mechanisms of toxicosis include generation of an unmeasured reactive metabolite causing hemolysis, preexisting erythrocyte fragility, or species-specific differences in hemolytic thresholds that were not assessed in the chicken erythrocyte model. More research is needed on the potential for toxicosis of azole antifungal drugs and carrier molecules in this and other avian species.

Panamanian golden frog (PGF) (Atelopus zeteki) is a critically endangered species. The Maryland Zoo in Baltimore houses two groups of PGF originating from distinct geographic locations as an assurance colony, with the goal of upholding genetics for future release of individuals back to their native environment. The purpose of this cross-sectional study was to characterize the prevalence of ocular abnormalities in these two zoo-housed populations of PGF as well as to establish normal parameters for selected diagnostic tests in these groups. Twenty-five females and 25 males were randomly selected from each group (100 PGF; 200 eyes in total) to undergo ocular examination using slit lamp biomicroscopy and direct ophthalmoscopy. Endodontic absorbent paper point test (EAPPT) and intraocular pressure (IOP) and Rose Bengal stain diagnostic tests were also performed. Reference ranges for tear production (EAPPT, 0.5-3 mm/min) and IOP (14-26 mmHg) were calculated in the nondiseased PGF eyes (n 1/4 160 eyes). Rose Bengal stain uptake was negative on all eyes. In total, 40 eyes of 30 PGF were found to have some form of ocular abnormality (28% of PGF, 20% of eyes). The most frequently observed ocular abnormalities were cataract (9% of PGF, 6% of eyes) and keratitis (nonlipid keratopathy; 10% of PGF, 5.5% of eyes). There was no significant difference in overall ocular abnormality prevalence between the two groups studied (P 1/4 0.37) or between the sexes (P 1/4 0.76). The median age of an eye with cataract and keratitis (nonlipid keratopathy) was 10.35 and 7.7 yr, respectively. Ocular abnormalities are common in these two populations of PGF. Documentation of these ocular abnormalities and establishment of diagnostic reference ranges have not previously been published and may be important for maintaining the health of this endangered species.

Antemortem serodiagnosis of aspergillosis remains challenging in Sphenisciformes. Protein electrophoresis, serology (antibody, antigen) by ELISA, and gliotoxin detection provide variable diagnostic value. In the present study, a commercially available Western blot (WB) validated for use in humans and dolphins was adapted for use with penguin samples. Using the same method and reagents, samples were analyzed from multiple institutions in the United States and one facility in France. This was inclusive of normal juvenile African penguins (Spheniscus demersus, n = 10) and various species of penguins in the United States with confirmed infection (n = 9) as well as 52 samples from Humboldt penguins (Spheniscus humboldti) in France. Cumulative WB scores (based on reactivity to different antigens) were found to be significantly higher in the group of penguins with confirmed infection (p < 0.0001). Significant differences were also observed between the clinically normal penguins in the two populations, with higher scores in the United States (median score 1.0, 95%CI [0-5], min 0, max 11) compared to France (median score 0,95%CI [0-0], min 0, max 5). The utilization of the WB as a diagnostic tool is inconclusive due to the use of samples from varying institutions, environmental background, age, and stages of infection. However, this tool may provide an overview of antigen reactivity in penguins infected with Aspergillus to help design a more robust serology assay and further understand the humoral immune response during infection.

Assessing quality of life in animals is an art as much as a science. Despite the use of questionnaires and keeper reports which consider several aspects of well-being, the process often remains subjective. Keepers have unique insights, and anecdotal observations can be enhanced with objective data. We combined the art and science of assessments in this study on a geriatric macaque (1.0 lion-tailed/pig-tailed (Macaca silenus/macaca nemestrina) hybrid), using historic data to inform management decisions. Following the unexpected death of his cage mate, his activity and engagement with keepers decreased, and new concerning behaviors presented. While the zoo worked to identify new social opportunities, we used these data to develop a plan to improve his quality of life (e.g., increase training sessions, enrichment, social interactions). After intense implementation, we saw a significant increase in activity level and engagement with keepers; the frequency of unexpected behaviors suggesting a lower quality of life, however, increased over time. Our data allowed us to objectively compare changes in behavior, enabling the zoo to make the most informed animal management decision possible. [Figure: see text].

The mass extinction of amphibians necessitates specialized programs to ensure species' survival. Maryland Zoo in Baltimore houses the largest assurance population of the critically endangered Panamanian golden frog (Atelopus zeteki). However, individuals in this population experience a tetany-like syndrome, characterized by rigid/inappropriately positioned limbs and difficulty hopping, swimming, and righting. In this study, a syndrome case definition was assigned and the associated clinical signs were described. Then, four different treatments were systematically assessed in order to find the most effective protocol for treatment and begin to elucidate its underlying causes. Eighty-three frogs fulfilled the case definition and were treated orally for 14 d with either calcium gluconate, magnesium chloride, supplemental gavage feeding, or combination of calcium, magnesium, and vitamin B complex. Frogs were tested with a defined protocol assessing hopping, righting, and swimming abilities. Testing was performed at symptom onset and repeated weekly until resolution occurred. Analyses revealed that combination treatment was significantly more effective in eliminating clinical signs of tetany syndrome. Results show the most effective way to treat this syndrome, but do not help elucidate the underlying cause. Future work will focus on examining factors (e.g., diet, husbandry) that may elicit the syndrome for a more complete understanding of its etiology.

The Panamanian golden frog (Atelopus zeteki) is a critically endangered species and currently is believed to survive and reproduce only in human care. Panamanian golden frog males are considerably vocal which may be an important component in their successful reproduction, though little is currently known about their calls. To better understand the behavior and vocal patterns of this species and to improve breeding efforts in the assurance colony, we employed individual sound recording of male advertisement calls and acoustic monitoring of a breeding colony to investigate variation in the vocal behavior of Panamanian golden frogs. The goal was to capture variability within and among frogs as well as patterns of periodicity over time. First, the advertisement calls from individual male Panamanian golden frogs were recorded, and acoustic parameters were analyzed for individual differences. Results suggest that male advertisement calls demonstrate individual- and population specificity. Second, data collected through a year-long acoustic monitoring of the breeding colony were investigated for circadian and circannual periodicity. Male vocal activity revealed a circadian periodicity entrained by the daily light schedule. Seasonal periodicity was also found with highest vocal activities between December and March. The finding of a seasonal periodicity is worth noting given that the population had been bred for 20 years under constant environmental conditions. Finally, results suggest that vocal activity was responsive to daily animal care activity. Vocal activity decreased substantially when personnel entered the room and engaged in animal husbandry activities. The findings illustrate the usefulness of acoustic monitoring to provide insight into animal behavior in a zoo setting in a key breeding colony of endangered animals, and calling pattern observations may be utilized to modify husbandry practices to improve Panamanian golden frog breeding success and general care.

Y Aspergillosis remains a difficult disease to diagnose antemortem in many species, especially avian species. In the present study, banked plasma samples from various avian species were examined for gliotoxin (GT), which is a recognized key virulence factor produced during the replication of Aspergillus species hyphae and a secondary metabolite bis(methyl)gliotoxin (bmGT). Initially, liquid chromatography-tandem mass spectrometry methods for detecting GT and bmGT were validated in a controlled model using sera obtained from rats experimentally infected with Aspergillus fumigatus. The minimum detection level for both measurements was determined to be 3 ng/ml, and the assay was found to be accurate and reliable. As proof of concept, GT was detected in 85.7% (30/35) of the samples obtained from birds with confirmed aspergillosis and in 60.7% (17/28) of samples from birds with probable infection but only in one of those from clinically normal birds (1/119). None of the birds were positive for bmGT. Repeated measures from birds under treatment suggests results may have prognostic value. Further studies are needed to implement quantitative methods and to determine the utility of this test in surveillance screening in addition to its use as a diagnostic test in birds with suspected aspergillosis.

Avian malaria, caused by Plasmodium spp. and transmitted by mosquitos, is a leading cause of mortality of captive penguins. Antimalarial drugs are currently used to control infections in penguins. However, the effectiveness of treatment reduces significantly by the time the clinical signs appear, while early and unnecessary treatment interferes with development of protective immunity. Therefore, for suppressing parasitemia without affecting the development of immunity in captive penguins, antimalaria drugs need to be administered at the right time, which requires reliable diagnostic tools that can determine the levels of circulating antimalaria antibodies. In the present study, we have developed an enzyme-linked immunosorbent assay (ELISA) diagnostic assay based on the merozoite surface protein 1 (MSP-1) of P. relictum isolate SGS1 to specifically detect and relatively quantify antimalaria antibodies in penguins. We expressed and purified a truncated P. relictum isolate SGS1 MSP-1 and optimized its biotinylation and subsequent conjugation to streptavidin alkaline phosphatase for signal generation in ELISA. We tested the assay by analyzing sera obtained from penguins at the Baltimore Zoo, from Spring through Fall, and found that levels of detectable antibodies against MSP-1 varied seasonally for individual penguins, consistent with the expected seasonal variations in avian malaria prevalence. Corroboratively, we analyzed the sensitivity of the assay by titrating positive sera and found that the signal intensity generated was serum concentration-dependent, thus validating the ability of the assay to detect and relatively quantify the levels of antimalaria antibodies in penguin sera.

Fifteen maned wolves (Chrysocyon brachyurus) were anesthetized a total of 43 times as part of a long-term ecology and health study in a remote region of northeastern Bolivia. We administered tiletamine-zolazepam (TZ) to wolves in box traps or free-ranging, from blinds or on foot, at a mean dosage of 4.6 mg/kg intramuscularly. Detailed anesthetic information was recorded in 24 of these events in 11 wolves (six males, five females), and wolves were monitored closely post procedure with very high frequency or global positioning system telemetry collars. Anesthetic induction was smooth and rapid in all cases, with a mean 6.4 min from injection to recumbency. Vital parameters were stable during the majority of procedures. As expected with this drug combination, recovery was long (mean time to standing 163 min [range: 80-235 min]) but smooth, and animals were monitored in most cases in box traps until stable for release. One case of apnea and prolonged recovery is reported. In two cases, wolves recovered normally but were found to move minimally in the 2.5-4 d postprocedure before resuming normal movements. Overall, TZ provided safe, stable immobilization of free-ranging maned wolves in remote and extreme field conditions, although postanesthesia monitoring via telemetry is recommended.

BACKGROUND:With Panamanian golden frogs (Atelopus zeteki; PGFs) likely extirpated from the wild, ensuring long-term sustainability of captive populations is crucial in order to conserve this critically endangered species. Unfortunately, PGFs display a unique reproductive behavior involving a prolonged period of amplexus leading to challenges in their successful captive propagation. The Maryland Zoo in Baltimore has observed high levels of mortality during the breeding season and suboptimal reproductive success leading to the use of hormone stimulation to aid in reproduction and health management.METHODS:This project aimed to develop induced ovulation and health management protocols by (1) evaluating different doses of gonadotropin releasing hormone analogue (GnRHa), (2) comparing the efficacy of GnRHa and GnRHa + metoclopramide, (3) determining latency periods and the effects of pulsed hormone sequences; and (4) establish if mortality is impacted by hormone therapy. Female PGFs (n = 174) were given GnRHa either in various concentrations (Experiment 1) or combined with metoclopramide (Experiment 2), and oviposition success, latency, and mortality were measured as binary response variables.RESULTS:Overall, the use of exogenous hormones significantly decreased mortality when compared to the control data of natural egg-laying females. GnRHa doses of 0.05 μg/g body weight produced similar ovulation rates compared to higher doses, and the addition of metoclopramide did not increase oviposition success compared to GnRHa alone. Lastly, results indicate the majority of female PGFs will release eggs within 48 h following the initial pulse of hormones with a small percentage ovipositing after a second pulse.CONCLUSION:Findings from this study will benefit captive management of PGFs by documenting the increased survival of females when given hormone stimulation and defining appropriate GnRHa doses and expected latency to spawning.

Currently, more than 20% (51/240) of zoos and aquariums accredited by the Association of Zoos and Aquariums house African penguins (Spheniscus demersus) in their collections. The African penguin Species Survival Plan (SSP) veterinary advisors regularly collect information from those facilities to characterize morbidity and mortality for this species and to collate preventative medicine and treatment regimens. These efforts resulted in more than 10 yr of collection of management data across the SSP, representing the care and management of more than a thousand birds. The most common morbidities reported included those of dermatologic (27%, 125/452 institutions) and musculoskeletal or neurologic (18%, 82/452 institutions) disease, while the most common causes of mortality were respiratory diseases (20%, 65/323 deaths) and systemic or multifactorial conditions (19%, 62/323 deaths). Aspergillosis cases accounted for 69% (45/65 deaths) of respiratory-related mortality and avian malaria cases comprised 31% (19/62 deaths) of mortality related to systemic diseases. Mortality was most commonly reported in geriatric birds, or those older than 15 yr of age (34%, 111/323 deaths). Reproductive related mortality was only defined in female birds, while other causes of death were more evenly distributed between sexes. Utilizing the SSP data to determine morbidity and mortality trends within this population provides important information to veterinary and animal care teams, allowing them to provide enhanced levels of care to the penguins housed at their institutions. By recognizing the most important diseases and causes of death in this species, management and healthcare resources can target conditions with the highest impact on the population.

Primaquine is an 8-aminoquinolone drug commonly used for the chemoprophylaxis and treatment of avian malarial infections in managed penguin populations worldwide. Little is known about its pharmacokinetic properties in avian species. The objective of this study was to describe the disposition of primaquine phosphate after a single oral dose in 15 healthy African penguins (Spheniscus demersus). A single tablet containing 26.3 mg of primaquine phosphate (equivalent to 15 mg primaquine base) was administered orally to each bird in a herring fish. Blood samples were collected prior to drug administration and at predetermined timepoints through 144 hr postadministration. Plasma was analyzed for drug concentration by high-performance liquid chromatography with ultraviolet detection. Mean maximum plasma concentration of primaquine phosphate was 277 ± 96 ng/ml at approximately 3.1 hr following oral administration. The mean disappearance half-life was 3.6 ± 1.6 hr. Plasma concentrations were below detectable limits in all but one penguin by 36 hr. A single oral administration of 26.3 mg of primaquine phosphate in African penguins resulted in a pharmacokinetic profile comparable to those attained in human studies. These results suggest that a dosing interval similar to human regimens may be of potential use in the prevention and treatment of avian malaria in penguins. Additional clinical studies are needed to determine the efficacy and safety of this regimen.

Failure of passive transfer of immunity (FPT) leads to increased calf morbidity and mortality and requires intensive, time-sensitive, and often expensive management for nondomestic ruminants. Without species-specific information with which to make informed decisions, neonatal data from domestic ruminants are often extrapolated to nondomestic zoo-housed species. To date, there have been no studies evaluating FPT in sitatunga (Tragelaphus spekii). The goal of the present study was to establish parameters to characterize adequate passive transfer in sitatunga calves and compare them to published reference intervals in other species. Medical records of 22 sitatunga calves (12 female, 10 male) were reviewed. Seventeen of these calves were defined as "healthy," having survived at least 60 days without colostrum administration or a plasma transfusion. Calf weight, serum glucose, serum gamma-glutamyl transferase (GGT), total protein (TP), globulin concentrations, and results of a zinc sulfate turbidity test (ZSTT) were noted where possible. Mean birth weight of healthy calves at 24 hr was 4.5 kg (range: 3.76.5 kg, n = 12). The mean blood glucose in healthy calves was 152 mg/dl (range: 80-182, n = 16), mean serum TP concentration was 5.9 g/dl (range: 4.9-7.5, n = 16), mean serum globulin concentration was 3.3 g/dl (range: 1.7-4.7, n = 17), and mean serum GGT concentration was 466 U/L (range: 91-1901, n = 16). A ZSTT was performed for 10 healthy calves, resulting in four negative ZSTT results despite having no clinical signs of FPT and the calves having been observed nursing before testing. Sitatunga appear to have lower values for normal FPT parameters than those developed for domestic cattle. This study illustrates the difficulty of cross-species comparisons, as even closely related species can vary greatly in biologic parameters.

The anatomy of the avian gastrointestinal (GI) tract is uniquely suited to each species' dietary requirements. African penguins (Spheniscus demersus) are charismatic and popular exhibit animals. As their prevalence grows, there is a need to understand their unique digestive tract to diagnose abnormalities. Reference material specific to the digestive tract of piscivores is scant, and knowledge of the GI tract of a healthy penguin is based on information from other birds. The purpose of this study is to determine the normal gross anatomy, transit time, and histopathologic structures of the penguin GI tract. Twelve clinically healthy penguins were selected for this study from the colony at the Maryland Zoo in Baltimore, which, at the time of this study, consisted of 55 birds. All penguins underwent a barium contrast study, and radiographic images were obtained until the entire GI tract was empty. Approximately 2 wk later, each penguin was anesthetized, and an endoscopic evaluation of the anterior GI tract was performed. Time from barium administration to defecation ranged from 17 to 70 min, and on average, barium clearance was 17.6 hr (range, 5-36 hr). Fluid from the ventriculus had an average pH of 2.75 and contained a mixed bacterial population. Koilin presence and thickness appreciated on endoscopy did not correspond with the thickness determined on histopathology. The results of this study provide a comparative baseline to use during diagnostic workups and help guide treatment decisions.

Abnormal molting, including partial or incomplete molt, arrested molt cycle, or inappropriate frequency of molt, is a primary concern for the managed African penguin (Spheniscus demersus) population and is documented across institutions. To identify factors associated with increased odds of abnormal molts and characterize intervention opportunities, a comprehensive survey evaluating numerous husbandry and medical parameters was created. Survey results represent 45 North American African penguin holding facilities and 736 unique animals. Of these individuals, 135 (18.3%) demonstrated an abnormal molt over the 5-yr study period (2012-2017). Increased odds ratios for abnormal molt included biologic (age, sex, etc.), geographic (elevation, latitude), and husbandry (exhibit design, diet, etc.) variables. The mean age of affected animals was 15.2 yr (1-45 yr, n =135) compared with 9.92 yr (4 mo-38 yr, n = 601) for unaffected animals. In addition, although statistically insignificant, males were overrepresented in the affected cohort compared with a near even distribution among unaffected animals. Identified factors with increased odds for abnormal molting included advanced age and facilities using freshwater pools. Normally molting penguins were more commonly found with saltwater pool access and natural lighting exposure. Anecdotal medical intervention attempts are discussed, although further research is needed to define their use. Of attempted interventions, subcutaneous 5.4-mg melatonin implants placed in anticipation of environmental molting cues showed the most promise at inducing catastrophic molt, with 14 of 17 (82.3%) of affected individuals molting normally following this treatment. Survey analysis indicated that abnormal molt is a complex, multifactorial process, and modifiable factors that may predispose animals to abnormally molt exist. Addressing these factors in future exhibit design may mitigate the prevalence of this condition. Despite these efforts, it is likely that medical interventions will be required to aid in the treatment of abnormal molting in this species.

A group of five juvenile Meller's chameleons (Trioceros melleri) experienced 100% mortality over a period of 1 mo due to ranavirus infection. The index case was found dead without premonitory signs. The three subsequent cases presented with nonspecific clinical signs (lethargy, decreased appetite, ocular discharge) and were ultimately euthanatized. The final case died after initially presenting with skin lesions. Postmortem examination revealed thin body condition in all five animals and mild coelomic effusion and petechiae affecting the tongue and kidneys of one animal. Microscopically, all animals had multifocal necrosis of the spleen, liver, and kidney; four of five animals had necrosis of the nasal cavity; and two of five had necrosis of adrenal tissue, bone marrow, and skin. Numerous basophilic intracytoplasmic inclusions were present in the liver of all animals and nasal mucosa of three of the five animals. Consensus polymerase chain reaction for herpesvirus and adenovirus were negative, whereas ranavirus quantitative polymerase chain reaction was positive. Virus isolation followed by whole genome sequencing and Bayesian phylogenetic analysis classified the isolates as a strain of frog virus 3 (FV3) most closely related to an FV3 isolate responsible for a previous outbreak in the zoo's eastern box turtle (Terrapene carolina carolina) group. This case series documents the first known occurrence of ranavirus-associated disease in chameleons and demonstrates the potential for interspecies transmission between chelonian and squamate reptiles.

An estimated 9.5-yr-old, male, captive American toad (Anaxyrus americanus) presented with bilateral cataracts of unknown duration. At the time of presentation, the animal continued to eat and behave normally. Lesions appeared to be static prior to brumation; however, following brumation, and 7 months after initial examination, the toad presented with progressive lesions and presumed blindness. Phacoemulsification was performed bilaterally under tricaine methanesulfonate immersion anesthesia. Although induction, maintenance anesthesia, and the phacoemulsification procedure were unremarkable, the animal died during recovery. This case illustrates the first reported phacoemulsification in an amphibian.