DETECTION OF GLIOTOXIN BUT NOT BIS(METHYL)GLIOTOXIN IN PLASMA FROM BIRDS WITH CONFIRMED AND PROBABLE ASPERGILLOSIS.

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.

CLINICOPATHOLOGIC EVALUATION OF AN EX SITU POPULATION OF ADULT AFRICAN WHITE-BELLIED PANGOLIN (PHATAGINUS TRICUSPIS).

Death due to illegal trade and trafficking serves as the largest threat to pangolins in their geographic range countries. Appropriate veterinary care is essential to the welfare of sick or debilitated pangolins. Given the challenge of comprehensive physical examination of this species due to its unique anatomic features and poorly studied physiology, laboratory diagnostics can provide essential information to guide appropriate medical care. This retrospective study included previously analyzed blood and urine samples collected from adult (n = 17) African white-bellied pangolins (Phataginus tricuspis) under managed care that were sampled over a 4-yr period as part of routine veterinary examinations. Samples from clinically normal animals were used to summarize results for this species. Pangolins had a lymphocyte-predominant leukogram with a mean neutrophil to lymphocyte ratio of 0.83 (min-max: 0.13-4.81). Seasonal, sex, and age differences were present for several hematologic and serum biochemical analytes. Urine samples had a median urine specific gravity (USG) of 1.040 (min-max: 1.006-1.060), a neutral-to-basic urine pH (6.0-8.6), and contained mainly struvite crystals or infrequently calcium oxalate dihydrate crystals. Winter samples had higher USG than summer samples. The clinicopathologic data provided herein offers applicable information useful for the clinical management of pangolins.

SAFETY OF AND HUMORAL IMMUNE RESPONSE TO THE MERIAL RECOMBITEK CANINE DISTEMPER VIRUS VACCINE IN MANED WOLVES (CHRYSOCYON BRACHYURUS).

This study evaluated the safety of and humoral response to the Merial Recombitek® recombinant canine distemper virus (rCDV) vaccine in maned wolves (n = 9, age 2-9 yr). All maned wolves had prior history of annual vaccination with the Merial Purevax® ferret rCDV vaccine. Serum neutralization (SN) to CDV was measured prior to initial vaccination with the rCDV Recombitek vaccine followed by a booster vaccination at 4-6 wk. Final SN titers were obtained at 13 wk post initial vaccination. The maned wolves developed no observable adverse side effects through the study. Pre-Recombitek vaccination SN titers ranged from negative to 1: 8. Postvaccination CDV titers ranged from negative to 1: 8, and were therefore below the range of that considered protective in domestic dogs.

Comprehensive Breeding Techniques for the Giant Panda.

The dramatic growth of the captive giant panda (Ailuropoda melanoleuca) population exemplifies how the application of scientific findings to animal care and reproductive management can improve conservation breeding outcomes. Detailed behavioral studies of giant panda estrus, pregnancy and cub rearing have demonstrated the importance of husbandry management that supports natural reproductive behavior to enhance breeding success. Natural breeding has been valuably augmented by the development of assisted reproductive techniques founded through detailed studies of the reproductive physiology of the giant panda and outlining fundamental information about reproductive seasonality, male fertility and characterization of the estrous cycle. The resultant holistic understanding of giant panda reproduction has improved reproductive success in the captive population to such an extent that it is now self-sustaining and provides surplus animals for reintroduction. Despite these significant advances, there are knowledge gaps and remaining challenges to be addressed. Pregnancy detection remains the single biggest challenge when determining if natural mating or assisted breeding have been successful. Because pregnancy can only be determined in the few weeks prior to parturition, there are gaps in understanding and detecting delayed implantation and early embryonic loss. Additionally, dynamic management practices and standard of care for reproductive assistance needs to be developed. Only large breeding centers in China have the ability to promote normal reproductive behaviors and allow mate choice for the giant panda. These challenges need to be addressed in the near future in order to maintain a self-sustaining, genetically diverse and behaviorally competent captive population. This chapter documents the development of successful giant panda managed breeding programs by focusing on three key areas, (1) the development of science-driven reproductive techniques to improve fecundity in a species where the mating system was poorly understood, (2) how targeted research and adaptive management of social settings surrounding estrus and breeding improved reproductive success, and (3) insights and solutions to challenges faced across the program's history with future directions for research.

ECHOCARDIOGRAPHIC PARAMETERS IN AFRICAN WHITE-BELLIED PANGOLINS (PHATAGINUS TRICUSPIS) WITHOUT CARDIAC DISEASE.

Relatively little is known about pangolin care and physiology, and it has become increasingly important to have a better understanding of normal health parameters for evaluation of free-ranging, rehabilitating, and managed populations. Echocardiograms were performed on six African white-bellied pangolins (Phataginus tricuspis) under professional care at a single institution. Because of the propensity of pangolins to roll up with only thick keratinized scales exposed, the pangolins were anesthetized with either isoflurane or sevoflurane. All echocardiograms were performed and evaluated by a board-certified cardiologist. None of the pangolins had any evidence of prior cardiac disease. All standard measurements were obtained. Four pangolins (66.7%) had trace to mild mitral regurgitation, six (100%) had trace to mild tricuspid regurgitation, and two (33.3%) had trace to mild pulmonic regurgitation. All examinations were determined to be normal, with no evidence of valvular structural abnormalities, cardiac dysfunction, chamber enlargement, or cardiac hypertrophy. This report broadens understanding of normal echocardiogram parameters in pangolin species and contributes techniques and baseline data for veterinarians caring for these vulnerable populations.

COMPLICATIONS ASSOCIATED WITH PREGNANCY AND PARTURITION IN AFRICAN WHITE-BELLIED PANGOLINS (PHATAGINUS TRICUSPIS).

There are no studies to date on the normal reproductive physiology of African white-bellied pangolins (Phataginus tricuspis). As a reclusive species, little is known about normal gestation, successful parturition, and potential complications during pregnancy. Ten female P. tricuspis were diagnosed as pregnant and monitored under professional care. Five developed complications during pregnancies or during parturition and are detailed in this case series. Dystocia occurred in two dams each, with malposition of a singleton fetus. Both dams were successfully treated with surgical intervention by caesarian section. Of the two individuals, one fetus was nonviable, but the other neonate survived and was reared by the dam to weaning. A third pregnant female died during pregnancy from septicemia resulting in death of the preterm fetus. The two additional dams of the five gave birth to full-term neonates. One fetus was stillborn with evidence of fetal distress, and the other died immediately after birth (perinatal death) with undetermined etiology. Based on this case series, complications associated with pregnancy occur in P. tricuspis, indicating the need for further study and close monitoring during impending parturition.

VENOUS BLOOD GAS AND SELECTED BIOCHEMICAL VALUES FROM AWAKE AND ANESTHETIZED WHITE-BELLIED PANGOLINS ( PHATAGINUS TRICUSPIS).

Pangolins are one of the most-trafficked endangered species and are rare in zoologic collections. Blood gas and select biochemical analysis was performed on nine African white-bellied tree pangolins ( Phataginus tricuspis). Paired blood samples were collected from the ventral tail vasculature prior to (T1) and after 10 min (T2) of sevoflurane anesthesia, and selected blood gas and biochemical parameters were measured using a portable blood gas analyzer. Median (min-max) values for corrected venous blood were pH = 7.372 (7.207-7.529) at T1 and 7.278 (7.124-7.418) at T2. Corrected values were PCO2 = 51.7 (29.6-69.0) mmHg at T1 and 58.431 (38.1-90.8) mmHg at T2. Clinicians should be aware that this species may hypoventilate while anesthetized with sevoflurane and develop a respiratory acidosis. Concomitant administration of respiratory depressants could compound this effect in a species where oro-tracheal intubation has not been described.

HEMATOLOGY AND SERUM BIOCHEMISTRY VALUES OF THE RED PANDA SUBSPECIES ( AILURUS FULGENS STYANI).

Health evaluations were conducted on individuals ( n = 62, age < 1 to 11 yr) from a captive red panda ( Ailurus fulgens styani) population at the Chengdu Research Base for Giant Panda Breeding in Sichuan Province, People's Republic of China. Individuals were anesthetized using combinations of ketamine, dexmedetomidine, diazepam, isoflurane, and tiletamine/zolazepam. Whereas physical exams revealed no abnormalities for 40% of red pandas, 24% had dental abnormalities and 18% had orthopedic abnormalities. As the red panda is the only member of the family Ailuridae, complete blood count and serum biochemistry samples were collected and used to develop reference intervals for this species. When hematologic and serum chemistry values of male and female red pandas were compared within this A. f. styani population, males had higher mean cell hemoglobin, mean cell hemoglobin concentration, and blood urea nitrogen concentrations, whereas females had higher alkaline phosphatase, cholesterol, and iron. Adult red pandas (≥2 yr) had higher lymphocyte percentage, total protein, globulin, and chloride levels, whereas juveniles (<2 yr) had higher white blood cell concentrations, neutrophil percentage and absolute neutrophil values, alkaline phosphatase, creatine kinase, cholesterol, potassium, total and ionized calcium, and phosphorus levels. Reference values and expected age and sex differences will assist with health management of the captive A. f. styani population.

OSTEOCHONDRAL AUTOGRAFT TRANSFER FOR TREATMENT OF STIFLE OSTEOCHONDRITIS DISSECANS IN TWO RELATED SNOW LEOPARDS ( PANTHERA UNCIA).

Two related female snow leopards ( Panthera uncia) were diagnosed with osteochondritis dissecans of the lateral femoral condyle via radiographs and computed tomography. Lesions were either bilateral (Patient 1) or unilateral (Patient 2). Surgical treatment was performed using an osteochondral autograft transfer system following cartilage flap removal. After a 6-wk recovery period, both animals had a markedly improved gait with mild pelvic limb stiffness. Patient 1 developed intermittent lameness 9 mo postsurgery and Patient 2 had mild lameness 3 yr postsurgery. Because of recurrent lameness, both animals were treated with platelet-rich plasma intra-articular injections and oral gabapentin (Patient 1) or oral meloxicam (Patient 2). Full incorporation of autografts was confirmed with computed tomography (both patients) and arthroscopy (Patient 1 only). Osteochondritis dissecans has been previously reported in snow leopards and these cases represent the first reported use of osteochondral autograft repair and platelet-rich plasma to treat the condition.

REPEATED ANESTHESIA IN A BLACK RHINOCEROS ( DICEROS BICORNIS) TO MANAGE UPPER RESPIRATORY OBSTRUCTION.

This report describes weekly repeated anesthesia in a 7-yr-old, 1,030 kg, female Eastern black rhinoceros ( Diceros bicornis michaeli), that was immobilized six times using a combination of 2 mg etorphine (0.002 mg/kg), 5 mg medetomidine (0.005 mg/kg), 25 mg midazolam (0.024 mg/kg), and 300 mg ketamine (0.29 mg/kg) delivered intramuscularly (IM) via remote dart to facilitate long-term medical care of a bilateral, obstructive Actinomyces sp. rhinitis. The drug combination described in this study resulted in reliable, rapid recumbency of the animal within 2-8 min after initial administration via dart and produced deep anesthesia for 34-78 min without supplemental anesthetic administration. Antagonist drugs (100 mg naltrexone [0.1 mg/kg] and 25 mg atipamezole [0.024 mg/kg] IM) produced reliable and uneventful recoveries in all the procedures. During each anesthetic procedure, the animal was intubated and provided intermittent positive pressure ventilation with a megavertebrate demand ventilator. Tachycardia and hypoxia noted after induction resolved after positive pressure ventilation with oxygen. This report provides useful information on a novel anesthetic protocol used repeatedly for intensive medical management in a black rhinoceros.

SURVEY OF ANAPLASMA PHAGOCYTOPHILUM ANTIBODIES IN CAPTIVE PRZEWALSKI'S HORSES (EQUUS FERUS PRZEWALSKII).

Anaplasma phagocytophilum (formerly Ehrlichia equi ) is a tickborne pathogen of domestic horses and the causative agent of equine granulocytic anaplasmosis. After the occurrence of clinical anaplasmosis in a Przewalski's horse ( Equus ferus przewalskii) housed at the Smithsonian Conservation Biology Institute in 2008, opportunistic serosurveillance of the herd was initiated. From 2008 to 2014, 57 serum samples were collected from 27 individuals (10 males; 17 females). Using indirect immunofluorescent antibody assays for anti- Anaplasma phagocytophilum antibodies, it was determined that prevalence was 53%. No significant sex differences were identified. A statistical association between increasing age and seropositive status suggests cumulative risk of exposure to Anaplasma phagocytophilum . After exclusion of four clinical cases of anaplasmosis, it was found that 22-57% of those sampled each year were seropositive and clinically normal, suggesting that the majority of Przewalski's horses develop subclinical or self-limiting anaplasmosis after exposure to A. phagocytophilum .

CLINICAL DISEASE ASSOCIATED WITH ANAPLASMA PHAGOCYTOPHILUM INFECTION IN CAPTIVE PRZEWALSKI'S HORSES (EQUUS FERUS PRZEWALSKII).

Anaplasma phagocytophilum is a tick-borne pathogen of domestic horses and the causative agent of equine granulocytic anaplasmosis. This case series describes three confirmed cases of clinical anaplasmosis, and a fourth case of presumptive anaplasmosis in Przewalski's horses ( Equus ferus przewalskii) housed at the Smithsonian Conservation Biology Institute from 2008 to 2014. Clinical signs varied among individuals with affected horses exhibiting lethargy, weakness, pyrexia, hypophagia, reluctance to move, or ataxia. Anaplasmosis cases were confirmed with a combination of identification of neutrophilic inclusions (morulae) on peripheral blood smear, positive polymerase chain reaction (PCR) testing of whole blood, or convalescent titers. All animals recovered after antimicrobial therapy with oxytetracycline. Diagnosis should be made by a combination of clinical signs plus identification of morulae or positive A. phagocytophilum PCR. Treatment is curative using oxytetracyline intramuscularly or intravenously followed by daily therapy with oxytetracyline or minocycline for 14-30 days. The authors recommend that A. phagocytophilum infection be included on any differential list for Przewalski's horses presenting with fever or ataxia within or near an enzootic area.

HEMATOLOGY, SERUM BIOCHEMISTRY, AND URINALYSIS VALUES IN THE ADULT GIANT PANDA ( AILUROPODA MELANOLEUCA).

The giant panda ( Ailuropoda melanoleuca) is a high-profile threatened species with individuals in captivity worldwide. As a result of advances in captive animal management and veterinary medicine, the ex situ giant panda population is aging, and improved understanding of age-related changes is necessary. Urine and blood samples were collected in April and July 2015 and analyzed for complete blood count, serum biochemistry, and biochemical and microscopic urine analysis for all individuals sampled ( n = 7, 7-16 yr of age) from giant panda housed at the China Research and Conservation Centre for the Giant Panda in Bifengxia, Sichuan Province, China. Hematology and serum biochemistry values were similar to those previously reported for giant panda aged 2-20 yr and to Species360 (formerly International Species Information System) values. Urine was overall dilute (urine specific gravity range: 1.001-1.021), acellular, and acidic (pH range: 6-7). This is the first report of hematologic and serum biochemistry, with associated urinalysis values, in the giant panda aged 7-16 yr.

GASTRIC DILATATION VOLVULUS IN ADULT MANED WOLVES (CHRYSOCYON BRACHYURUS).

Gastric dilatation volvulus (GDV) was identified in six adult maned wolves ( Chrysocyon brachyurus ) housed at the Smithsonian Conservation Biology Institute in Front Royal, Virginia, and the Dickerson Park Zoo in Springfield, Missouri. Four individuals were found dead in their enclosures, and GDV was diagnosed postmortem based on radiographic and necropsy findings. Two individuals were diagnosed with GDV antemortem, with one wolf surviving following surgical intervention and supportive management. A concurrent splenic torsion was identified in three of six cases. Although GDV has been well documented in domestic dogs and is known to occur in maned wolves, objective, detailed case descriptions have been limited in this species. This report represents the first description of a surviving case of GDV in the maned wolf. Thorough species-specific documentation of any acutely fatal condition, such as GDV, is imperative for proper case recognition and medical management, with profound implications for species recovery efforts.

NOVEL RADIOGRAPHIC TECHNIQUE FOR PREGNANCY DETECTION IN THE MANED WOLF (CHRYSOCYON BRACHYURUS) WITHOUT ANESTHESIA.

Maned wolves ( Chrysocyon brachyurus ) maintained in ex situ populations challenge veterinarians and managers with high neonatal mortality and parental incompetence. These challenges led to the development of a novel diagnostic approach for pregnancy detection using radiographic imaging without anesthesia or sedation. To do this, a specialized crate was constructed to easily contain a single maned wolf, allowing the capture of lateral projection radiographic images of the abdomen prior to and throughout a 66-day pregnancy (days 20, 34, 48, and 55 of 66). Radiographs taken at days 48 and 55 postbreeding showed evidence of neonatal skeleton mineralization, confirming pregnancy with two pups. The dam gave birth at day 66 to two pups. This technical report describes a novel approach without anesthesia for successful radiographic pregnancy detection and determination of litter size in the maned wolf, a midsize carnivore, using a specially constructed crate.

CONGESTIVE HEART FAILURE ASSOCIATED WITH PREGNANCY IN OKAPI (OKAPIA JOHNSTONI).

Acute signs associated with cardiovascular disease occurred in three pregnant okapi ( Okapia johnstoni ) during early to midgestation and progressed to congestive heart failure. Congestive heart failure was diagnosed antemortem using echocardiography and plasma cardiac troponin levels. Clinical signs included decreased activity, hyporexia, tachypnea, dyspnea, flared nostrils, and productive coughing with copious amounts of foamy nasal discharge. Parenteral and oral treatment with furosemide, enalapril, and spironolactone controlled clinical signs in the three okapi allowing each to carry out one pregnancy to term. Two okapi carried the first pregnancy to term after showing signs, while one okapi aborted the first calf and gave birth to a healthy calf in a subsequent pregnancy. Subsequent pregnancy in one okapi ended with abortion and associated dystocia and endometritis. Following parturition, clinical signs associated with heart failure resolved in all three individuals; serial echocardiography in two individuals showed improvement in fractional shortening and left atrial size and all three okapi showed markedly decreased pleural effusion and resolution of pulmonary edema. However, subsequent pregnancies in all three okapi induced respiratory distress and recurrence of congestive heart failure; one okapi died from congestive heart failure associated with subsequent pregnancy. This case series describes the clinical presentation and pathologic findings of congestive heart failure during pregnancy in adult okapi.

FATAL GASTRIC DILATION IN TWO ADULT BLACK-FOOTED FERRETS (MUSTELA NIGRIPES).

Acute gastric dilation resulting in death was identified in two adult black-footed ferrets (Mustela nigripes) housed at the Smithsonian Conservation Biology Institute in Front Royal, Virginia. Both individuals were adult males (3 and 5 yr) and previously clinically healthy prior to the event. The etiology of gastric dilation in both cases could not be definitively determined, and necropsy revealed severe cardiovascular compromise secondary to bloat. Limited literature is available regarding a syndrome of this type in adult black-footed ferrets. Differential diagnoses considered included gastric dilatation volvulus (GDV), severe gastric distention of unknown origin, and gastric outflow obstruction. Given the severity of this syndrome and the findings in these two cases, acute gastric dilation should be considered in black-footed ferrets presenting with acute abdominal distention, respiratory distress, and cardiovascular compromise.

ACUTE PHASE PROTEINS AS A MARKER OF RESPIRATORY INFLAMMATION IN PRZEWALSKI'S HORSE (EQUUS FERUS PRZEWALSKII).

Acute phase proteins are sensitive markers of inflammation, which are highly conserved across taxa. Although the utility of these proteins are becoming well defined in human and domestic animal medical fields, their role in nondomestic species remains unclear. In this communication, a 20-yr-old Przewalski's horse was presented for unresolving aspiration pneumonia, which cultured a unique Actinomyces-like bacteria. Despite waxing and waning clinical signs and minimal changes on baseline hematologic analysis, protein electrophoresis, serum amyloid A, and surfactant protein D serum concentrations showed changes that more accurately reflected the clinical severity of this case.

HEMATOLOGY AND BIOCHEMISTRY IN CAPTIVE WHITE-NAPED CRANES (GRUS VIPIO).

In this retrospective study, 36 hematologic and biochemistry samples from white-naped crane (Grus vipio) were analyzed. All birds were housed at a single institution, and samples were obtained between 1990 and 2013. All cranes were healthy at the time of sample collection, and ages ranged from 1 mo to 33 yr old. Cranes below 18 mo old were considered juveniles. Comparisons were made between means for juveniles and adults and for males and females. Significant increases in adults (P < 0.05) were found in the following variables (differences shown in parentheses): packed cell volume (8.3%), mean cell volume (28 fl), monocyte percentage (3.2%), absolute monocyte count (200 × 10³/ml), total solids (1.0 g/dl), albumin (0.5 g/dl), and sodium (3 mM/L). Significant decreases in adults (P < 0.05) were found in phosphorus (1.7 mg/dl), creatine phosphokinase (1,146 U/L), alkaline phosphatase (451 U/L), lactate dehydrogenase (149 U/L), and glucose (25 mg/dl). Results from adult male and female cranes were also compared; calcium levels were higher in females (4.68 mg/dl, P < 0.05). Plasma electrophoresis and radioimmunoassay for bile acids were performed on 25 banked serum or plasma samples; results from juvenile and adult samples were also compared. Significant increases in adult birds were found in total protein (1.2 g/dl, P < 0.05) and in all absolute values for all protein types; however, no difference was found when protein fractions were compared. Bile acids were found to decrease in adults (19.9 µM/L, P < 0.05).

Critical care for a hypothermic and hypoglycemic white rhinoceros (Ceratotherium simum simum) calf.

A 3-day-old, 57.3-kg, male white rhinoceros (Ceratotherium simum simum) neonate presented laterally recumbent with comatose mentation, cold extremities, and severe hypothermia, hypoxemia, and hypoglycemia. Critical care support was initiated with aggressive fluid and warming support as well as dextrose and oxygen supplementation. After initial stabilization, additional complications arose in subsequent weeks including intermittent diarrhea, dry skin with loss of epidermal layers, urticaria on the head, and superficial wounds on the face, ears, feet, and penis. There is a lack of critical care information for rhinoceros calves. However, due to similarities to other Perissodactylids, some general guidelines for critical equid neonatal care were implemented. The calf was hand-raised until weaning and is now a subadult white rhinoceros with no abnormalities.