DILATED CARDIOMYOPATHY IN SLENDER-TAILED MEERKATS (SURICATA SURICATTA) UNDER HUMAN CARE.

Meerkats (Suricata suricatta) housed at two accredited zoological institutions in the United States were evaluated via echocardiography, thoracic radiography, and blood biomarkers-taurine and feline N-terminal pro-B-type natriuretic peptide-to determine the prevalence and severity of dilated cardiomyopathy (DCM) in both populations. In total, 24 meerkats were evaluated and 7 were diagnosed with DCM based on the following parameters: left ventricular internal diameter at end diastole > 1.30 cm, left ventricular internal diameter at end systole > 1.10 cm, and a fractional shortening of <18%. Echocardiographic parameters were identified and reported for normal and affected meerkats, whereas thoracic radiographs were not useful for screening for DCM. Meerkats with DCM were treated with pimobendan and/or benazepril and furosemide if indicated. Seven meerkats died during the study period, with the majority exhibiting myocardial fibrosis. Of the blood parameters tested, elevated taurine levels were associated with DCM. Further research is necessary to characterize the etiology of DCM in meerkats.

MANAGEMENT OF SUSPECTED DILATED CARDIOMYOPATHY WITH PIMOBENDAN IN TWO LEOPARD SHARKS (TRIAKIS SEMIFASCIATA).

Two adult male leopard sharks (Triakis semifasciata) under managed care were diagnosed with suspected dilated cardiomyopathy. Clinical signs included lethargy, inappetence, and regurgitation. On cardiac ultrasound, fractional shortening was 14% and 10%, respectively (versus 21%-31% in four healthy conspecifics). Ventricular end-diastolic diameter to body weight ratio was 1.72 cm/kg in Case 1 (versus 0.52-1.24 cm/kg in four conspecifics). These results collectively suggested a dilated cardiomyopathy. Treatment was implemented with oral pimobendan at 0.3 mg/kg q48h for 1 mon. The pimobendan dose was increased to 0.5 mg/kg 3/wk, following plasmatic dosage of pimobendan and its metabolite. After 3 mon, fractional shortening increased to 38% and 20%, respectively, sharks regained a normal appetite, and body weight increased by 50% in one individual. After 2 yr, both individuals remained clinically normal, and no adverse effect was noted with pimobendan administration. Pimobendan plasma concentration suggested that this medication was well absorbed in this species.

Establishment of a 2-dimensional echocardiographic protocol and reference parameters for clinically healthy southern stingrays (Hypanus americanus).

OBJECTIVE: To establish an echocardiographic technique and 2-dimensional reference parameters for southern stingrays (Hypanus americanus). A second objective was to compare echocardiographic measurements obtained from animals of different sex, size, environment, handling technique, and position. ANIMALS: 84 presumed healthy, wild, semiwild, and aquarium-housed southern stingrays. PROCEDURES: Animals, anesthetized and manually restrained, were positioned in dorsal recumbency, and echocardiography was performed. A subset of this population was also imaged in ventral recumbency for comparison. RESULTS: Echocardiography was feasible, and reference parameters were established for this species. While some standard measurements could not be assessed due to body habitus, all valves, chambers, and the conus were clearly visualized in the majority of animals. Statistical significance was reached for some variables when comparing animals from different environments and handling methods, but these differences were not considered clinically relevant. The data were therefore separated into 2 subsets of echocardiographic reference parameters based on disc width since some of the measurements were dependent on body size. This approach mostly separated the sexes due to strong sexual dimorphism. CLINICAL RELEVANCE: Limited information is available regarding cardiac disease in elasmobranchs; most of the available information on cardiac physiology focuses on a few shark species. Two-dimensional echocardiography is a noninvasive tool utilized to evaluate cardiac structure and functionality. Southern stingrays are one of the most commonly displayed elasmobranchs in public aquaria. This article expands on the growing body of information regarding veterinary care in elasmobranchs and provides clinicians and researchers with another diagnostic modality to utilize in screening for health/disease.

Outcomes from an anti-gonadotropin-releasing hormone immunotherapeutic trial in large flying foxes (Pteropus vampyrus).

OBJECTIVE: The anti-GnRH immunotherapeutic product Improvest was administered to intact male large flying foxes (Pteropus vampyrus) under managed care for androgen mitigation, leading to a decrease in agonistic behaviors, falls, and injuries from conspecific attention. ANIMALS: 12 males were included in this study. PROCEDURES: Eleven bats received subcutaneous (SC) Improvest interscapular, and 1 animal received Improvest SC in its leg. Assessments included clinical presentation, treatment, behavior, and urine and fecal glucocorticoid metabolites and testosterone (T5) concentrations. RESULTS: Eleven of the 12 bats developed reactions, which included facial edema, localized irritation, swelling of the head and neck, and pruritus with varying degrees of skin ulceration and subsequent necrosis. Three of the animals required extensive treatments, and the 1 animal who received the injection in its leg was unaffected. Posttreatment, fecal glucocorticoid metabolite and/or T5 values were at or below the nonbreeding season baseline for 3 successive breeding seasons, and there was a reduction in agonistic interactions, falls, and injuries. CLINICAL RELEVANCE: A behavioral characteristic of this species is to focus on areas of irritation that exacerbated the extent of the skin wounds. Some cases required medical, surgical, and behavioral intervention. Large flying foxes may be particularly sensitive to this immunotherapeutic when given subcutaneously in the interscapular region. Despite this reaction, the positive long-term effects on behavior and multiyear reduction of hormones suggest that the use of this immunotherapeutic warrants further investigation, although the results should be taken into consideration with other factors such as handling, treatments, chronicity of lesions.

Initial Characterization of Male Southern Stingray (Hypanus americanus) Reproductive Parameters and Preliminary Investigation of Sperm Cryopreservation.

This study investigated the reproductive biology and sperm cryopreservation of ex situ southern stingrays (Hypanus americanus) by semen collection and characterization and the development and validation of an enzyme-linked immunoassay for plasma total testosterone. Semen was collected in March and June using a manual massage technique, and the ejaculates were assessed for volume, pH, osmolarity, motility, status (0-5 scale: 0 = no forward progression, 5 = rapid linear progression) and total sperm count. Semen was extended in Hank's elasmobranch ringer solution containing 10% DMSO, 10% glycerol or 5% glycerol with 5% N-methylformamide and cryopreserved using a conventional freezing method (~-50 °C/min) or a modified slow freezing method (~-3 °C/min). Body condition was scored from 1-5 and was noted to be low in March (1.93 ± 0.07) due to feeding practices and increased by June (2.93 ± 0.05) after dietary corrections were made. A concomitant increase (p < 0.05) in plasma total testosterone concentration and sperm motility was noted between March (8.0 ± 7.2 ng/mL, 5.71 ± 2.77%) and June (97.3 ± 11.3 ng/mL, 51.4 ± 14.3%). Samples cryopreserved using a modified slow freeze method (~-3 °C/min) had higher post-thaw motility and plasma membrane integrity than conventionally cryopreserved samples. Data indicate that southern stingray sperm morphometrics adheres to those of other elasmobranch species and that a slow cooling rate may be an avenue of research to improve southern stingray sperm survival during cryopreservation.

Simulated heatwave and fishing stressors alter corticosteroid and energy balance in neonate blacktip reef sharks, Carcharhinus melanopterus.

The increasing frequency and duration of marine heatwaves attributed to climate change threatens coastal elasmobranchs and may exacerbate existing anthropogenic stressors. While the elasmobranch stress response has been well studied, the role of the unique corticosteroid-1α-hydroxycorticosterone (1α-OHB)-in energy balance is not understood. Therefore, 1α-OHB's utility as a stress biomarker in elasmobranch conservation physiology is equivocal. Here, we analyse the roles of corticosteroids, 1α-OHB and corticosterone, and metabolites, glucose and 3-hydroxybutyrate (3-HB), in response to stress in a protected tropical shark species, the blacktip reef shark (Carcharhinus melanopterus). Wild-caught neonates were exposed to ambient (27°C) or heatwave conditions (29°C) and subsequently a simulated fishing stressor (1 min air exposure). Blood samples were taken prior to temperature exposure, prior to air exposure, and 30 min, 1 h, 24 h, and 48 h post-air exposure at treatment temperatures. Plasma 1α-OHB was elevated for 48 h in 27°C-exposed sharks but declined over time in 29°C-exposed sharks. Plasma 1α-OHB was not correlated with either metabolite. Plasma glucose was higher and plasma 3-HB was lower in 29°C-exposed sharks. In a separate experiment, blood samples were collected from both neonate and adult sharks immediately following capture and again 5 min later, and analysed for corticosteroids and metabolites. Plasma 1α-OHB increased in neonates within 5 min, but neonates displayed lower plasma 1α-OHB and higher glucose concentrations than adults. We conclude that 1α-OHB does not serve as a classic glucocorticoid role in C. melanopterus under these stressors. Furthermore, we show for the first time, ontogenetic differences in plasma 1α-OHB. Ultimately, our findings provide insights into hormonal control of energy mobilization during stress in C. melanopterus, particularly during simulated heatwave conditions, which seem to alter both endocrine and energy mobilization. Further work is needed to determine the utility of 1α-OHB as a biomarker for the mobilization of energy during a stress event in elasmobranchs.

Energy and corticosteroid mobilization following an induced stress response in an elasmobranch fish, the North Pacific spiny dogfish (Squalus acanthias suckleyi).

The dominant corticosteroid in elasmobranchs, 1α-hydroxycorticosterone (1α-OHB), has a described role in mineral regulation but a presumptive role in energy balance. Energy demand in vertebrates following exposure to a stressor typically involves an immediate but transient release of glucocorticoids as a means of mobilizing available energy stores, usually in the form of glucose. Although a glucocorticoid role for 1α-OHB would be expected, direct glucocorticoid function of this steroid has yet to be reported in any elasmobranch. In addition, elasmobranchs also utilize the metabolite ß-hydroxybutyrate (ß-HB), which is thought to replace the role fatty acids play in most vertebrates as a predominant fuel source in extrahepatic tissues. To determine the mobilization of metabolites and corticosteroids during a stress event, North Pacific spiny dogfish, Squalus acanthias suckleyi, were cannulated and held in a darkened isolation box to recover (24-48 h) before being subjected to an acute air exposure or corticosterone injection. Dogfish were then serially blood sampled at nine timepoints over 48 h. Glucose, ß-HB, 1α-OHB, corticosterone, as well as lactate, pH, and osmolality were quantified in plasma samples. All measured variables increased in control and treatment groups within 48 h from the start of experimentation, and ß-HB and 1α-OHB remained elevated for the duration of the experiment. There was no linear correlation between glucose and 1α-OHB, but there was a weak (R2 = 0.230) although significant (p = 0.001), positive correlation between ß-HB and 1α-OHB. Interestingly, there were also significant correlations between increasing circulating glucose and corticosterone (R2 = 0.349; p < 0.001), and decreasing ß-HB and corticosterone concentrations (R2 = 0.180; p = 0.008). Our data suggest that following successive stressors of capture, surgery, and confinement, 1α-OHB was not correlated with circulating glucose, only weakly correlated with circulating ß-HB concentrations (R2 = 0.230; p = 0.001), and that corticosterone may also serve a role in energy mobilization in this species.

Reference Intervals for Blood Analytes of Adult Aquarium-Housed Russian Sturgeon Acipenser gueldenstaedtii.

Russian Sturgeon Acipenser gueldenstaedtii are an important, critically endangered, roe-producing species. Despite a wealth of knowledge pertaining to other members of family Acipenseridae, there is very limited published information regarding baseline blood analytes in Russian Sturgeon. The objectives of this study were (1) to establish reference intervals for a suite of hematological and biochemical data and (2) to compare plasma chemistry data to two point-of-care (POC) cartridges, tested on the VetScan iSTAT 1 analyzer, that use heparinized whole blood for the assessment of clinically normal, aquacultured adult Russian Sturgeon sedated with eugenol (AQUI-S 20E) at a single institution. Reference intervals are reported. The calculated hematocrit measured by the POC analyzer tended 4-5% lower than the spun packed cell volume, confirming the importance of spun packed cell volume as a reliable measurement of red blood cell mass. Various analytes, notably whole-blood urea nitrogen, glucose, sodium, total carbon dioxide, chloride, ionized calcium, and anion gap, were significantly different by both POC cartridges. This study successfully produced reference intervals for blood analytes in adult Russian Sturgeon under managed care and creates a foundation for future studies into the effects of extrinsic and intrinsic factors and variations of analytical methodologies on blood analytes in this species.

Pharmacokinetics of Ketoprofen in Nile Tilapia (Oreochromis niloticus) and Rainbow Trout (Oncorhynchus mykiss).

The objective of this study was to document the pharmacokinetics of ketoprofen following 3 mg/kg intramuscular (IM) and intravenous (IV) injections in rainbow trout (Oncorhynchus mykiss) and 8 mg/kg intramuscular (IM) injection in Nile tilapia (Oreochromis niloticus). Plasma was collected laterally from the tail vein for drug analysis at various time intervals up to 72 h following the injection of ketoprofen. In trout, area under the curve (AUC) levels were 115.24 µg hr/mL for IM and 135.69 µg hr/mL for IV groups with a half-life of 4.40 and 3.91 h, respectively. In both trout and tilapia, there were detectable ketoprofen concentrations in most fish for 24 h post-injection. In tilapia, there was a large difference between the R- and S-enantiomers, suggesting either chiral inversion from R- to S-enantiomer or more rapid clearance of the R-enantiomer. AUC values of the S- and R-enantiomers were 510 and 194 µg hr/Ml, respectively, corresponding to a faster clearance for the R-enantiomer. This study shows that there were very high plasma concentrations of ketoprofen in trout and tilapia with no adverse effects observed. Future studies on the efficacy, frequency of dosing, analgesia, adverse effects, and route of administration are warranted.

Practical Management of Iron Overload Disorder (IOD) in Black Rhinoceros (BR; Diceros bicornis).

Critically endangered black rhinoceros (BR) under human care are predisposed to non-hemochromatosis Iron Overload Disorder (IOD). Over the last 30 years, BR have been documented with diseases that have either been induced by or exacerbated by IOD, prompting significant efforts to investigate and address this disorder. IOD is a multi-factorial chronic disease process requiring an evidence-based and integrative long-term approach. While research continues to elucidate the complexities of iron absorption, metabolism, and dysregulation in this species, preventive treatments are recommended and explained herein. The aim of this report is to highlight the accumulated evidence in nutrition, clinical medicine, and behavioral husbandry supporting the successful management of this disorder to ensure optimal animal health, welfare, and longevity for a sustainable black rhinoceros population.

Ultrasonographic and hormonal characterization of reproductive health and disease in wild, semiwild, and aquarium-housed southern stingrays (Hypanus americanus).

OBJECTIVE: To characterize physical examination, plasma biochemical, and ultrasonographic findings in aquarium-housed, managed semiwild, and wild southern stingrays (Hypanus americanus) with and without reproductive disease. ANIMALS: Southern stingrays from aquarium (n = 48), lagoon (managed semiwild; 34), and wild (12) habitats. PROCEDURES: Limited, opportunistic prosections were performed of presumed anatomically normal wild southern stingrays and compared with findings for aquarium-housed stingrays with reproductive disease. Ultrasonographic video data from both groups were used to assign a score (1 to 5) indicating increasing severity of ovarian and uterine reproductive disease. Plasma total 17ß-estradiol, estrone, progesterone, and testosterone concentrations were measured with enzyme immunoassays validated for use in southern stingrays. RESULTS: Ultrasonographic ovarian scores were significantly correlated with uterine scores. No reproductive disease was detected in semiwild or wild stingrays, but 65% (31/48) of aquarium-housed stingrays had developing or advanced reproductive disease (ie, ultrasonographic ovarian or uterine score of 4 or 5). Significant correlations were identified between ovarian and uterine disease status and plasma concentrations of all steroid hormones except testosterone. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that ultrasonography and plasma hormone concentrations may be useful in the identification of reproductive disease and determination of disease severity in southern stingrays.

Evaluation of Comprehensive Coelomic Fluid Analysis through Coelomic Pore Sampling as a Novel Diagnostic Tool in Elasmobranchs.

The objectives of this study were to describe a minimally invasive coelomic fluid sampling technique in elasmobranchs, to characterize the coelomic fluid composition in clinically normal and abnormal animals, and to compare findings from wild and managed populations. Fluid was collected via the coelomic pore in 89 individuals from 16 species spanning clinically normal and abnormal patients within a managed population (n = 54), a semi-managed open-lagoon population (n = 18), and a wild population (n = 17). Biochemical and cytological fluid analyses were performed on all samples, and bacterial and fungal culture, protein electrophoresis, and cholesterol electrophoresis were performed on a subset of samples. The presence of a variable volume of colorless to white and clear to slightly turbid coelomic fluid was consistent with a normal finding; however, the cytological and chemical makeup of coelomic fluid was found to provide additional clinically relevant information. The coelomic fluid from some of the abnormal samples (n = 37) contained white blood cells (n = 15) and concurrent bacteria (n = 7), the latter suggestive of bacterial coelomitis. Yolk was identified in both clinically normal and abnormal females. Of the biochemical parameters tested, calcium, chloride, cholesterol, osmolality, phosphorus, salinity, sodium, specific gravity, total protein, and urea nitrogen have clinical utility. Abnormal samples were mostly associated with reproductive disease, but to a lesser extent with coelomitis and hemocoelom. The wild and semi-managed groups had biochemical differences presumably reflective of the higher salinity of ocean water compared with that in the managed habitat. Aerobic bacteria were identified in normal (n = 7) and abnormal (n = 11) animals. Positive bacterial culture without inflammation may be normal. This study contributes to a further understanding of elasmobranch coelomic fluid analysis and its use as a diagnostic modality for the evaluation of elasmobranch health.

Seasonal dynamics of agonistic behavior and hormones in an ex situ all-male colony of large flying foxes.

Large flying foxes (Pteropus vampyrus) are a socially complex species. In situ colonies typically comprise thousands of individuals in small harems of one male to many females. In ex situ environments, all-male colonies are becoming more common due to a surplus of males in the population. There is limited information describing the hormonal and behavioral patterns of all-male colonies during the breeding season. We assessed seasonal changes in hormones and behavior in an all-male colony of 12 large flying foxes at Disney's Animal Kingdom® . We validated hormone assays using morning urine and fecal samples to assess seasonal changes in excreted immunoreactive testosterone and glucocorticoid metabolites. We collected behavior data using an all-occurrence method, recording agonistic behaviors related to territorial defense (hooking, biting, wing flexing, vocalizing, and wrestling), and sexual behavior (mounting and frontal grabbing). Results indicated that (i) we could reliably measure testosterone and glucocorticoid metabolites concentrations from fecal and urine samples collected from individual bats; (ii) there were distinct relationships between changes in levels of agonism and hormone concentrations throughout the year; and (iii) three agonistic behaviors (chasing, wrestling, and open-mouth threat) peaked prior to the increase in testosterone and glucocorticoid hormones measured during the breeding season. These three behaviors could potentially be used as early indicators to signal the onset of the breeding season and allow time to implement ex situ management changes to reduce the incidence of agonism between individuals.

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species.

PHARMACOKINETIC EVALUATION OF MELOXICAM AFTER INTRAVENOUS AND INTRAMUSCULAR ADMINISTRATION IN NILE TILAPIA (OREOCHROMIS NILOTICUS).

Critically evaluating the pharmacokinetic behavior of a drug in the body provides crucial information about how to effectively treat a patient. Pharmacokinetic studies that exist in fish have primarily focused on drugs used to treat infectious disease, with minimal attention given to analgesic drugs. The objective of this study was to determine the pharmacokinetics of meloxicam (1 mg/kg) in Nile tilapia ( Oreochromis niloticus ) (n = 12). A single dose of meloxicam was administered either i.v. or i.m. Blood samples were obtained at predetermined times after drug injection. Plasma meloxicam concentrations were determined by a validated liquid chromatography/mass spectrometry method, and noncompartmental pharmacokinetic analysis was performed. The mean peak plasma concentration after i.m. injection was 1.95 µg/ml. The mean terminal half-life of meloxicam after i.v. and i.m. administration was 1.36 and 1.8 hr, respectively. The area under the plasma concentration-versus-time curve extrapolated to infinity was 11.26 hr·µg/ml after i.v. administration and 5.72 hr·µg/ml after i.m. administration. Bioavailability of meloxicam after i.m. administration was approximately half that of i.v. administration. Elimination was rapid in both the i.m. and i.v. routes of administration, suggesting that maintaining clinically relevant plasma concentrations may be difficult using this dose. This study represents the first pharmacokinetic evaluation of a nonsteroidal anti-inflammatory drug in a fish species, and further studies evaluating efficacy are needed.

MANAGEMENT OF ENDOMETRIOSIS IN TWO CAPTIVE MANDRILLS (MANDRILLUS SPHINX).

Endometriosis has been reported in humans, great apes, and Old World monkeys. Although cases are noted anecdotally in Mandrillus spp., and a previously reported case was noted on postmortem examination, to the authors' knowledge, no previous reports of case management have been published in the peer-reviewed literature. This paper describes the medical and surgical management of endometriosis in two mandrills (Mandrillus sphinx).

A SEROLOGIC AND POLYMERASE CHAIN REACTION SURVEY OF EQUINE HERPESVIRUS IN BURCHELL'S ZEBRAS (EQUUS QUAGGA), HARTMANN'S MOUNTAIN ZEBRAS (EQUUS ZEBRA HARTMANNAE), AND THOMSON'S GAZELLES (EUDORCAS THOMSONII) IN A MIXED SPECIES SAVANNAH EXHIBIT.

Reports of equine herpesvirus (EHV) 1 and EHV-9 causing clinical disease in a wide range of species have been well documented in the literature. It is thought that zebras are the natural hosts of EHV-9 both in the wild and in captive collections. Concerns about potential interspecies transmission of EHV-1 and EHV-9 in a mixed species savannah exhibit prompted serologic and polymerase chain reaction surveys. Eighteen Burchell's zebras ( Equus quagga ), 11 Hartmann's mountain zebras ( Equus zebra hartmannae), and 14 Thomson's gazelles ( Eudorcas thomsonii ) cohabitating the same exhibit were examined for EHV-1 virus neutralization titers, and evidence of virus via EHV 1-5 polymerase chain reactions. None of the animals had previous exposure to vaccination with EHV-1 or EHV-4. All tested zebras had positive EHV-1 titers, ranging from 4 to 384. All zebras and Thomson's gazelles had negative polymerase chain reaction results for all targeted equine herpesviruses. EHV-9-specific assays are not available but EHV-1, EHV-4, and EHV-9 cross-react serologically. Positive serology results indicate a potential latent equine herpesvirus in the zebra population, which prompted initiation of an equine herpesvirus vaccine protocol, changes in pregnant zebra mare management, and equine herpesvirus polymerase chain reaction screening prior to shipment to or from the study site.