Uneeda K. Bryant

Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky

REASON FOR PERFORMING STUDY An emerging problem of equine herpesvirus-1 (EHV-1) infection in horses in the USA is a high-mortality myeloencephalopathy that commonly occurs where large numbers of horses are stabled. EHV-1 isolates recovered from recent neurological outbreaks represent a mutant virus strain that possesses enhanced neuropathogenicity. A central question of EHV-1 myeloencephalopathy is the latency carriage rate for these mutants of EHV-1 in USA horse populations. OBJECTIVE To estimate the prevalence of neuropathogenic strains of EHV-1 as latent infections in the Thoroughbred broodmare population of central Kentucky. METHODS Submandibular lymph nodes (SMLN) were collected during post mortem examination of 132 Thoroughbred broodmares. Total DNA purified from SMLN tissue was tested for the presence of latent EHV-1 DNA by an ultrasensitive magnetic bead-based, sequence-capture, nested PCR method. Differentiation of active from latent infections by EHV-1 was achieved by detection of transcripts of EHV-1 glycoprotein B by reverse transcription PCR. RESULTS Latent EHV-1 DNA was detected in the SMLN tissues of 71 (54%) of the 132 mares submitted for necropsy. Thirteen (18%) of the 71 latently infected horses harboured the neuropathogenic biovar of EHV-1. Of the 13 horses latently infected with an ORF30 mutant strain of EHV-1, 11 also carried a latent, wild-type strain of the virus in their SMLN tissues. CONCLUSIONS Neuropathogenic strains of EHV-1 have established a significant presence in the Thoroughbred broodmare population of central Kentucky as latently infected carrier horses. The data also indicate that a highly sensitive DNA detection method is required to identify many instances of EHV-1 latency. POTENTIAL RELEVANCE The presence of a relatively large biological reservoir of latent, neuropathogenic EHV-1 has the potential for posing emerging equine health and economic threats to the future prosperity of the USA horse industry.

OAS1 Polymorphisms Are Associated with Susceptibility to West Nile Encephalitis in Horses

West Nile virus, first identified within the United States in 1999, has since spread across the continental states and infected birds, humans and domestic animals, resulting in numerous deaths. Previous studies in mice identified the Oas1b gene, a member of the OAS/RNASEL innate immune system, as a determining factor for resistance to West Nile virus (WNV) infection. A recent case-control association study described mutations of human OAS1 associated with clinical susceptibility to WNV infection. Similar studies in horses, a particularly susceptible species, have been lacking, in part, because of the difficulty in collecting populations sufficiently homogenous in their infection and disease states. The equine OAS gene cluster most closely resembles the human cluster, with single copies of OAS1, OAS3 and OAS2 in the same orientation. With naturally occurring susceptible and resistant sub-populations to lethal West Nile encephalitis, we undertook a case-control association study to investigate whether, similar to humans (OAS1) and mice (Oas1b), equine OAS1 plays a role in resistance to severe WNV infection. We identified naturally occurring single nucleotide mutations in equine (Equus caballus) OAS1 and RNASEL genes and, using Fishers Exact test, we provide evidence that mutations in equine OAS1 contribute to host susceptibility. Virtually all of the associated OAS1 polymorphisms were located within the interferon-inducible promoter, suggesting that differences in OAS1 gene expression may determine the hosts ability to resist clinical manifestations associated with WNV infection.

Halicephalobus gingivalis-associated meningoencephalitis in a Thoroughbred foal

A 13-week-old Thoroughbred colt from central Kentucky was euthanized after an acute onset of ataxia, blindness, head tremors, leaning to the right, recumbency, and seizures. Microscopically, there was a verminous meningoencephalitis characterized by an eosinophilic and granulomatous inflammatory reaction primarily affecting the cerebellum. Dispersed within regions of inflammation were numerous cross and longitudinal sections of intact and degenerative small nematodes. The nematodes had dorsoflexed ovaries and ventroflexed vulvas, which are distinguishing features of Halicephalobus gingivalis. Intact nematodes, compatible with H. gingivalis, also were recovered and identified from portions of the brain that had been frozen for 5-week post-necropsy examination via tissue maceration and additional laboratory techniques.

Acute Deterioration and Death with Necrotizing Enteritis Associated with Lawsonia intracellularis in 4 Weanling Horses

An 8-month-old Thoroughbred colt was evaluated in October 2010 with a less than 1-day history of inappetence. Physical examination in the field revealed throat latch edema, lethargy, and fever (103.8°F) (ref 99–101.5°F). A complete blood count revealed leukocytosis (18.0 × 10/lL; ref 5.0–12.6 × 10/lL) with a relative neutropenia (49%; ref 55–65%) and lymphopenia (26%; ref 35–45%), as well as a toxic left shift (25% bands; ref 0–5%). Serum biochemistry abnormalities included hypoproteinemia (3.3 g/dL; ref 6–7.9 g/dL), hypoalbuminemia (1.2 g/dL; ref 3.4–4.1 g/dL), and an increased BUN (45 mg/dL; ref 11–26 mg/dL), along with other abnormalities (Table S1). Lawsonia intracellularis-induced equine proliferative enteropathy (EPE) was suspected because of the combination of hypoproteinemia, hypoalbuminemia, inappetence, and the autumn presentation. Treatment consisted of intravenous oxytetracycline (6.6 mg/kg IV q24hr), flunixin meglumine (1 mg/kg IV q12h), oral omeprazole (1 mg/kg PO q24hr), dexamethasone (0.1 mg/kg IV q24hr), intravenous crystalloid fluids (10 mL/kg IV bolus once), and intravenous colloids (10 mL/kg IV bolus once). Despite treatment, the weanling was euthanized within 48 hours of presentation because of continued deterioration and signs of pulmonary disease characterized by nasal discharge, epistaxis, and tachypnea. Blood work submitted the morning of euthanasia revealed a worsening leukocytosis (35.4 9 10/lL) with an unchanged differential, as well as continued hypoproteinemia (3.5 g/dL), hypoalbuminemia (1.1 g/ dL), and an increased BUN (59 mg/dL). Case 2

Rhabdomyosarcoma in 8 Horses

This multi-institutional report describes 8 cases of rhabdomyosarcoma in horses. Four neoplasms were in the tongue and other areas of the mouth or head, 2 were in the abdominal wall, and 1 each was in right shoulder muscles and heart. Four rhabdomyosarcomas that were less than 10 cm in diameter were treated by surgical excision or radiation with no recurrence. Two neoplasms greater than 10 cm in diameter in the abdominal wall and the right shoulder were considered inoperable and led to decisions to euthanize the horses. Two neoplasms were incidental findings at necropsy. All the neoplasms were classified as embryonal except for 1 pleomorphic rhabdomyosarcoma. These 8 cases were evaluated with 9 published case reports of equine rhabdomyosarcoma. For all cases, the most common sites were limb muscles (5/17) and tongue (4/17). Metastasis was reported in 4 of the previously published cases; none was found in this study.

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.

Renal Mucus Gland Cystadenomas in a Horse

A 35-year-old horse was submitted to the necropsy service at the University of Kentucky Livestock Disease Diagnostic Center. At necropsy, multiple 1–4-cm-diameter cystic structures were incidentally identified unilaterally in the right renal medulla and the cortex. On histologic examination, the cystic structures compressed the normal renal architecture, were lined by tall columnar epithelium that formed occasional papillary projections, and contained large amounts of mucicarmine and periodic acid–Schiff-positive mucinous material. The masses were diagnosed as renal mucus-gland cystadenomas. This tumor should be considered as a differential diagnosis when cystic structures are identified in the equine kidney.

Diagnostic value of tissue monensin concentrations in horses following toxicosis

Two separate incidents of monensin exposure in horses resulting in toxicosis provided insight into the diagnostic value and interpretive criteria of various biological samples. In case 1, 25 horses broke into a shed and ingested feed that was supplemented with 800 g/ton (880 µg/g) of monensin. Within 48 hr, 1 horse had died, 2 developed cardiac arrhythmias, lethargy, and recumbency, and another was euthanized due to severe deterioration. Minimal histologic lesions were noted in the horse that died peracutely, while another showed characteristic lesions of acute cardiomyocyte degeneration and necrosis. Stomach content, heart, liver, urine, and serum revealed various detectable concentrations of monensin in clinically affected and unaffected horses with known exposure. In case 2, a pastured horse had access to a mineral mix containing 1,600 g/ton (1,760 µg/g) of monensin. Within 48 hr, the horse became symptomatic and was euthanized because of severe respiratory distress. Histologic cardiac lesions were minimal but detectable amounts of monensin were found in blood, heart, liver, and stomach contents. In both cases, monensin toxicosis was confirmed with toxicological analysis. These cases demonstrate an overall lack of correlation of monensin concentrations in various biological samples with clinical outcome. However, serum, urine, blood, liver, heart, and stomach content can be tested to confirm exposure. More importantly, the consistently higher concentrations found in heart tissue suggest this is the most useful diagnostic specimen for postmortem confirmation of toxicosis in horses especially in cases in which associated feed cannot be tested for monensin or in cases with no histologic lesions.

Anomalous Origin of the Left Coronary Artery from the Pulmonary Artery (ALCAPA) in Four Calves

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA), or Bland-White-Garland syndrome, is a rare congenital malformation described in children and adults. In this condition, the left coronary artery, which normally originates from the left coronary sinus in the aorta, instead originates from the pulmonary trunk, which results in retrograde flow of blood away from the myocardium into the lower-pressure pulmonary artery. Myocardial hypoxic-ischemic injury results in cardiac dysfunction, failure, and eventually in patient death if not surgically repaired. This report describes gross and microscopic findings in 4 beef calves with ALCAPA. All the calves had a history of being found dead with few or no premonitory signs, 2 shortly after sudden strenuous exercise. Gross necropsy lesions suggestive of heart failure included cardiomegaly with atrial and ventricular dilation and/or ventricular hypertrophy, and hepatomegaly. Dissection of each heart revealed the origin of the left coronary artery arising in the pulmonary trunk above the anterior cusp of the pulmonic valve. No other cardiac anomalies were identified. Histopathologic changes in the heart included cardiomyocyte degeneration; mineralization; and fiber loss, with replacement by fibrous connective tissue, predominantly in the left ventricular papillary muscle and the interventricular septum. Changes observed in the liver and lungs, including hepatomegaly, sinusoidal congestion, centrilobular fibrosis, and pulmonary congestion, edema, and intra-alveolar pigment-laden macrophages were consistent with heart failure.

Fatal bromethalin intoxication in 3 cats and 2 dogs with minimal or no histologic central nervous system spongiform change

Use of the neurotoxic rodenticide bromethalin has steadily increased since 2011, resulting in an increased incidence of bromethalin intoxications in pets. Presumptive diagnosis of bromethalin toxicosis relies on history of possible rodenticide exposure coupled with compatible neurologic signs or sudden death, and postmortem examination findings that eliminate other causes of death. Diagnosis is confirmed by detecting the metabolite desmethylbromethalin (DMB) in tissues. In experimental models, spongiform change in white matter of the central nervous system (CNS) is the hallmark histologic feature of bromethalin poisoning. We describe fatal bromethalin intoxication in 3 cats and 2 dogs with equivocal or no CNS white matter spongiform change, illustrating that the lesions described in models can be absent in clinical cases of bromethalin intoxication. Cases with history and clinical signs compatible with bromethalin intoxication warrant tissue analysis for DMB even when CNS lesions are not evident.