Equine herpesvirus 1 and 4

Vet Clin Equine 20 (2004) 631–642

Equine herpesvirus 1 and 4 Stephen M. Reed, DVM, Ramiro E. Toribio, DVM, PhD Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, 601 Vernon Tharp Street, Columbus, OH 43210, USA

Equine herpesvirus 1 (EHV-1) infection in horses is responsible for upper respiratory infection in young horses and abortion, usually in late gestation, although recent work by Smith et al [1] and Allen et al [2] has demonstrated uterine or placenta damage without abortion during early gestation as well as myeloencephalopathy, generally in adult horses. Equine herpesvirus 4 (EHV-4) had been thought to be a subtype of EHV-1, but through the use of molecular characterization techniques, it has been demonstrated to be a distinct viral species that can also cause abortions and respiratory disease. EHV-4 is responsible for upper respiratory infection in young horses and may rarely cause other problems, such as abortion [2–4]. Both viruses are capable of inducing myeloencephalopathy, although EHV-1 is clearly the more common cause of this form of the disease [4,5]. The respiratory disease is often silent or difficult to detect. Despite this fact, the respiratory form of EHV-1 may be observed as an outbreak of fever and nasal discharge in a group of young horses. Disease may occur in individual horses or, sometimes, as case clusters. EHV-1 is highly contagious and is often responsible for significant economic losses as a result of abortion, neurologic disease, respiratory problems, loss of use, and even death. Outbreaks of this disease have been recognized for centuries among domestic horse populations. An outbreak of any form of EHV-1 has a significant economic impact on the farm, veterinary hospital, or other venue where the outbreak occurs. The economic influence of an outbreak of EHV-1 or EHV-4 is a result of increased expense for medical treatment, lost time for training and performing, and death. Virology Herpesviridae are divided into three groups (a, b, and c) based on host range, reproductive cycles, cytopathology, and genome structure. A total of E-mail address: [email protected] 0749-0739/04/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cveq.2004.09.001

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S.M. Reed, R.E. Toribio / Vet Clin Equine 20 (2004) 631–642

eight herpesviruses have been isolated from Equidae. Five have been described in horses and three in donkeys, one of which is similar to EHV-1 [2,4,6–8]. EHV-1, equine coital exanthema virus (EHV-3), and EHV-4 are typical a-herpesviruses, whereas equine cytomegalovirus (EHV-2) and EHV-5 are c-herpesviruses. The a-herpesviruses have a wide host range, short reproductive cycle, and most importantly, the capacity to establish latent infections. EHV-1, EHV-3, and EHV-4 (a-herpesviruses) can be distinguished from EHV-2 and EHV-5 (c-herpesviruses) and from each other by use of DNA fingerprinting, polymerase chain reaction (PCR) testing, and several immunologic tests [3,4,8,9]. Genetic and biologic markers help to track the viruses and explain the changes in virulence and may partially explain the inability to determine when and why cases of neurologic disease occur. EHV-1 and EHV-4 are ubiquitous in the equine population. The complete DNA sequence for EHV-1 and EHV-4 has been published [9,10]. These viruses have a double-stranded DNA genome consisting of 145 to 150 kilobase (kb) encoding for 76 unique genes [10]. Sequence analysis by Telford et al [10] showed that there is from 55% to 84% DNA homology between EHV-1 and EHV-4 depending on the gene and that the homology at the amino acid level ranged from 55% to 96%. The highest homology at the protein level between EHV-1 and EHV-4 was found in proteins associated with replication, packaging, and capsid formation. EHV-1 and EHV-4 contain glycoproteins that are important for cell attachment, entry, and cell-to-cell dissemination. These glycoproteins are also important in inducing host immune responses. Upper respiratory infection is the most common manifestation of EHV-1 infection in weanlings, yearlings, and 2-year-olds, whereas infection of pregnant mares can produce late-gestation abortion, stillbirth, and weak neonatal foals. Appropriate vaccination can reduce the incidence of abortion in mares. Slater et al [6] demonstrated that EHV-1 is a neurotrophic aherpesvirus that can cause neurologic signs in horses as a result of vasculitis, thrombosis, and necrosis. EHV-1 and EHV-4 can cause upper respiratory tract infection and, occasionally, cell-associated viremia, abortion, and paralysis. Tissues that can become infected with EHV-1 include the lining of the uterus; male genital tract; intestine; and the conjunctiva of the eye, spinal cord, and brain. In most instances after exposure, horses demonstrate mild respiratory disease consisting of cough, fever, and mild anorexia. It seems that certain strains of the virus that are more likely to cause the paralytic disease have a mutation in the DNA polymerase gene (N. Davis-Poynter, BVSc, MRCVS, PhD, unpublished data presented at the Havermeyer workshop on EHV-1, Tuscany, Italy, July 2004) [11]. Epidemiology EHV-1 is important to the equine industry because of the economic impact that results from multiple abortions, multiple cases of equine


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respiratory disease, and occasional outbreaks of myeloencephalopathy. The earliest recognized outbreak of paralytic disease associated with EHV-1 was recognized in 1966 [12]. Respiratory and abortion outbreaks have resulted in detailed tracking of this virus worldwide. Through this surveillance, variations in the composition of the nucleic acid have been detected [13]. EHV-1 and EHV-4 are found in most equine populations worldwide, and evidence of this exposure can be detected by examination of serologic samples collected from horses at shows, racetracks, or stables. Detection of antibodies after abortions or neurologic disease on premises may last for a long time, reportedly ranging from 1 to 4 years [14,15]. Recently, the neurologic form of this disease has seemed to be predominant, although perhaps the significant numbers of horses that have died in association with these outbreaks and the large economic losses at several farms, racetracks, and riding schools have been the reasons for much public recognition of this problem. In these outbreaks, large numbers of horses have been infected and unusually high numbers of horses have experienced severe neurologic signs and have died [12,13,15–20]. In past episodes, the neurologic form of the disease has seemed to be more sporadic, often affecting only a few animals within an infected herd [21]. These recent outbreaks of the neurologic form of the disease have seemed more similar to the ‘‘abortion storms’’ that have been previously described [13,20]. In a recent exposure at a riding school in Findlay, Ohio, more than 90% of 138 horses were affected with fever and nasal discharge, and more than 42 of these horses developed neurologic disease, several of which were severely infected and died or were euthanized [20].

Etiology and pathogenesis Since the first definitive association between EHV-1 and myeloencephalopathy [12], examples of similar cases have been documented worldwide. In recent instances, myeloencephalopathy has occurred as an outbreak of cases, although sporadic disease has been reported. The natural spread of this disease is through inhalation, and infection first develops on the mucosal surface of the respiratory tract. Nasal aerosols from infected horses are the most common route of infection; however, direct contact with infected aborted fetuses or placental tissues may also serve as a possible source of infection. Spread of infection may occur in three ways: direct cell-to-cell spread, hematogenous spread through infected peripheral blood monocytes, and neural spread in which the virus is considered to be neurotrophic and endotheliotropic and results in vasculitis. Once the host has contracted the virus, it rapidly enters the respiratory epithelium and associated lymphoreticular tissues and lymphocytes and circulates throughout the body in virus-infected phagocytes. EHV-1 and EHV-4 contain several glycoproteins [22]. The glycoproteins of herpesviruses play a role in development of infection, attachment, and entry into

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host cells and cell-to-cell spread [23]. Glycoproteins on the surface of the viral membrane bind to the host cell surface and allow fusion of the virus to the host cell [24]. Replication occurs in the nucleus, and protovirions derive their envelope from the inner lamella of the nuclear membrane. Virus particles bud from the cell surface and result in necrosis of the respiratory epithelial cells. The presence of intranuclear inclusion bodies is characteristic of equine herpesviruses. Once the virus is within the white blood cell, it seems to be able to circulate without destruction despite high circulating antibody titers. In this location, the virus can disseminate to other tissues, including the central nervous system [25,26]. The neural lesions are a result of the vasculitis caused by infection of the endothelia of small blood vessels, leading to thrombosis. This infection and cellassociated viremia occur in the presence of high antibody titers. Vasculitis and thrombosis damage the microcirculation of affected areas, resulting in hypoxia and eventual neuron cell death. Similar mechanisms seem to play a role in damage of the placenta and malnutrition of the fetus, leading to abortions. Equine herpesvirus targets the reproductive, respiratory, and central nervous systems in the horse and is excellent at evading all parts of the immune system. Clinical signs depend on which system is affected after exposure. The virus is usually transmitted from horse to horse by inhalation and establishes initial infection within epithelial cells lining the upper respiratory tract. Direct horse-to-horse contact as well as aerosol contamination of fomites may also result in spread of the disease. After direct infection, lytic damage occurs to respiratory mucosal cells. This results in serous nasal discharge, which may contain high concentrations of the virus. Over time, the mucus discharge may change in character and consistency and often signals the presence of secondary bacterial infections. Around this time, horses are often febrile and have nasal hyperemia and congestion, and some horses show petechial hemorrhages. In addition, horses may show generalized edema of the legs and along the ventrum of the body. Because the virus also targets endothelial cells lining blood vessels, thrombo-occlusive necrotizing vasculitis of the nervous system may develop, resulting in clinical signs of myeloencephalopathy. Initial transfer of the virus is to underlying lamina propria of the mononuclear cells of regional lymph nodes of the respiratory system. The virus amplifies in leukocytes of the respiratory tract lymph nodes and is able to enter the peripheral blood stream within monocytes and CD4þ and CD8þ T lymphocytes. Here, the cell-associated viremia disseminates the virus to distant sites, such as the pregnant uterus, the central nervous system, and other organs. Infection of endothelial cells initiates an inflammatory cascade, leading to hemorrhage and necrosis and eventual abortion or myeloencephalopathy. The virus concurrently gains access to the neurons of the trigeminal ganglion [26]. It seems that the neurologic form of this disease is associated with infection of the endothelia of small blood vessels, leading to vasculitis,


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thrombosis, and necrosis. The result of vasculitis and thrombosis is localized tissue hypoxia and neuronal death. The vascular endothelium is the site of viral replication of EHV-1. The vasculitis caused by EHV-1 may be a result of two mechanisms. The first is caused by direct damage to the endothelial cells lining small blood vessels. The second may be caused by the formation and deposit of immune complexes of EHV-1 virus and antibody (Arthus-type reaction). The finding of endothelial necrosis is not limited to the vessels of the nervous system but occurs in many other sites throughout the body. The neurologic signs are a result of the vasculitis, hemorrhage, edema, necrosis, and ischemia that can result from the virus having a profound endothelial tropism. Thus, the deficits correspond to the site of damage, but, in general, the ischemia and necrosis cause the most severe damage to the gray matter. The propensity of certain viral strains to induce neurologic signs does not seem to be the result of those viruses having specific neurotropism, although some recent work using specific pathogen-free ponies resulted in chorioretinopathy and neural lesions, suggesting neurotropism with this virus [27,28]. Similar events are responsible for abortions and stillbirths in mares. Abortions most often occur during late gestation, although some changes recognized in early stages of pregnancy have been attributed to this virus [25]. Abortion, similar to myeloencephalopathy, is a result of infection and damage to several systems, including respiratory epithelial cells, followed by infection of leukocytes and endothelium of blood and lymphatic vessels within the lamina propria of the upper respiratory system. After infection, EHV-1 can establish latency and protect itself from the antiviral effects of the cytotoxic T lymphocytes and neutralizing antibodies [26]. Estimated to be found in up to 50% of the adult equine population [24], the latent form of EHV-1 virus hides in the leukocytes of the lymphoreticular system or the trigeminal ganglia. At times, the reactivated virus may result in viral shedding in clinically normal horses, referred to as ‘‘silent shedders.’’ In a recent experiment by Professor Klaus Osterrieder of Cornell University, it was demonstrated that EHV-1 membrane gp2 is a major determinant of virulence. When the truncated version of the gp2 gene was inserted into virulent RacL 11 virus, the virulent virus became attenuated [26]. Similarly, if this glycoprotein is inserted into an avirulent strain of EHV-1, such as Ky A, it is rendered more virulent. Immunity After infection, horses seem to be resistant to reinfection for a short time, although resistance to reinfection may be gained after repeated infections [7]. Infection with EHV-1 results in a transient suppression of the immune system, specifically the peripheral blood monocytes, likely through the action of cytokines, such as transforming growth factor-b and prostaglandins, which mediate this immunosuppression. Soboll et al [29] and O’Neill et al [30]

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previously reported that after active EHV-1 infection, there is an expansion of virus-specific cytotoxic lymphocytes capable of detecting and destroying virus-infected cells. Study of the mechanisms responsible for protective immunity is important in the development of new vaccines. Immunity seems to occur as a result of blockage to new infection at the respiratory epithelium by means of the production and secretion of virus-neutralizing antibodies into the airway lumen. In addition, exposure to EHV-1 virions in the peripheral blood is thought to activate cytotoxic T-lymphocytes capable of destroying EHV-1– infected cells [26]. Cell-mediated immunity is seemingly more important than humoral immunity because of the high degree of cell association in infection and the ability to have cell-to-cell infection without release of virions. History and clinical signs Clinical signs include acute fever, inappetence, and depression combined with serous nasal discharge and cough. The spread of this virus is slower than that of influenza because EHV-1 is better transmitted by contact than by aerosolization. Abortion and fatal neonatal disease can result; abortions may occur at any stage of gestation but seem to take place most often late in pregnancy. Foals may become infected in utero and die, or they are born weak and often die. Neurologic signs are often preceded by fever or upper respiratory disease during the 2 weeks before the onset of neurologic signs. The clinical signs observed as a result of EHV-1 myelitis can be quite variable and, in the authors’ experience, may include unilateral lameness because of involvement of the brachial plexus, as observed in one case. In most EHV-1–affected horses, viral infection results in symmetric ataxia and weakness of the pelvic limbs, along with urinary incontinence, loss of sensation, and motor deficits around the tail and perineal area of one or more horses on the premises. At times, there may be involvement of the vestibular region or other cranial nerves. Affected horses often begin with minor neurologic gait deficits, which rapidly progress to significant clinical signs. Diagnostic evaluation Although genetic and biologic characterization is used for diagnosis of the neurologic form, in general, a prospective diagnosis can be made based on clinical signs alone. To confirm the diagnosis, a history of antecedent or concurrent upper respiratory tract disease in the affected horses or other horses on the premises, xanthochromic cerebrospinal fluid containing an elevated quantity of protein, and identification of a three- to fourfold rise in anti–EHV-1 neutralizing antibody or complement fixation titers in serum samples collected 7 to 21 days apart are also useful. In addition, histologic


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evaluation of nervous tissue from an affected horse should show the classic vasculitis changes; in some cases, virus isolation from the buffy coat, nasal swabs, or postmortem samples is helpful. In summary, diagnosis is based on virus isolation (cell culture), serologic typing, identification by restriction endonuclease analysis of DNA, or PCR. PCR can provide rapid identification of EHV-1 and EHV-4 DNA from primers derived from conserved regions of the genome, which amplify products of similar size for each virus. To separate EHV-1 from EHV-4, one needs restriction endonuclease digestion or hybridization using virus-specific probes [27,31,32]. A positive cerebrospinal fluid titer is of limited usefulness and most likely reflects disruption of the blood-brain barrier as a result of vasculitis. Other valuable tests include immunohistochemistry or immunofluorescence tests. In many cases, however, it is impossible to achieve a definitive diagnosis without benefit of a postmortem examination. Differential diagnoses should include equine protozoal myelitis, polyneuritis equi, cervical vertebral stenotic myelopathy, equine degenerative myeloencephalopathy, trauma, and viral diseases like the togaviral encephalitides and West Nile virus encephalomyelitis. Ruling out the specific conditions described is helpful to support a diagnosis of EHV-1 myeloencephalopathy. Treatment and prognosis The management of horses with suspected EHV-1 myelitis or myeloencephalopathy should be directed at achieving a safe environment and providing excellent nursing care. Attention should be given to maintaining good hygiene; for some horses, judicious use of a sling may also be important. Because the disease may be transmitted from horse to horse, isolation of affected horses is essential even though horses are not thought to be highly contagious at the time of the neurologic signs. The level of care necessary is dependent on the severity of clinical signs. Frequent aseptic evacuation of the bladder may be required in horses with bladder dysfunction. An indwelling Foley catheter that is attached to a simplex or other fluid removal tube can be used in mares and stallions. This is then glued together and sutured to the leg of the horse to allow continuous drainage at a site low enough on the leg so as to prevent urine scald. In stallions, placement has been made through a perineal urethrostomy site. Prophylactic antibiotics are essential to combat the problems associated with the development of cystitis. We have observed a Quarter Horse stallion that was recumbent for such a long time that it developed pressure necrosis of several sites on the body, which became infected, leading to septicemia and eventual seeding of the central nervous system with bacterial organisms. Rectal evacuation may also be required in some affected horses. Use of acyclovir for the treatment of EHV-1 is indicated and has recently been shown to be beneficial in the treatment of EHV-1 myeloencephalopathy

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at a dose of 10 mg/kg administered orally five times a day [33]. Symptomatic treatment using nonsteroidal anti-inflammatory drugs (eg, phenylbutazone, flunixin meglumine) may be helpful. It is the authors’ opinion that use of intravenous dimethyl sulfoxide (DMSO) at a rate of 0.9 gm/kg as a 10% solution is helpful. The osmolality of this solution is approximately 1660 mosm/L. This is routinely used once daily for 3 days and then once every other day for three to four additional treatments or longer as necessary. Administration of corticosteroids, such as dexamethasone (0.05–0.1 mg/kg administered intravenously) or prednisolone (1 mg/kg/d) is also useful. Larger doses of corticosteroids have sometimes been recommended, but it is important to maintain as short a course as possible. Careful use of nonsteroidal anti-inflammatory agents is essential, because many of the horses may be unable to drink, making dehydration a serious complication. The daily water needs for an affected horse should be 60 to 80 mL/kg. Along with water, it is important to feed a gruel or, if the horse can eat, to provide a highly palatable source of energy and protein daily. The prognosis is guarded to favorable if one is willing and able to provide long-term management for the horse. Treatment of neurologic disease and the complications, such as cystitis, urine scald, inability to rise, and constipation or fecal incontinence, sometimes enters the picture and is an important consideration when informing the owner of his or her long-term commitment. Horses that have been exposed to infected horses but have not developed any clinical signs within 21 days of the potential exposure are unlikely to do so. The longer the time after exposure without evidence of clinical signs, the less likely the horse is to develop disease or transmit the virus to other horses.

Control and prevention The source of EHV-1 is usually horses that are inapparent carriers. The virus is transmitted by close contact via aerosol exposure, usually by infected respiratory secretions, although fetal tissues, placenta, and uterine fluids from mares that have aborted are also infective and need to be disposed of in a safe and proper fashion to prevent exposure from this source. Common airspace enhances transmission, and in experimental infections, the disease has occurred over a distance of 35 ft. Fomite transmission is possible. Virus can be transmitted via organic material on clothes, shoes, or tack or even on material inside stalls, trailers, water buckets, or feed. Riders should be encouraged to wear leather or rubber boots that can be disinfected before entering or leaving a stall or the barn. Ask all visitors to use foot baths before entering or leaving the facility, remembering to include regular visitors like veterinarians and farriers as well. One of the most cost-effective approaches for the management of infectious disease is the use of vaccines. At this time, there is no vaccine that claims to protect


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against the neurologic form of the disease. In several reports associated with recent outbreaks as well as in several textbook publications, anecdotal information has reported an association between the neurologic form of the disease and frequent vaccination [34]. Although there has not been a clear definition of ‘‘frequent vaccination,’’ in a recent outbreak, most of the horses had been vaccinated every 60 days, some for as long as 10 years. Some of the difficulties associated with use of vaccines are recognition of the appropriate antigen(s) for developing an immune response, achievement of high antibody concentrations at the appropriate site, and knowing whether one is attempting to prevent the infection or the disease, to name only a few. The best vaccine would develop protection at the site of infection rather than prevent disease. There are no vaccines that prevent latency; to do this, one must have high mucosal immunity present at all times to block transmission. The question of appropriate vaccination strategies to reduce the likelihood of neurologic disease in an EHV-1 outbreak is complex. Vaccination may reduce the level of viremia in infected horses, which, in turn, could reduce the amount of virus shed by the infected horses and help to reduce the risk of all manifestations of EHV-1. Cases of EHV-1 myeloencephalopathy occur despite regular vaccinations administered at short intervals, indicating probable variants of EHV-1 within the equine population. Although no currently marketed vaccines provide protection against the neurologic form of the disease, the risk of viral shedding may be decreased in herds of vaccinated horses. Therefore, frequent vaccination is important, with recommended revaccination at 60to 90-day intervals. Current vaccination recommendations for use of an EHV-1 vaccine are directed at prevention of abortion in pregnant mares. In addition to isolation of affected horses and vaccination of unexposed horses, reduction in the incidence of disease may be helped by thorough disinfection procedures. Elimination of all organic material from the area is required, along with cleaning of contaminated surfaces with soap and water. Disinfection may be accomplished with a solution of 1 part bleach (sodium hypochlorite) to 10 parts water. Surfaces should be allowed to dry to reduce the chance for further spread of virus. Prevention of new cases can be reduced by isolation of all new arrivals, sick horses, and horses returning from a show or other competition for 2 to 3 days. In situations in which there is suspicion of infected horses, isolation for up to 7 days should decrease the chance of developing an infection. If horses were exposed to EHV-1, isolation of all incontact horses for a period of 21 days is essential or ideal. Case study at The Ohio State University After the admission of six horses (one was dead on arrival) from the University of Findlay in middle to late January 2003 for emergency treatment of neurologic disease, The Ohio State University Veterinary Teaching Hospital experienced fever or neurologic disease in several horses in the

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hospital, in two horses that left the hospital, and in one horse that was in close contact with a horse that had been in the hospital. After signs of ataxia suggestive of EHV-1 developed in one hospitalized horse, the hospital endeavored to identify and notify owners of horses that were in the hospital between the dates of January 18 and February 11. At this same time, the hospital initiated an intensive investigation to determine the source of infection for these animals. It was determined that six horses developed fever alone and four horses developed fever and incoordination during this period, and all were tested for EHV-1. One horse that was negative on PCR of a nasal swab recovered uneventfully, and in two horses that were negative for all tests for EHV-1, the fever resolved uneventfully as well. One horse that was negative for EHV-1 on PCR of blood and respiratory secretions had acute and convalescent titers for EHV-1 that were identical at a ratio of 1:80, indicating no recent exposure. This horse was treated with antimicrobials and recovered. On PCR, one horse that was positive for EHV-1 in blood but negative on respiratory secretions had EHV-1 isolated from the blood, demonstrating an acute 1:160 and convalescent 1:640 rise in titers, indicating recent exposure. This horse remained at our hospital for 21 days while it recovered uneventfully. There was no evidence of neurologic disease in this horse, and it was returned home. Another horse that was negative for EHV-1 on PCR and virus isolation of blood and respiratory secretions demonstrated rising acute (1:10) and convalescent (1:>640) titers, indicating recent exposure to this virus. This horse did have evidence of neurologic disease, although it had recently undergone myelography and ventral stabilization surgery to correct vertebral canal stenosis. Within 120 days after leaving our hospital, the horse entered a training program and remains in race training to date. Three research horses belonging to The Ohio State University that were housed in a separate ward of the hospital also became febrile during this period. No positive PCR test results were noted, but serologic testing suggested recent exposure to EHV-1. All febrile horses, whether they were owned by clients or were research horses owned by The Ohio State University, were maintained in the hospital for 21 days beyond the last recognition of fever in any horses within the group. There were four horses that developed fever and incoordination while hospitalized during this period. One horse was presented for evaluation of fever and cellulitis of one limb; this horse was confirmed to have EHV-1 as the cause of the ataxia. Another horse was presented for osteoarthritis of the cervical vertebral facets, which would explain the ataxia but not the fever. A third horse was presented for a laceration over the right carpus and became febrile and severely ataxic within 8 days after discharge from our hospital. The final horse had been presented for hematuria and developed ataxia and fever shortly after discharge. This horse recovered; however, a pasture mate of this horse developed incoordination as a result of EHV-1 and had to be euthanized.


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As a result of this experience, it became obvious to our faculty that horses demonstrating signs of neurologic disease as a result of EHV-1 remain contagious to naive horses. Therefore, all horses with neurologic disease that have a recent history of being febrile or come from a farm with a history of respiratory disease, abortion, or an outbreak of neurologic disease are admitted into isolation facilities. In addition, any horses with clinical signs, such as ascending paralysis, poor tail tone, or urinary and fecal incontinence, are suspected to have EHV-1 until proven otherwise and are also maintained in an isolation facility.

Acknowledgments The authors thank Karen Bolten for provision of technical assistance.

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