Prevalence of equine herpesvirus-1 and equine herpesvirus-4 infections in equidae species in Turkey as determined by ELISA and multiplex nested PCR

In this report we examined the presence of specific antibodies against equine herpesvirus type 1 (EHV-1), and equine herpesvirus type 4 (EHV-4) in several equidae, including mules, donkeys, horses. The presence of EHV-1 and EHV-4 in respiratory diseases of equids, and ability of multiplex nested polymerase chain reaction (PCR) screening in simultaneous diagnosis of horses acutely infected by EHV-1 and EHV-4 were also investigated. Sera from 504 horses, mules and donkeys sampled were tested for the presence of EHV-1 and EHV-4 specific antibodies. Blood samples taken from 21 symptomatic horses and nasal swabs taken from 40 symptomatic horses were tested for the presence of EHV-1 and EHV-4 by a multiplex nested PCR. A total of 14.3% (3/21) of buffy coat samples and 32.5% (13/40) nasal swab samples were found to contain EHV-1 DNA, while 19% (4/21) buffy coat samples and 22.5% (9/40) nasal swab samples were found to be positive for EHV-4 DNA. By species, 14.5% of horses, 37.2% of mules and 24.2% of donkeys tested were EHV-1 seropositive. EHV-4 specific antibodies were detected in 237 (81.7%) of 290 horse sera tested. Results from this investigation demonstrate that EHV-1 and EHV-4 are prevalent throughout the equid population, and that donkeys and mules might also represent an important source of infection for other equids. We also showed that the multiplex nested PCR assay might be useful for diagnosis of mixed respiratory infections in horses due to EHV-1 and EHV-4.     

Antemortem detection of latent infection with neuropathogenic strains of equine herpesvirus-1 in horses

OBJECTIVE: To evaluate a technique for identifying horses latently infected with neuropathogenic strains of equine herpesvirus-1 (EHV-1). ANIMALS: 36 adult mares, 24 of which were experimentally infected as weanlings with neuropathogenic or nonneuropathogenic EHV-1. PROCEDURES: Mandibular lymph node (MLN) tissue was obtained from each horse via biopsy during general anesthesia. Purified DNA from MLNs was tested for EHV-1 DNA by use of a magnetic bead, sequencecapture, nested PCR assay. For MLNs that contained EHV-1 DNA, the 256-bp DNA fragments amplified via sequence-capture nested PCR were sequenced to determine the nucleotide at the polymorphic site that determines pathotype (ie, neuropathotype [G(2254)] or non-neuropathotype [A(2254)]). RESULTS: Latent viral DNA was detected in 26 of the 36 (72%) mares tested. Neuropathogenic and nonneuropathogenic EHV-1 genotypes were detected in the latently infected horses. In each mare previously infected with known EHV-1 pathotypes, the open reading frame 30 genotype of latent EHV-1 was identical to that of the strain that had been inoculated 4 to 5 years earlier. Latent viral DNA was detected in 10 of the 12 mares that were inoculated as weanlings with neuropathogenic strains of EHV-1. The detection rate of the sequence-capture PCR method for EHV-1 latency was double that of conventional nested or realtime PCR assays performed on the same MLN DNA preparations. CONCLUSIONS AND CLINICAL RELEVANCE: The magnetic bead, sequence-capture, nested PCR technique enabled low-threshold detection of DNA from latent neuropathogenic strains of EHV-1 in MLN specimens from live horses. The technique may be used to screen horses for latent neuropathogenic EHV-1 infection.     

Development of a real-time polymerase chain reaction assay for rapid diagnosis of neuropathogenic strains of equine herpesvirus-1.

This communication reports the development and performance assessment of a rapid diagnostic test for identifying horses actively infected with the neurovirulent pathotype of equine herpesvirus-1 (EHV-1). The test is a real-time polymerase chain reaction (PCR)-based assay that uses EHV-1 pathotype-specific TaqMan(R) reporter probes for discrimination between neuropathogenic and non-neuropathogenic strains of EHV-1 in equine blood or nasal swabs. The diagnostic performance of the new technique was evaluated by testing specimens collected from 234 horses involved in recent outbreaks of EHV-1 myeloencephalopathy at three separate thoroughbred racetracks and one large riding/boarding stable. Side-by-side comparison of the EHV-1 pathotyping results yielded by the new single-step, PCR-based allelic discrimination technique (24-hour turn-around-time) with those generated by a multi-step, conventional nested PCR followed by nucleotide sequencing of the amplified DNA (4-day turn-around-time) revealed complete agreement between the 2 test methods. The ability to rapidly identify horses infected with neuropathogenic strains of EHV-1 using a single-step, PCR-based method has significant implications for future diagnostic evaluation of suspect animals.     

Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR

Background: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Objectives: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Animals: Fifteen horses experimentally infected with EHV-1.
Methods: Experimental study : Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
Results: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI ( P < .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86–100) and 27% (95% CI: 20–35).
Conclusions and Clinical Importance: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C _T values are provided as well as justification of a minimum 10-day quarantine period.     

Frequency of latent equine herpesvirus type-1 infection among a sample of horses in the central North Island of New Zealand

ABSTRACT

Aim: To estimate the frequency of infection with equine herpesvirus type-1 (EHV-1) among horses from the central North Island of New Zealand, including the frequency of detection of the D₇₅₂ genotype.

Methods: Samples of retropharyngeal lymph nodes (RLN) and submandibular lymph nodes (SLN) were dissected from the heads of 63 horses that were humanely killed for various unrelated reasons between March and November 2015. DNA extracted from these tissues was subjected to enrichment for EHV-1 sequences by hybridisation with biotin-labelled EHV-1 specific probe, followed by recovery of EHV-1 sequences on streptavidin-coated magnetic beads. Enriched samples were tested for the presence of EHV-1 using nested quantitative real-time PCR. The EHV-1 amplicons were sequenced to determine the genotype of the virus.

Results: The median age of the horses was 6 (min 2, max 30) years, and 47/63 (75%) were Thoroughbreds. EHV-1 DNA was detected in RLN samples from 6/63 (10%) horses, and three of these horses were also positive for EHV-1 DNA in SLN. The remaining horses were negative for EHV-1 DNA in both RLN and SLN samples. The N₇₅₂ genotype was detected in all positive samples and the D₇₅₂ genotype was not detected in any of the samples.

Conclusions: EHV-1 continues to circulate among horses in New Zealand. The frequency of latent EHV-1 infection among sampled horses may have been underestimated due to the sensitivity limit of the assay or because of the limited anatomical sites sampled in the study. Lack of detection of the D₇₅₂ genotype suggests that infection with this genotype is not common in horses in New Zealand.

Clinical Relevance: If live animals are tested for EHV-1 using SLN biopsy it should be kept in mind that negative results do not rule out the presence of latent EHV-1 infection at other sites inaccessible for testing. The RLN appear to be the preferred sample for detection of EHV-1 DNA in horses following recent euthanasia.     

Identification of Equine Herpesvirus 5 in Horses with Lymphoma

Equine multinodular pulmonary fibrosis, equine herpesvirus 5 (EHV-5), and multicentric lymphoma were discovered in one patient. Review of gamma herpesvirus activity in humans revealed a propensity for lymphoproliferative disorders associated with infection. The objective was to determine the frequency of EHV-5 in lymphoma tissues and compare with the frequency found in the lymph nodes of clinically normal horses. Case control investigation of lymphoma-positive tissues and analysis via polymerase chain reaction (PCR) for EHV-5 was performed on 12 horses. Prospective collection and PCR analysis of lymph nodes (mesenteric or submandibular) for EHV-5 was performed on 21 control horses. Thirteen samples of lymphoma-positive tissues and fluid were submitted for PCR analysis for EHV-5. Of these, 67% was positive. In the control horse population, 14% was positive for EHV-5 (P = .004). Neoplastic samples positive for EHV-5 were classified as T-cell rich B-cell lymphoma (three), T-cell lymphoma (one), one was nondifferentiated, and two were not stained. Gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as Kaposi sarcoma and Burkitt lymphoma. This study reveals an increased frequency of EHV-5 (gamma herpesvirus) in horses diagnosed with lymphoma compared with healthy control horses. Although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia is unknown, EHV-5 may be an etiologic agent associated with the development of some types of equine lymphoma.     

Prevalence of latent alpha-herpesviruses in Thoroughbred racing horses

The objective of this study was to detect and characterize latent equine herpes virus (EHV)-1 and -4 from the submandibular (SMLN) and bronchial lymph (BLN) nodes, as well as from the trigeminal ganglia (TG) of 70 racing Thoroughbred horses submitted for necropsy following sustaining serious musculoskeletal injuries while racing. A combination of nucleic acid precipitation and pre-amplification steps was used to increase analytical sensitivity. Tissues were deemed positive for latent EHV-1 and/or -4 infection when found PCR positive for the corresponding glycoprotein B (gB) gene in the absence of detectable late structural protein gene (gB gene) mRNA. The EHV-1 genotype was also determined using a discriminatory real-time PCR assay targeting the DNA polymerase gene (ORF 30). Eighteen (25.7%) and 58 (82.8%) horses were PCR positive for the gB gene of EHV-1 and -4, respectively, in at least one of the three tissues sampled. Twelve horses were dually infected with EHV-1 and -4, two carried a latent neurotropic strain of EHV-1, six carried a non-neurotropic genotype of EHV-1 and 10 were dually infected with neurotropic and non-neurotropic EHV-1. The distribution of latent EHV-1 and -4 infection varied in the samples, with the TG found to be most commonly infected. Overall, non-neurotropic strains were more frequently detected than neurotropic strains, supporting the general consensus that non-neurotropic strains are more prevalent in horse populations, and hence the uncommon occurrence of equine herpes myeloencephalopathy.     

Study of equid herpesviruses 2 and 5 in Iceland with a type-specific polymerase chain reaction

The horse population in Iceland is a special breed, isolated from other horses for at least 1000 years. This provides an exceptional opportunity to investigate old and new pathogens in an inbred herd with few infectious diseases. We have developed a high sensitivity semi-nested PCR to study equid gammaherpesviruses 2 and 5 (EHV-2 and 5) in Iceland. The first PCR is group specific, the second type-specific, targeting a 113bp sequence in the glyB gene. DNA isolated from white blood cells and 18 different organs was tested for the presence of EHV-2 and 5. This was done in adult horses and foals, healthy and with various enteric infections. Both virus types were easily detected in all types of organs tested or EHV-2 in 79% cases and EHV-5 in 63%. In DNA from PBMC or buffy-coat EHV-2 was found in 20% cases and EHV-5 in 10%, all except one positive were foals. Co-culture of PBMC on fetal horse kidney cells was efficient for detecting EHV-2 but not for EHV-5. We verify here for the first time infections with EHV-2 and 5 in horses in Iceland and show that both viruses are common.     

First Report on the Frequency and Molecular Detection of Neuropathogenic EHV-1 in Turkey

There has been an increase in outbreaks of neuropathogenic equine herpesvirus-1 (EHV-1) in the United States and Europe. However, the presence and frequency of neuropathogenic EHV-1 in Turkish horses are not known at present. This study aimed to investigate the frequency of EHV-1 and neuropathogenic strains of EHV-1 in the Marmara Region of Turkey. Samples were analyzed for the presence of EHV-1 and neuropathogenic EHV-1 by real-time PCR TaqMan probe assays. Overall detection rate of EHV-1 was 45.5% (51 of 112). The detection rates were 70.5% (24 of 34) in aborted fetuses, 53.3% (8 of 15) in neonatal deads, 66.6% (4 of 6) in foals, 40% (2 of 5) in dead mares, and 25% (13 of 52) in living mares. Overall detection rate of neuropathogenic EHV-1 was 7.8% (4 of 51), and the real-time PCR results were confirmed by sequencing. Neuropathogenic strains of EHV-1 were detected in the brain and lung of two mares with neurological disease but without a history of abortion, in the brain of a foal that died of respiratory disorder, and in the nasal swab from a mare with a history of abortion. On histopathology, nonpurulent meningoencephalitis, hemorrhages, and vasculitis were seen in the brain. In conclusion, results of this study indicated, for the first time, that the neuropathogenic EHV-1 is circulating in the Marmara Region of Turkey. The results of this study also show that the current risk for non-neuropathogenic strains is high, whereas risk for the neuropathogenic EHV-1-G₂₂₅₄ strain seems to be low. As outbreaks of EHV-1 continue in the Marmara region of Turkey, surveillance for neuropathogenic EHV-1 genotype should be maintained.     

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.     

Lack of Detectable Equine Herpesviruses 1 and 2 in Paraffin-Embedded Specimens of Equine Sarcoidosis

Background: Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2).
Objective: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis.
Animals: Eight horses with sarcoidosis, reported previously.
Methods: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with EHV-1 and EHV-2. For both herpesviruses the target was their respective glycoprotein B gene. Positive controls consisted of DNA from viral cultures of culturettes from naturally occurring respiratory infections of EHV-1 and EHV-2.
Results: The PCR analyses for both equine herpesviruses' DNA were negative in all 8 horses.
Conclusion: The failure to detect DNA from EHV-1 and EHV-2 in paraffin-embedded skin of these 8 horses does not discount EHV-1 or EHV-2 as causing some cases of ES, but lends support to the presumably multifactorial etiologic nature of the disease.     

Prevalence of equine herpesvirus type 2 (EHV-2) DNA in ocular swabs and its cell tropism in equine conjunctiva

Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased horses were positive. To define the main virus reservoir ocular tissue from 13 randomly selected horses without pathological evidence of ocular disease were analysed by nested PCR. In two horses optic nerve, lacrimal gland and conjunctiva, in further two cases lacrimal gland and conjunctiva and in four horses the conjunctiva only were EHV-2 PCR positive. For specifying the target cell we focused on conjunctivae and selected 3 out of 15 clinically healthy slaughterhouse horses positive for EHV-2 by PCR. In situ hybridisation on sections of these paraffin embedded conjunctivae localized viral genome in histiocyte-like cells of the submucosa. Immunohistochemical staining with an EHV-2 or S100 specific polyclonal antiserum demonstrated that Langerhans cells were co-localized in the same region of the sample section where virus positive cells were detected. Furthermore, we concluded that detection of viral antigen revealed a productive virus infection.     

Study of equid herpesviruses 2 and 5 in Iceland with a type-specific polymerase chain reaction

The horse population in Iceland is a special breed, isolated from other horses for at least 1000 years. This provides an exceptional opportunity to investigate old and new pathogens in an inbred herd with few infectious diseases. We have developed a high sensitivity semi-nested PCR to study equid gammaherpesviruses 2 and 5 (EHV-2 and 5) in Iceland. The first PCR is group specific, the second type-specific, targeting a 113 bp sequence in the glyB gene. DNA isolated from white blood cells and 18 different organs was tested for the presence of EHV-2 and 5. This was done in adult horses and foals, healthy and with various enteric infections. Both virus types were easily detected in all types of organs tested or EHV-2 in 79% cases and EHV-5 in 63%. In DNA from PBMC or buffy-coat EHV-2 was found in 20% cases and EHV-5 in 10%, all except one positive were foals. Co-culture of PBMC on fetal horse kidney cells was efficient for detecting EHV-2 but not for EHV-5. We verify here for the first time infections with EHV-2 and 5 in horses in Iceland and show that both viruses are common.     

Prevalence of equine herpesvirus-1 infection among Thoroughbreds residing on a farm on which the virus was endemic

OBJECTIVE: To determine the incidence of equine herpesvirus-1 (EHV-1) infection among Thoroughbreds residing on a farm on which the virus was known to be endemic. DESIGN: Prospective cohort study. ANIMALS: 10 nonpregnant mares, 8 stallions, 16 weanlings, 11 racehorses, and 30 pregnant mares and their foals born during the 2006 foaling season. PROCEDURES: Blood and nasopharygeal swab samples were collected every 3 to 5 weeks for 9 months, and placenta and colostrum samples were collected at foaling. All samples were submitted for testing for EHV-1 DNA with a PCR assay. A type-specific EHV-1 ELISA was used to determine antibody titers in mares and foals at birth, 12 to 24 hours after birth, and every 3 to 5 weeks thereafter. RESULTS: Results of the PCR assay were positive for only 4 of the 1,330 samples collected (590 blood samples, 590 nasopharyngeal swab samples, 30 placentas, and 30 colostrum samples), with EHV-1 DNA detected in nasal secretions from 3 horses (pregnant mare, stallion, and racehorse) and in the placenta from 1 mare. Seroconversion was detected in 3 of 27 foals during the first month of life. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that there was a low prevalence of EHV-1 infection among this population of Thoroughbreds even though the virus was known to be endemic on the farm and that pregnant mares could become infected without aborting. Analysis of nasopharyngeal swab samples appeared to be more sensitive than analysis of blood samples for detection of EHV-1 DNA.     

Mitigation of pyrexia by a Th-1-biased IgG subclass response after infection with equine herpesvirus type 1 in horses pre-immunized with inactivated vaccine

The immunoglobulin G (IgG) subclass response was investigated in horses with or without pyrexia after natural infection with equine herpesvirus type 1 (EHV-1) in the field. All horses were kept at the training centers of the Japan Racing Association and were immunized with an inactivated EHV-1 vaccine before EHV-1 infection. An IgG subclass response dominated by IgGa and IgGb was induced in horses without pyrexia after EHV-1 infection. In contrast, horses that developed pyrexia showed increased IgGc and IgG (T) subclass production in addition to IgGa and IgGb. Although inactivated EHV-1 vaccines are considered to induce a mainly Th-2-biased response, these results indicated that the responses in horses inoculated with inactivated EHV-1 vaccine were not uniform, and that horses with a Th-1-biased response were likely to be protected from pyrexia.     

应用多重PCR检测和区分3个型的马疱疹病毒

针对马疱疹病毒（EHV）的EHV-1、EHV-2和EHV-4糖蛋白B基因序列，设计、合成了3对特异性引物进行多重PCR，不仅可以在数小时内分别检测这3个型的EHV，而且在同一反应系统内可以清晰地区分EHV-1、EHV-2和EHV-4，其PCR产物大小分别为226、333、570bp，符合预期的片段大小，序列分析证实与已发表的序列一致；该检测方法的灵敏度达到10^3 TCID50；分别从血清学阳性但病毒分离为阴性的1匹进口马组织样品和一些出口前检疫马的鼻咽样品检测到EHV-1和EHV-4特异性核酸。     

Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a gamma-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an "alveolar-like" architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera-specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine gamma-herpesvirus, EHV-5.     

Clinical observations and management of a severe equine herpesvirus type 1 outbreak with abortion and encephalomyelitis

Latent equine herpesvirus type 1 (EHV-1) infection is common in horse populations worldwide and estimated to reach a prevalence nearing 90% in some areas. The virus causes acute outbreaks of disease that are characterized by abortion and sporadic cases of myeloencephalopathy (EHM), both severe threats to equine facilities. Different strains vary in their abortigenic and neuropathogenic potential and the simultaneous occurrence of EHM and abortion is rare. In this report, we present clinical observations collected during an EHV-1 outbreak caused by a so-called “neuropathogenic” EHV-1 G₂₂₅₄/D₇₅₂ polymerase (Pol) variant, which has become more prevalent in recent years and is less frequently associated with abortions. In this outbreak with 61 clinically affected horses, 6/7 pregnant mares aborted and 8 horses developed EHM. Three abortions occurred after development of EHM symptoms. Virus detection was performed by nested PCR targeting gB from nasal swabs (11 positive), blood serum (6 positive) and peripheral blood mononuclear cells (9 positive) of a total of 42 horses sampled. All 6 fetuses tested positive for EHV-1 by PCR and 4 by virus isolation. Paired serum neutralization test (SNT) on day 12 and 28 after the index case showed a significant (≥ 4-fold) increase in twelve horses (n = 42; 28.6%). This outbreak with abortions and EHM cases on a single equine facility provided a unique opportunity for the documentation of clinical disease progression as well as diagnostic procedures.