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.     

Role of equine herpesviruses as co-infecting agents in cases of abortion, placental disease and neonatal foal mortality

Herpesviral infections frequently occur in horses. The objective of this study was to investigate the possible association of equine herpesviruses (EHV-1, EHV-2, EHV-3, EHV-4, EHV-5) with other causes of abortion, neonatal mortality or placental disorder. Sixty-seven abortions, 22 stillbirths, 14 cases of neonatal foal mortality and 3 cases of placental disease were investigated for infectious and non-infectious causes. Type-specific nested PCR assays and virus isolation were performed to detect EHV infections. A cause of fetal loss or placental disease was reached in 68 out 116 (58.7 %) cases. Twenty-seven cases were positive for EHV, and 22/27 (81.5 %) were positive for EHV-1 (16 neuropathogenic and 6 non-neuropathogenic strains), 4 (14.8 %) for EHV-2 and 3 (11.1 %) for EHV-5. The association between EHV infections and other etiological agents was statistically significant (two sided P?=?0.002). The odds ratio of EHV DNA associated with other diagnoses, especially with bacterial infection and premature placental separation, was 10.88 (95 % confidence interval: 2.15–55.16). EHV-1 was the main viral cause of pregnancy loss in this study, also associated with other etiological agents, including EHV-2 and EHV-5. The latter viruses in particular need to be more fully investigated to elucidate what role either or both may play as co-infecting agents with other established infectious causes of reproductive disease.     

A new field strain of equine abortion virus (equine herpesvirus-1) among Kentucky horses

From restriction endonuclease characterization of the DNA of 317 isolates of equine abortion virus (equine herpesvirus-1; EHV-1) from 176 epizootically unrelated outbreaks of equine virus abortion occurring over 24 years in Kentucky, an epizootic pattern and variation of the virus have emerged. Two electropherotypes of EHV-1 (1P and 1B) accounted for greater than 90% of the nonvaccine-related abortion isolates examined. From 1960 to 1981, EHV-1 1P was the predominant isolate circulating in the central Kentucky area and the cause of greater than 80% of EHV-1-related abortions. In 1981, the occurrence of isolate 1B-related abortions began to increase and since 1982, 1B has become the most frequently recovered isolate of EHV-1 from aborted fetuses.     

A retrospective study of sporadic bovine abortions, stillbirths, and neonatal abnormalities in Atlantic Canada, from 1990 to 2001

In a retrospective study on 265 cases of sporadic bovine abortions, stillbirths, and neonatal deaths in Atlantic Canada (1990 to 2001), an etiological diagnosis was made in 117 cases (44.2%). The cases were divided into 2 groups: 234 abortions, and 31 stillbirths and neonatal deaths. Identified causes of abortion were bacteria (24.4%), fungi (6.8%), viruses (6.0%), protozoa (Neospora spp.) (2.1%), congenital anomalies (0.4%), and miscellaneous conditions (1.3%). In addition, placentitis without demonstrable infectious agents was observed in 17 (7.3%). Of the 31 cases of stillbirth and neonatal death, identified causes were dystocia (22.5%), congenital anomalies (22.5%), meconium aspiration syndrome (16.1%), and miscellaneous conditions (6.5%). No etiological diagnosis was made in 59% of abortions and 32.4% of stillbirths and neonatal deaths. The 3 most common identifiable causes of abortion in this study were bacterial, fungal, and viral infections.     

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.     

Pathogenesis and clinical signs of equine herpesvirus-1 in experimentally infected ponies in vivo.

Equine herpesvirus-1 (EHV-1) causes respiratory disease, neonatal death, abortion and neurologic disease. The main purpose of this study was to identify viral antigen in respiratory tract samples by immunoperoxidase staining. Six pony foals were selected on the basis of demonstrating seronegativity to EHV-1 by virus neutralization and housed in isolation. They were infected experimentally by administering EHV-1 nebulized ultrasonically through a face mask. Successful infection was clinically apparent as each of the foals had febrile responses, nasal discharge, and enlarged submandibular lymph nodes. Sporadic coughing was also heard. EHV-1 was isolated from nasopharyngeal swabs of 4/6 ponies and seroconversion was demonstrated in all foals. Bronchoscopic examination of the large airways revealed hyperemia. The incidence of recovery of Actinobacillus suis from nasopharyngeal swabs increased initially, with recovery of Streptococcus zooepidemicus isolates predominating at 3 wk post-infection. Cytology brushes were used to sequentially sample the respiratory tract of the infected ponies at the nasopharynx, mid-trachea and the mainstem bronchus. Bronchoalveolar lavage provided lung cells. Immunocytochemistry techniques were applied to both types of samples to locate EHV-1 antigen. Indirect immunoperoxidase staining of samples utilizing monoclonal antibodies specific for EHV-1 demonstrated viral antigen associated with cellular debris, primarily in the nasopharyngeal samples on days 3-9 post-infection.     

Equine herpes myeloencephalopathy

The neurologic form of EHV-1 infection appears to be the result of central nervous system infarction caused by vasculitis, which is initiated in endothelial cells of small blood vessels. The etiologic agent is equine herpesvirus-1, subtype 1. There is some evidence to suggest that the neurologic form of the disease actually results from reactivation of a previous infection. Whether the vasculitis that causes the central nervous system injury is the direct result of the infection or an immune response to the infection has not been determined. The clinical signs are rapid in onset, nonprogressive, and many horses may improve. The diagnosis must often remain tentative, particularly in horses that recover, because there is no single reliable confirmatory test. The prognosis is generally good, although recovery may be slow and incomplete. Supportive therapy is essential, and administration of corticosteroids may be useful. There is no specific therapy for the virus or for the vasculitis. Currently no vaccine can be claimed to protect against the central nervous system form of the disease. Vaccination is recommended, however, to reduce the incidence of respiratory disease, abortion, and neonatal death on the farm. Repeated vaccination is necessary to maintain presumably protective antibody concentrations. Vaccination every 3 to 4 months may decrease the incidence of EHV-1 infection on the farm and therefore may indirectly prevent the occurrence of the neurologic form of the disease.     

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.     

Infection of central nervous system endothelial cells by cell-associated EHV-1

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.     

Abortion of virologically negative foetuses following experimental challenge of pregnant pony mares with equid herpesvirus 1.

From 1988 to 1991, 51 pregnant pony mares were challenged intranasally or by aerosol with an isolate of EHV-1 (AB4) originally recovered from a quadriplegic mare. This resulted in 32 abortions, occurring from 9 to 29 days after infection. In 14 of the early abortions (Days 9-14), EHV-1 was not demonstrated in the foetal tissues by virus isolation or immunostaining despite no other non-viral cause for the abortion being evident. Application of the polymerase chain reaction to foetal tissues from 9 of these cases also proved negative. One of the 14 mares was destroyed immediately after abortion, and post-mortem examination revealed severe and widespread vasculitis, thrombosis and secondary ischaemic damage in the endometrium with replication of EHV-1 in endothelial cells. These findings suggest that EHV-1 abortion can occur due to endometrial damage without the establishment of a foetal infection.     

Equine herpesvirus type 1 (EHV-1) replication in primary murine neurons culture

Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were isolated from aborted fetuses: Rac-H, a reference strain isolated by Woyciechowska in 1959 (Woyciechowska 1960) and Jan-E isolated by Bańbura et al. (Bańbura et al. 2000). Upon infection of primary murine neuronal cell cultures with Jan-E or Rac-H strains, a cytopathic effect was observed, manifested by a changed morphology and disintegration of the cell monolayer. Positive results of immunofluorescence, nPCR and real-time PCR tests indicated high virus concentration in neurons, meaning that both EHV-1 strains were likely to replicate in mouse neurons in vitro without the need for adaptation. Moreover, we demonstrated that some neurons may survive (limited) virus replication during primary infection, and these neurons (eight weeks p.i.) harbour EHV-1 and were still able to transmit infection to other cells.     

A simple and rapid immunoperoxidase test for the identification of equine herpes virus-1

Equine herpesvirus-1 (EHV-1) is an alphaherpesvirus which is a major cause of abortion in mares and it is of a considerable economic importance worldwide (Crabb and Studdert, 1996). EHV-1 has been isolated in New Zealand from cases of abortion and perinatal mortalities (Hutton and Durham, 1997; Horner, 1989). Recently, a herpesvirus was isolated from a lung sample from an aborted foetus and this virus was identified as EHV-1 by the immunoperoxidase technique with the use of a monoclonal antibody specific for EHV-1.     

Viraemia and abortions are not prevented by two commercial equine herpesvirus-1 vaccines after experimental challenge of horses

Eighteen horses, vaccinated on a number of occasions over a period of 12 to 20 months with either a live equine herpesvirus-1 (EHV-1) or an inactivated EHV-1 vaccine, were challenged by the intranasal instillation of the subtype 1 virus isolated from the 1983 outbreak of abortion and paralytic disease at the Lipizzan Stud, Piber, Austria. The prechallenge serum titres of all vaccinated horses were remarkably low, although most horses had received their last vaccine dose only 3 weeks before test-infection. Higher titres were obtained with the inactivated product than with the live virus vaccine. However, no obvious differences were found between the two vaccines in their ability to prevent disease, in that all vaccinated and two 'sentinel' horses became infected and developed viraemia and some degree of clinical disease after challenge; five of the 10 in-foal mares aborted.     

Equine Herpesvirus-I Infection in Horses: Recent Updates on its Pathogenicity,Vaccination, and Preventive Management Strategies

Equine herpesvirus-1 (EHV-1) is one of the most common and ubiquitous viral pathogens infecting equines, particularly horses worldwide. The EHV-1 is known to induce not only humoral but also cellular immune responses in horses. Respiratory distress, abortion in pregnant mares, neurological disorders, and neonatal foal deaths represent EHV-1 infection. Despite the limited success of inactivated, subunit, live, and DNA vaccines, over the past few decades, vaccination remains the prime preventive option to combat EHV-1 infection in horses. However, current vaccines lack the potentiality to protect the neurological form of infections in horses. There is desperate necessity to search effectual EHV-1 vaccines that may stimulate not only mucosal and systemic cellular immunity but also humoral immunity in the horses. This review highlights the state of knowledge regarding EHV-1 biology, EHV-1 pathogenesis, and disparate vaccines studied in the past to prevent EHV-1 infection. The review also underlines the best management strategies which certainly need to be adopted by veterinarians in order to avoid and prevent EHV-1 infection and outbreak in horses in the future.     

Immune responses to commercial equine vaccines against equine herpesvirus-1, equine influenza virus, eastern equine encephalomyelitis, and tetanus

Horses are commonly vaccinated to protect against pathogens which are responsible for diseases which are endemic within the general horse population, such as equine influenza virus (EIV) and equine herpesvirus-1 (EHV-1), and against a variety of diseases which are less common but which lead to greater morbidity and mortality, such as eastern equine encephalomyelitis virus (EEE) and tetanus. This study consisted of two trials which investigated the antigenicity of commercially available vaccines licensed in the USA to protect against EIV, EHV-1 respiratory disease, EHV-1 abortion, EEE and tetanus in horses. Trial I was conducted to compare serological responses to vaccines produced by three manufacturers against EIV, EHV-1 (respiratory disease), EEE, and tetanus given as multivalent preparations or as multiple vaccine courses. Trial II compared vaccines from two manufacturers licensed to protect against EHV-1 abortion, and measured EHV-1-specific interferon-gamma (IFN-gamma) mRNA production in addition to serological evidence of antigenicity. In Trial I significant differences were found between the antigenicity of different commercial vaccines that should be considered in product selection. It was difficult to identify vaccines that generate significant immune responses to respiratory viruses. The most dramatic differences in vaccine performance occurred in the case of the tetanus antigen. In Trial II both vaccines generated significant antibody responses and showed evidence of EHV-1-specific IFN-gamma mRNA responses. Overall there were wide variations in vaccine response, and the vaccines with the best responses were not produced by a single manufacturer. Differences in vaccine performance may have resulted from differences in antigen load and adjuvant formulation.     

马疱疹病毒1型SYBR Green Ⅰ荧光定量PCR检测方法的建立

为建立马疱疹病毒Ⅰ型(EHV-1)的检测方法,本研究以EHV-1 gB基因的一段保守区域(1207 bp～1509 bp)作为检测的目的片段设计引物,通过对其反应条件的优化,建立了特异性检测EHV-1的SYBR Green I 荧光定量PCR方法.实验结果表明:该方法检测目的基因的灵敏度下限为10拷贝/μL,比常规PCR方法高100倍;与马疱疹病毒4型(EHV-4)及其他马传染病病原体无交叉反应;组内及组间的变异系数均小于2％.该方法检测速度快及高敏感性的特点为马鼻肺炎的防制提供了有力保障,同时也为进一步开展马鼻肺炎相关的研究提供了有效的辅助检测方法技术.     

Equid Herpesvirus-Associated Abortion in Poland between 1977-2010

Equid herpesvirus 1 (EHV-1) isolation and direct immunofluorescence were performed on tissues from 452 aborted and stillborn foals, which were submitted to the Pathology Department of Wroc?aw University of Environmental and Life Sciences, Poland, from different farms in Poland between 1977 and 2010. Although case submissions were received year-round, the number of submitted abortions peaked in the month of January, and then gradually decreased until June. EHV-1-associated abortion or neonatal death was diagnosed in 106 cases. At the beginning of the investigations (late 70s), there were a few cases of the EHV-1 abortions, but in the first half of the 80s, EHV-1 was not isolated. Until 1990, the percentage of abortion ranged from 0% to 50%, reaching its peak in 1984. A similar situation was observed during the next two decades. In 1991, 1998, 2007, and 2008, there was no isolation, and the highest rate (50%) was noticed in 1996 and 2000. The results of this study indicate that EHV-1 in Poland remains an important cause of pregnancy loss in horses, despite extensive vaccinations.     

Neonatal Equine Herpesvirus Type 1 Infection on a Thoroughbred Breeding Farm

Of 17 foals born on a Thoroughbred breeding farm between March and April 1995, infection with equine herpesvirus type 1 (EHV-1) was associated with neonatal morbidity in 5 foals, 3 of which died or were euthanized. Morbidity and mortality were associated with pulmonary inflammation, and EHV-1 was identified in the lungs of the 3 foals that died. All neonatal EHV-1 infections occurred in foals of mares housed in the same pasture and barn. No other clinical manifestations of EHV-1 infection (eg, abortion, neurologic disease, or respiratory disease) occurred during this outbreak. Three foals were treated with acyclovir (1 died, 2 survived), which may have influenced the clinical outcome in the surviving foals.     

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.     

Serological responses and clinical outcome after vaccination of mares and foals with equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) vaccines

Equine herpesvirus type 1 and type 4 (EHV-1 and EHV-4) cause infections of horses worldwide. While both EHV-1 and EHV-4 cause respiratory disease, abortion and myeloencephalopathy are observed after infection with EHV-1 in the vast majority of cases. Disease control is achieved by hygiene measures that include immunization with either inactivated or modified live virus (MLV) vaccine preparations. We here compared the efficacy of commercially available vaccines, an EHV-1/EHV-4 inactivated combination and an MLV vaccine, with respect to induction of humoral responses and protection of clinical disease (abortion) in pregnant mares and foals on a large stud with a total of approximately 3500 horses. The MLV vaccine was administered twice during pregnancy (months 5 and 8 of gestation) to 383 mares (49.4%), while the inactivated vaccine was administered three times (months 5, 7, and 9) to 392 mares (50.6%). From the vaccinated mares, 192 (MLV) and 150 (inactivated) were randomly selected for serological analyses. There was no significant difference between the groups with respect to magnitude or duration of the humoral responses as assessed by serum neutralization assays (median range from 1:42 to 1:130) and probing for EHV-1-specific IgG isotypes, although neutralizing responses were higher in animals vaccinated with the MLV preparation at all time points sampled. The total number of abortions in the study population was 55/775 (7.1%), 9 of which were attributed to EHV-1. Seven of the abortions were in the inactivated and two in the MLV vaccine group (p=0.16). When foals of vaccinated mares were followed up, a dramatic drop of serum neutralizing titers (median below 1:8) was observed in all groups, indicating that the half-life of maternally derived antibody is less than 4 weeks.