The occurrence of equine arteritis virus in Australia

This paper reports the first isolation of equine arteritis virus (EAV) in Australia and serological evidence of exposure to EAV in Australian horses. Twelve Standardbred stallions imported from North America were found to shed EAV in semen. One hundred and seven stallions were tested for serum antibodies to EAV and 73% of Standardbred stallions tested were seropositive as compared to 8% of Thoroughbred stallions. Serum antibody was detected in 71% of Standardbred mares, 6% of Standardbred racehorses and 1% of Thoroughbred mares and racehorses. Examination of stored serums demonstrated that EAV had been present in Australia since at least 1975.     

Lateral transmission of equine arteritis virus among Lipizzaner stallions in South Africa

REASONS FOR PERFORMING STUDY: A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. OBJECTIVES: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. METHODS: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF 5-specific primers, subjected to sequence and phylogenetic analysis. RESULTS: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stallions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from bedding or fomites contaminated with semen. CONCLUSIONS: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. POTENTIAL RELEVANCE: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV- infected semen.     

Epizotiology and phylogeny of equine arteritis virus in hucul horses

The aim of the study was to determine the situation of equine arteritis virus (EAV) infections in hucul horses. A total of 176 horses (154 mares and 22 stallions) from the biggest hucul horse stud in Poland were tested. Antibodies against EAV were detected in 97 (55.1%) horses. The EAV seroprevalence among mares was 53.2% while in stallions - 68.2%. The percentage of positive mares increased with their age, thus amongst the mares of less than 2 years of age the percentage was 32.5%, while in the group of 3-5 years old increased to 59.4% and in the mares in the age of 6-10 years and older than 10 years 89.5% and 95% were seropositive, respectively. Among 11 seropositive stallions five were supposed to be shedders of EAV with their semen. It is likely that those persistently infected stallions were the reservoirs of the virus in the stud. Genetic studies using of ORF5 gene showed high homology between the viruses detected in the semen of those stallions what suggested lateral transmission between the stallions sharing the same stable. Persistent infection in an immature stallion, which has not yet been used for breeding, was established as a result of infection via respiratory route. Phylogenetic analysis confirmed that all hucul viruses shared the same ancestor and as most of EAV strains dominating in Polish horse population belonged to the European origin EAV subgroup (EU-1).     

Abortogenic viruses in horses

Viral causes of abortion include equine viral arteritis (EVA) and infection with equine herpesviruses‐1 and ‐4 (EHV‐1 and EHV‐4). Transmission of equine arteritis virus (EAV) occurs through respiratory, venereal or transplacental routes. Horizontal respiratory transmission of EAV results from exposure to infective nasopharyngeal secretions from acutely infected horses. For this transmission to occur, direct and close contact between horses is necessary. Venereal infection is an efficient method of transmission, with seroconversion of 85 to 100% of seronegative mares bred to virus shedding stallions. Asymptomatic carrier stallions are the essential natural reservoir of equine arteritis virus. Equine herpesviruses‐1 and ‐4 infect a susceptible host, replicate and establish a lifelong latent infection without any associated clinical signs. Reactivation of latent infections can result from factors such as stress and intercurrent disease. The control of these diseases is by implementation of appropriate management and hygiene measures, supplemented by vaccination and, in the case of EVA, by the identification of persistently infected stallions, which can be removed from breeding or continue to be bred to if managed under controlled conditions to prevent the risk of an outbreak of the disease.     

Seroprevalence of antibodies against equine arteritis virus in horses residing in the United States and imported horses

OBJECTIVE: To compare seroprevalence of antibodies against equine arteritis virus (EAV) in horses residing in the United States with that of imported horses. DESIGN: Serologic survey. SAMPLE POPULATION: Serum samples from 364 horses on 44 equine operations in California and 226 horses imported from various countries. PROCEDURE: Serum samples were collected from each imported horse and from up to 20 horses on each operation. For resident horses, the number of sampled horses on each operation was determined on the basis of the number of horses on the operation. Samples were tested for antibodies against EAV by use of a serum neutralization test. RESULTS: 1.9% of resident horses and 18.6% of imported horses were seropositive to EAV, including 16.1% of imported stallions. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the EAV seroprevalence of horses residing in California is considerably lower than that of imported horses, including imported stallions.     

Equine arteritis virus: an overview.

The causative agent of the respiratory disease equine viral arteritis is a small, single-stranded RNA virus with a genome organization and replication strategy related to that of coronaviruses and toroviruses. Clinical signs of infection in horses vary widely and severe infection can lead to pregnant mares aborting. Infected horses generally make good recoveries but stallions may become semen shedders of equine arteritis virus (EAV). These carrier stallions play an important role in the dissemination and perpetuation of EAV. Laboratory tests exist to detect virus and the equine immune response to infection. However, vaccines are not currently licensed in the UK to combat viral arteritis, the incidence of which may increase due to changes in European legislation.     

Veterinary recommendations for the handling of equine virus arteritis (EVA) in practical breeding care

The equine virus arteritis (EVA) consistently epidemically varying throughout the different breeds of the horse breeding countries is up to now only of lower significance by means of the typical clinical manifestation as well as an abortion causing factor. The susceptibility of the sexual mature stallions against the equine arteritis virus (EAV) causes different infection response which may lead to some restrictions in their use in natural breeding especially in the artificial insemination. In a certain not precisely predictable part of the stallion population EAV infection will cause a transient or permanent virus presence in the accessorial apparatus of the genital tract with transient or permanent shedding of the virus via seminal secretions. This makes the stallion to one of the dominant factors of the propagation of the field virus. The use of EAV shedding stallions in natural breeding or AI is very risky and only justifiable under certain precautions and additional measurements e.g. in EAV-seropositive or vaccinated mares. A consistent progress in the defeat of the disease can be expected from vaccination of the seronegative stallions with dead or inactivated live vaccines as they are considered to be able to prevent the establishing of EAV shedder status.     

Equine viral arteritis control scheme: a brief review with emphasis on laboratory aspects of the scheme in New Zealand

AIM: To review laboratory aspects of the equine viral arteritis (EVA) control scheme in New Zealand between 1989 and 2002. METHODS: The optimisation and performance of the virus neutralisation test (VNT) for equine arteritis virus (EAV) antibody, and the cell culture test to detect EAV in semen were analysed. Laboratory data and control scheme results were reviewed. RESULTS: Using optimised tests, it has been shown that antibody prevalence in Standardbred horses has steadily declined from 54% to <20%. Prevalences in Thoroughbred horses have remained at a low level of around 3%. The number of horses shedding EAV (all Standardbreds) has steadily declined from a maximum at any one time of 20 to the current figure of three. CONCLUSION: Eradication of EVA from the horse population in New Zealand is achievable in the near future.     

Status of equine viral arteritis in Kentucky, 1985

Clinical cases of equine arteritis virus infection have not been diagnosed in Kentucky since 1984, and there has been no indication that any of the horses involved in the 1984 epizootic have since been responsible for spread of the disease to horses in other states or other countries. Cases of abortion caused by naturally acquired infection with this virus have not been confirmed in 1984 or 1985. Neither field nor vaccine strains of equine arteritis virus have been shown to induce teratologic abnormalities or the carrier state in foals born to infected or vaccinated mares. The carrier stallion appears to have played a major epidemiologic role in the dissemination and perpetuation of the virus. A commercial modified live equine viral arteritis vaccine was found to be safe and efficacious for stallions and mares. The disease can be controlled by immunizing the stallion population and by restricting the breeding of equine arteritis virus-shedding stallions to vaccinated or seropositive mares, followed by an appropriate period of isolation from other nonvaccinated Equidae.     

Immune potency of lyophilized, killed vaccine for equine viral arteritis and its protection against abortion in pregnant mares

Immune potency test was conducted in horses by inoculating a killed vaccine for equine viral arteritis (EVA) which had been freeze-dried and contained aluminum hydroxide adjuvant. Serum neutralizing (SN) antibody to equine arteritis virus (EAV) was detected at maximal titers of 1:80 to 1:640, 1 to 2 weeks after 2-dose vaccination of 6 female horses. However, 6 pregnant mares inoculated with the vaccine which had been kept in storage for 1 year at 4°C produced much higher titers ranging from 1:320 to 1:1280. A maximal mean titer of 1:199.5 occurred in the 1st and 2nd week after 2-dose inoculation with the nonpreserved vaccine, whereas a maximal mean titer of 1:794.3 occurred in the 2nd week using the preserved vaccine. The horses showed no systemic or local adverse reactions clinically or hematologically after vaccination. Four of the 6 vaccinated pregnant mares were exposed to the Bucyrus strain of EAV but resisted challenge exposure, while 3 nonvaccinated control pregnant mares revealed acute EVA causing abortion and death. Isolation of EAV was positive from the body tissues of the aborted and dead fetuses and their dams, but was negative from the vaccinated mares. No significant rise of SN antibody titers was detected in the vaccinated mares following challenge exposure, suggesting that the vaccine can protect against EAV infection in pregnant mares and prevent abortion or death.     

Evidence for absence of equine arteritis virus in the horse population of New Zealand

AIM: To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand.

METHODS: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA. Horse breeds were categorised as Thoroughbred, Standardbred or other.

RESULTS: A total of 7,157 EVA serological test records (from import and export testing, EVA control scheme testing and TAD investigations) were available for analysis between 2005 and 2011. For the three breed categories a seroprevalence of ≤1.6% at the 95% confidence level was determined for each category. Between 2001 and 2011, as part of the EVA control scheme, the EVA status of 465 stallions was determined to be negative. During 2005–2011 EVA was excluded from 84 TAD investigations.

CONCLUSIONS: There was no evidence of equine arteritis virus being present in the general horse population outside of carrier stallions managed under the EVA control scheme.

CLINICAL RELEVANCE: Equine arteritis virus is absent from the general horse population of New Zealand.     

Detection of equine arteritis virus (EAV) in stallions--a contribution to the improvement of EAV diagnosis

Serum samples from 72 stallions were examined for the occurrence of antibodies against equine arteritis virus, of which 41 animals (57%) were found to be positive. 32 of the seropositive stallions were then screened for persistent EAV infection, before and after the breeding season. Semen samples were investigated by RT-PCR followed by dot blot hybridization and nested PCR, and by virus isolation on cell cultures as well. The carrier state was virologically confirmed in 11 of 32 stallions (34%) during the first and in 9 of 20 (45%) during the second investigation. RT-PCR followed by confirmatory methods was more sensitive when compared to virus isolation on cell cultures. It is suggested to implement the national and EU directives by recommending RT-PCR as a routine diagnosis of EAV in semen samples.     

Effective removal of equine arteritis virus from stallion semen

REASONS FOR PERFORMING STUDY: A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. OBJECTIVES: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. METHODS: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing technique. The sperm preparations were tested by PCR for the presence of EAV. The procedure was repeated using an ejaculate from a known shedding stallion, testing processed and unprocessed aliquots by PCR and virus isolation. RESULTS: Virus-free sperm preparations were obtained using the double sperm processing technique. The 'swim-up' step is apparently required to ensure complete virus removal. CONCLUSIONS: The double semen processing technique is potentially a useful and simple tool for the removal of EAV from the semen of shedding stallions. POTENTIAL RELEVANCE: The inclusion of density gradient centrifugation and 'swim-up' in protocols for the processing of semen for artificial insemination could help prevent the transmission of viral diseases carried in semen, such as EAV.     

Single‐Layer Centrifugation Reduces Equine Arteritis Virus Titre in the Semen of Shedding Stallions

Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single‐layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll‐E, a species‐specific colloid were evaluated for their ability to separate spermatozoa from virus in ejaculates from carrier stallions. The three types of SLC significantly reduced the virus titre in fresh semen at 0 h and in stored semen at 24 h (p < 0.001) but did not completely eliminate the virus. Sperm motility parameters such as total motility and progressive motility were significantly increased after colloid centrifugation, whereas curvilinear velocity and amplitude of lateral head deviation were decreased, and the remainder (straight line velocity, average path velocity, straightness, linearity, wobble and beat cross‐frequency) were not significantly affected by the processing. Although virus titres were reduced in the SLC samples, significant levels of infectivity still remained, especially in stallions shedding large amounts of virus. It remains to be determined whether SLC‐processed sperm samples from stallions shedding low virus titres retain sufficient equine arteritis virus to cause infection in mares through artificial insemination.     

Equine viral arteritis (EVA): A potential trapdoor for the practicing veterinary surgeon in the United Kingdom

Equine viral arteritis (EVA) is a notifiable disease in the UK with potentially serious consequences for horse owners and breeders. Legislation exists under ‘The Equine Viral Arteritis Order 1995’ to protect the UK from this contagious equine disease. The UK is at risk of introduction of EVA through importation of infected horses or semen. There is however much misunderstanding regarding the importance of EVA and occasionally misinterpretation, specifically on how to protect and manage stallions with vaccination. Issues with changing vaccine datasheet recommendations and vaccine availability have resulted in stallions being inappropriately vaccinated or vaccinations lapsing. This article is aimed at the practicing veterinary surgeon, to update them as to the current status of EVA vaccination and disease screening in horses in the UK and how to avoid some common pitfalls.     

Equine viral arteritis

Equine viral arteritis is reviewed with specific reference to clinical features, etiology, transmission, diagnosis, epidemiology, and current methods for the control of this disease. There is evidence of variation in pathogenicity among strains of equine arteritis virus. Virus transmission occurs primarily by the respiratory and venereal routes during the acute phase of the infection. The long-term carrier stallion appears to play a major epidemiological role in dissemination and perpetuation of the virus. Unlike the stallion, the carrier state has yet to be demonstrated in the mare or foal. A commercial modifiedlive equine arteritis virus vaccine has been shown to be safe and efficacious for stallions and mares. The disease can be controlled by identification and isolation of carrier stallions, immunization of seronegative stallions, and by restricting the breeding of equine arteritis virus-shedding stallions to equine arteritis virus vaccinated or seropositive mares.     

Transmissibility and abortogenic effect of equine viral arteritis in mares

A group of 14 pregnant mares was exposed via contact to 4 mares bred to stallions infected with equine viral arteritis virus. There was a demonstrable febrile response in each donor mare and in 12 of the pregnant mares. All 18 mares became seropositive after exposure. Equine viral arteritis virus was isolated from the nasopharynx of 5 pregnant mares, but not from the donor mares. Ten of the pregnant mares aborted, and virus was isolated from fetal specimens or placenta of 8.     

Molecular epizootiology of equine arteritis virus isolates from Poland

Phylogenetic analysis was performed on the sequences of 44 Polish isolates of equine arteritis virus that were isolated from the semen of stallions from national and private studs, collected during 2001--2005. These sequences were also compared with 41 reference strains previously described and commonly used in phylogenesis. On the basis of the nucleotide sequence analysis of the ORF5 gene, encoding the glycoprotein GP5, it was demonstrated that the Polish EAV isolates belonged to two subgroups and showed the closest relationship to the European strains. Similar results were obtained using the nucleotide sequences of the ORF7 gene. The nucleotide identity between the ORF5 and ORF7 sequences of all Polish isolates was in the range of 80.1-99.0% and 93.6-100%, respectively. The analysis of genetic diversity within the ORF5 sequences enabled a retrospective epizootic investigation. This study suggested that some of the EAV shedding stallions were probably infected before they were moved to Poland.