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.     

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.     

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.     

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.     

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.     

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).     

Serological surveillance of equine viral arteritis in the United Kingdom since the outbreak in 1993

Serological analysis of blood samples submitted to the Animal Health Trust showed that during 1995, 185 of 9203 unvaccinated horses (2.0 per cent) tested positive for antibodies to equine arteritis virus (EAV), and that during 1996, 46 of 8851 unvaccinated horses (0.52 per cent) tested positive. During both years thoroughbreds were the predominant breed tested and only a small proportion of these (<0.3 per cent), consisting predominantly of imported mares, were seropositive. In contrast, among standardbred horses, from which samples were actively solicited in 1995, 84 of 454 (18.5 per cent) were seropositive. Among standardbreds there was a difference in prevalence between types of horses, with 3.7 per cent of racing horses, 25 per cent of non-racing horses and 41 per cent of stallions testing seropositive. Investigations of seropositive stallions identified during 1994 and 1995 demonstrated that clinically inapparent equine viral arteritis (EVA) had occurred previously in the UK. Of 50 seropositive stallions, nearly half were standardbreds and nearly all had been imported from either North America or the European Union. Whether 34 seropositive stallions were shedding virus in their semen was established either by test mating, by the serology of the covered mares, or by investigation by MAFF following the introduction of the Equine Viral Arteritis Order 1995. Nine of the stallions (26.5 per cent) were identified as presumptive shedders of EAV in semen and among specific breeds, viral shedding was identified in six of 15 (40 per cent) standardbreds and three of nine (33 per cent) warmbloods. In contrast with the outbreak of EVA in the UK in 1993, no signs of disease typical of EAV infection were reported during these investigations, even in mares test mated to stallions shedding the virus.     

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.     

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.     

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.     

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.     

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.     

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.     

Study on the epidemiology of equine arteritis virus infection with different diagnostic techniques by investigating 96 cases of equine abortion in Hungary

The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.     

Interstate Equine Semen and Embryo Shipment Regulations in the United States and Their Implications on Control of Disease Transmission

Outbreaks of both equine viral arteritis and contagious equine metritis (CEM) in the United States have occurred in recent years. Shipped semen has been implicated in disease transmission and rapid spread. A survey was performed of state regulations regarding testing for equine arteritis virus (EAV) and CEM before interstate semen or embryo shipment. Results demonstrated lack of any requirements in 31 states. Four states had regulations regarding EAV; 17 required a Certificate of Veterinary Inspection (CVI) to accompany a semen or embryo shipment. Ten states required a negative equine infectious anemia test, primarily as a requirement of a CVI versus because of risk of dissemination of disease. No states required CEM testing. A comparison of state department of agriculture or veterinary medical association Web sites and direct communication with state veterinarians or their offices demonstrated contradictory information in six states. The lack of uniform regulations concerning CEM and EAV testing for shipped semen and embryos, and the threat they pose to the equine breeding industry and horse health, should be alarming to veterinarians and horse owners. Routine testing of animals before shipment of semen or embryos and veterinary involvement in collection and breeding activities are appropriate and necessary to help prevent future outbreaks and protect equine health.     

Responses of horses vaccinated with avirulent modified-live equine arteritis virus propagated in the E. Derm (NBL-6) cell line to nasal inoculation with virulent virus

Nineteen horses with no prior experience with equine arteritis virus (EAV) were inoculated IM with an avirulent live-virus vaccine against equine viral arteritis; the vaccinal virus had been passaged serially 131 times in primary cell cultures of equine kidney, 111 times in primary cell cultures of rabbit kidney, and 16 times in an equine dermis cell line (EAV HK-131/RK-111/ED-16). Three or 4 of the vaccinated horses each, along with appropriate nonvaccinated controls, were inoculated nasally with virulent EAV at each of months 3, 6, 9, 12, 18, and 24 after they were vaccinated. The following was concluded: Vaccination did not induce clinical signs of disease in any horse and, thus, seemed safe for use in the field. All vaccinated horses (n = 19) developed serum-neutralizing antibodies to EAV. Fourteen of the vaccinated horses were completely protected from clinical arteritis when exposed to large doses of virulent EAV. Four were partially protected, and one had little or no protection. Six of 13 nonvaccinated horses died of acute arteritis, and the remaining 7 horses experienced severe signs of disease, but survived the infection. All horses (n = 32), whether vaccinated or not, became infected when inoculated nasally with virulent EAV. Virus was recovered from 17 of the 19 vaccinated horses, and all 19 had a secondary humoral immune response. The duration and severity of thermal reaction and persistence of virus were more transitory in vaccinated horses than in the nonvaccinated controls. Protection afforded by this vaccine can persist for at least 24 months, the maximal time after horses were vaccinated that immunity was challenged in the present study.(ABSTRACT TRUNCATED AT 250 WORDS)