Genomic variability among globally distributed isolates of equine arteritis virus.

Equine arteritis virus (EAV), a non-arthropod borne togavirus, has been shown to have a global distribution. To date, no major antigenic variation has been demonstrated between EAV isolates from different geographic origins. In this study, the genomic RNA of EAV isolates obtained from horses of different breeds in various countries around the world was oligonucleotide fingerprinted. Comparisons of these fingerprints were used to determine the extent of genomic variation among such isolates. Comparisons among isolates from North American horses revealed, for the most part, oligonucleotide homologies of less than 60%. Only 29 of the 98 comparisons revealed greater than 60% oligonucleotide homology. Nonetheless, several comparisons indicated a close epidemiologic relationship between isolates from horses of different breeds located in different states. Though all European isolates were of Standardbred origin and were from horses located in northern European countries, the majority had oligonucleotide homologies of less than 60%. Where oligonucleotide homology was apparent, it was, with one exception, greater than 70%. The two isolates from New Zealand had 93.2% oligonucleotide homology. This is indicative of an extremely close epidemiologic relationship. Comparisons between EAV isolates from around the world revealed oligonucleotide homologies between viruses from North America, Europe and New Zealand. In several instances, this homology was greater than 70% and in one case greater than 80%. No oligonucleotide homology was evident in comparisons involving the virus from South Africa. The high level of genomic conservation between certain EAV isolates of disparate geographic origins may reflect dissemination of the virus associated with the international movement of horses. The extent of genomic variation demonstrated between most of the EAV isolates used in this study confirms the need for further investigation of genomic heterogeneity among strains of this virus before techniques that rely upon nucleic acid hybridization can be effectively applied as diagnostic procedures.     

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.     

Differentiation of strains of equine arteritis virus of differing virulence to horses by growth in equine endothelial cells

OBJECTIVE: To compare growth characteristics of strains of equine arteritis virus (EAV) of differing virulence to horses in rabbit kidney (RK)-13 cells and equine endothelial cells (EECs) cultured from the pulmonary artery of a foal. SAMPLE POPULATION: 13 strains of EAV, including 11 field isolates of differing virulence to horses; the highly virulent, horse-adapted Bucyrus strain; and the modified-live virus (MLV) vaccine derived from it. PROCEDURE: The growth characteristics of the 13 strains were compared in EECs and RK-13 cells. Viral nucleoprotein expression, cytopathogenicity, and plaque size were compared to determine whether growth characteristics of the 13 strains were predictive of their virulence to horses. RESULTS: Cytopathogenicity, viral nucleoprotein expression, and plaque size induced by all 13 viruses were similar in RK-13 cells, whereas virulent strains of EAV caused significantly larger plaques in EECs than did the avirulent strains of EAV. Paradoxically, the highly attenuated MLV vaccine and 1 field isolate of EAV caused plaques in EECs that were larger than those caused by any of the other viruses, and sequence analysis confirmed the field isolate of EAV to be indistinguishable from the MLV vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: With the notable exception of the MLV vaccine, growth of the various strains of EAV in EECs was predictive of their individual virulence to horses. Thus, EECs provide a relevant and useful model to further characterize determinants of virulence and attenuation amongst strains of EAV.     

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)     

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.     

The effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-viral protein contaminants were found to be closely associated with EAV in that they co-purified with the virus during gradient centrifugation.     

Assessment of correlation between in vitro CD3+ T cell susceptibility to EAV infection and clinical outcome following experimental infection

In a recent study, we demonstrated that the virulent Bucyrus strain (VBS) of EAV could infect in vitro a small population of CD3(+) T lymphocytes from some but not all horses. Furthermore, we have shown that a common haplotype is associated with this in vitro CD3(+) T cell susceptibility/resistance phenotype to EAV infection. In this study, we investigated whether the differences in the susceptibility or resistance of CD3(+) T cells in vitro correlate with the outcome and severity of clinical signs in vivo. Thus, horses were divided into two groups based on their CD3(+) T cell susceptible or resistant phenotype. Following experimental inoculation with the recombinant VBS of EAV, horses were assessed for presence and severity of clinical signs, duration and magnitude of virus shedding, as well as production of proinflammatory and immunomodulatory cytokines in peripheral blood mononuclear cells using real-time quantitative RT-PCR. The data showed that there was a significant difference between the two groups of horses in terms of cytokine mRNA expression and evidence of increased clinical signs in horses possessing the in vitro CD3(+) T cell resistant phenotype. This is the first study to provide direct evidence for a correlation between variation in host genotype and phenotypic differences in terms of the extent of viral replication, presence and severity of clinical signs and cytokine gene expression caused by infection with virulent EAV.     

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.     

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).     

West Nile virus infection of Thoroughbred horses in South Africa (2000-2001)

REASONS FOR PERFORMING STUDY: West Nile virus (WNV) infection is endemic in southern Africa. With the recent emergence of WNV infection of horses in Europe and the USA the present study was performed to estimate the risk of seroconversion to WNV in a cohort of 488 young Thoroughbred (TB) horses. OBJECTIVES: To estimate the risk of seroconversion to WNV among a cohort of South African TB yearlings sold at the 2001 National Yearling Sales (NYS) and to determine whether the risk varied geographically. Two horses were also infected with a recent South African isolate of WNV to evaluate its virulence in horses. METHODS: Serum samples were collected from the cohort of 488 TB yearlings at the 2001 NYS. Serum samples that were collected from the same horses at the time that they were identified were sourced from our serum bank. Sera from 243 of the dams that were collected at the time that the foals were identified were also sourced from our serum bank. These sera were subjected to serum neutralisation (SN) tests for antibody to WNV. RESULTS: Approximately 11% of yearlings seroconverted to WNV on paired serum samples collected from each animal approximately 12 months apart. Studfarms with WNV-seropositive yearlings were widely distributed throughout South Africa and SN tests on sera from their dams indicated that exposure to WNV was even more prevalent (75%) in this population. Neurological disease was not described in any of the horses included in this study and 2 horses inoculated with a recent lineage 2 South African isolate of WNV showed no clinical signs of disease after infection and virus was not detected in their blood. CONCLUSIONS: Infection of horses with WNV is common in South Africa, but infection is not associated with neurological disease. POTENTIAL RELEVANCE: In contrast to recent reports from Europe, North Africa, Asia and North America, the results of our field and experimental studies indicated that exposure of horses to the endemic southern African strains of WNV was not associated with neurological disease.     

Isolation of pure Babesia equi and Babesia caballi organisms in splenectomized horses from endemic areas in South Africa

Both Babesia equi and Babesia caballi are endemic in large parts of South Africa. Attempts were made to obtain pure local isolates of both B. equi and B. caballi for the purpose of developing serological tests to study the epidemiology of equine babesiosis in this country. The indirect fluorescent antibody test was used to screen horses for B. equi and B. caballi in an endemic area. Seven horses and 3 donkeys between 3 and 36 months of age that tested negative were subsequently splenectomized. The splenectomy operation was performed through the abdominal approach. A 100% survival rate was achieved through this method, probably because it reduced the risk involved in the operation. Blood collected from naturally infected horses and passaged in fully susceptible splenectomized horses and a donkey, under laboratory conditions, produced 2 isolates of Babesia caballi and 1 of B. equi. Microscopical and serological examinations confirmed that these were pure isolates.     

马传染性贫血病毒弱毒疫苗S2基因在体内变异分析

为研究马传染性贫血病毒(EIAV)驴白细胞弱毒疫苗EIAVDLV121的S2基因在马体内的变异规律,本研究选用4匹成年马,其中2匹(#1、#2)接种EIAVDLV121,另外2匹作为对照.免疫后监测马体温、血小板含量以及病毒载量结果显示,免疫马未出现马传染性贫血体征.通过RT-PCR方法检测病毒S2基因在感染马体内不同时期的基因序列,结果显示,免疫马体内EIAVDLV121 S2蛋白的突变主要发生在氨基酸第17位、22位、39位、41位、51位和55位.另外,#1马免疫后70 d以及#2马免疫后第14 d和第28 d检测疫苗毒S2蛋白序列与EIAVDLV121亲缘关系较近,而#1马免疫后第42 d、第112 d和第140 d的疫苗毒S2蛋白序列与EIAVDLV121的致病性亲本强毒株EIAVDN40亲缘关系最近.本研究结果有助于对EIAV及EIAV疫苗株在马体内感染进程的研究.     

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.     

The serologic response of horses to equine arteritis virus as determined by competitive enzyme-linked immunosorbent assays (c-ELISAs) to structural and non-structural viral proteins

In an effort to further characterize the humoral immune response of horses to equine arteritis virus (EAV), direct and competitive enzyme-linked immunosorbent assays (c-ELISAs) were developed using monoclonal and polyclonal anti-sera to structural (G(L), N and M) and non-structural (nsp1) viral proteins. A nsp1-specific monoclonal antibody was produced to facilitate development of a c-ELISA to this protein. Data obtained using the various c-ELISAs confirm that the M protein is a major target of the antibody response of horses to EAV. However, none of the c-ELISAs that were developed were as sensitive in detecting EAV-specific antibodies in horse sera as the existing serum neutralization test.     

Three cases of virus isolation from horse fetuses diagnosed with equine arteritis virus (EAV) abortion from stud farms with different breeds]

Three cases of abortions were diagnosed as caused by Equine Arteritis Virus (EAV) by isolation and typing of this virus from the respective fetuses. All 3 abortions were single cases, one occurring on a stud with Iceland Ponies, one with Warmbloods, one with Lipizzaner horses. On each stud horses of the respective breed were kept exclusively, therefore there existed no epidemiologic link. By means of seroneutralization tests performed on in contact horses it could be shown, that EAV had only been introduced recently into the stud with the Iceland Ponies. An extraneous mare stabled temporarily for covering by the stud's stallion could be incriminated for introducing EAV. By means of post-abortion serology it could be demonstrated that the Warmblood stud had been harbouring EAV for a longer period of time. Likewise, the Lipizzaner stud could be shown to have been persistently infected, this time on pre-abortion serums stored frozen at our Institute. On both these studs preexisting neutralizing antibodies accounted for the single case of abortion and prevented serial abortions. By investigating frozen serums taken in earlier years we could show that the Lipizzaner stallions had reacted positively to EAV for several years already. However, the gestation period of the aborting mare allowed to exclude EAV-positive semen transmitted on copulation as cause of its abortion. Both the Iceland Pony stallion as well as the Warmblood stallion could be excluded as sources of infection for the respective aborting mares as both repeatedly were seronegative.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Comparison of the microhardness of enamel, primary and regular secondary dentine of the incisors of donkeys and horses

The microhardness of the enamel, primary dentine and regular secondary dentine of seven donkey and six horse incisors was determined with a Knoop indenter at the subocclusal and mid-tooth level. The mean microhardnesses of the donkey incisor enamel, primary dentine and secondary dentine were 264.6 63.00 and 53.6 Knoop Hardness Number, respectively. There was no significant difference between the microhardness of the enamel and primary dentine on the incisors of the donkeys and horses, but the microhardness of the regular secondary dentine of the donkeys' incisors at the mid-tooth level was slightly but significantly less than that of the horses. There was also a difference in the microhardness of the secondary dentine between the subocclusal and mid-tooth levels in both donkey and horse incisors.