Carrier Stallions Research Articles

Monitoring for Equine Viral Arteritis has decreased its prevalence in European breeding sires

Equine viral arteritis (EVA) can induce a persistent carrier state in stallions and shedding of the virus with semen can spread EVA and cause abortions in seronegative pregnant mares. In the early 1990s, obligatory testing for EVA of stallions used for artificial insemination was implemented within the European Union (Council Directive 92/65/EEC). In addition, vaccination programs for young stallions and exclusion of carrier stallions from certain breed registry events were implemented in Warmblood sport horses. Information on the efficacy of these actions on the prevalence of EVA in reproductively active stallions are not yet available in Europe. This study retrospectively analyzed results of serological and virus antigen testing for EVA in sires (n=308, one sample set per sire, EVA-vaccination status unknown) of different age and breed referred to the Centre for Artificial Insemination of Vetmeduni Vienna for semen preservation between 2001 and 2021. For analysis, stallions were grouped by age (1-3, 4-10, 11-16, >16 years) and breed. The EVA antibody titer was determined by serum neutralization test and semen was analyzed for EVA virus by PCR and/or virus isolation test. Statistical analysis was performed by chi2-test, Kruskal-Wallis test or Mann-Whitney U-test using the IBM SPSS 28.0 statistics software. Of 308 stallions tested, 14.9% (n=46) were EVA seropositive and in 12 stallions EVAvirus was detected in semen (26% of seropositive and 3.8% of all tested stallions, respectively). The incidence of seropositive stallions (22% of sires tested during 2001-05 to 4.1% during 2016-21) and of stallions shedding EVA virus via semen (8.5% of all serologically tested stallions 2001-05 to 1.4% during 2016-21) decreased over time (p<0.05). No difference among breeds with regard to occurrence of seropositive stallions was detected. There was a tendency towards more Standardbred and Lipizzaner stallions (14% and 13%, respectively) shedding EVA virus in semen compared to all other breeds (<3%, p=0.074 among groups). Differences in the seroprevalence of EVA antibodies existed among stallions from different age groups (p<0.01) with the highest percentage of seropositive stallions in 11-16 year-stallions (45.7%). No differences among age groups with regard to virus shedding existed. The EVA antibody titer increased with age (p<0.01), reflecting a higher possibility of repeated virus challenge in older stallions. Antibody titer was not associated with carrier state. In conclusion, monitoring of stallions for EVA in combination with vaccination programs resulted in a decrease of the prevalence of seropositive and virus-shedding stallions during the past 20 years in a European stallion population and can therefore be considered successful.

Epidemiological aspects of some equine viral diseases

Although different equine viruses’ outbreaks have been recorded. However, the most important ones in are the African horse sickness virus (AHSV), equine influenza virus (EIV), equine viral arteritis (EVA), Equine infectious anaemia virus (EIAV), and equine herpes viruses (EHV). To combat these diseases, it is imperative to understand their epidemiological aspects. So, the current review aims to highlight some epidemiological aspects including; causative agents, clinical forms, history, prevalence and geographical distribution, source of infection, and methods of transmission. The AHSV mainly causes pulmonary, and cardiac forms with high morbidity and mortality rates in Africa. The EIV is found all over the world and results in respiratory signs. The EVA has low morbidity and mortality rates and is mainly found in the Americas and Europe, its significance is due to the reproductive problems as abortion in mares and subfertility in stallions. The EIAV has low morbidity and mortality rates and causes long time course disease mainly of fever, and chronic anaemia or death. The EHVs are the current most important pathogens due to their endemicity all over the world and their high morbidity. It causes respiratory, abortion, neonatal, and sometimes neurological manifestations. Aerosols and body excretions are the main sources of infection with EIV, EVA, and EHV. Venereal EVA transmission occurs through natural breeding or artificial insemination with the semen of infected or carrier stallions. The spreading of arboviruses is greatly affected by the vector activity like the AHSV which transmitted by the the Culicoide. imicola biting midges, and the EIAV by family Tabanidae. In general, it is recommended to take all epidemiological measures, including vaccinations and vector control, to limit the spread of such diseases and reduce economic losses.

Quantifying the effect of Warmblood Fragile Foal Syndrome on foaling rates in the German riding horse population.

Awareness of breeders of Warmblood Fragile Foal Syndrome (WFFS) increased after a widely discussed case in the USA in 2018. The hereditary connective tissue disorder, first described by a US research group in 2011 and for which a commercial genetic test exists since 2013, is caused by a point mutation in the PLOD1 gene, inherited autosomal recessively. Extension of molecular genetic testing and reporting of test results of organized horse breeders to their studbooks implies new opportunities for analyses. In Germany, data are centrally accessible through the integrated equine data base allowing comprehensive and population-wide investigation of the role of WFFS. The objective of this study was statistical testing for associations between WFFS and reproductive performance of German riding horses and quantifying possible differences between WFFS carriers and non-carriers, also in respect of performance traits. For this purpose, covering data from 2008 to 2020 were provided by ten German studbooks, so almost 400,000 coverings and resulting foaling rates were available for multiple analyses of variance with general and mixed linear models using procedures GLM, MIXED and HPMIXED of SAS software (version 9.2). Published breeding values of stallions were used for respective comparisons of riding horse performance. Assuming a WFFS carrier frequency of 9.5-15.0% in Warmblood horses, Hardy Weinberg principle implied an expected difference of 2.4-3.7% in the foaling rates of carrier and non-carrier stallions. Our results provided statistical evidence of detrimental effects of WFFS on the reproductive performance of Warmblood horses with about 2.7% lower average foaling rate in carriers of the mutant allele than in WFFS free sires, if mated to an average mare population. Indications of favorable dressage performance of WFFS carriers were found. Reported WFFS cases indicate only the tip of the iceberg and assessing the impact of WFFS on reproduction requires consideration of premature foal losses.

Equine arteritis virus long-term persistence is orchestrated by CD8+ T lymphocyte transcription factors, inhibitory receptors, and the CXCL16/CXCR6 axis.

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a “hub” gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract.

Intrahost Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.IMPORTANCE EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus.

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion.IMPORTANCE Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8+ T and CD21+ B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract.

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation.IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.

Equine viral arteritis in breeding and sport horses in central Spain

Equine viral arteritis (EVA) may have a high economic impact on breeding stud farms due to the occurrence of EVA-associated abortion outbreaks and the ability of the virus to persist in carrier stallions. While the consequences of EVA in premises with sport horses are usually less severe, the first confirmed outbreak of EVA in Spain occurred in a riding club in Barcelona, but no data on the seroprevalence of EVA in sport horses have been reported in Spain. Given the importance of both Spanish Purebred (SP) breeding horses and sport horses for Spain's equine industry, the aim of this study was to determine and compare the seroprevalence of EVA in these two horse populations in central Spain. Serum samples from 155 SP breeding horses residing in 16 stud farms and 105 sport horses of different breeds housed in 12 riding clubs, collected between September 2011 and November 2013, were tested using a commercial EVA antibody ELISA test with a 100% sensitivity, and confirmed by seroneutralisation (SN) test. EVA seroprevalence in SP breeding horses was higher 21.1% (95% CI 15.3–26.8%) than that in sport horses (6.7%, 95% CI 1.89–11.45%). However, the primary use (breeding vs. sport) was not significantly associated with seropositivity to Equine Arteritis Virus (EAV), suggesting that different management factors do not affect EVA circulation in these two horse populations.

Sites of persistent equine arteritis virus infection in the reproductive tract of the long-term carrier stallion

Sites of persistent equine arteritis virus infection in the reproductive tract of the long-term carrier stallion

Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10–70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.

Development of recombinant VHH nanobodies as an alternative for clearance of equine arteritis virus in carrier stallions

Development of recombinant VHH nanobodies as an alternative for clearance of equine arteritis virus in carrier stallions

Contagious Equine Metritis: Efficacy of US post-entry testing protocols for identifying carrier stallions and mares

tissue in 2002. In 2003, three species of Amycolatopsis and in 2009 two species of Streptomyces were identified in association with NPA cases. Amycolatopsis spp. (48.7%) and Crossiella equi (28.9%) were the most prominent nocardioforms identified in a recent abortion outbreak. This infection leads to late abortions, stillbirths and premature foaling. Premature foals sometimes die shortly after birth. Themode of transmission of this syndrome is not known. To date, nocardioform Actinomycetes have only been isolated from placental tissue. Most reported cases are from central Kentucky (Figure 1) but cases have also been diagnosed in Florida, Italy, and South Africa. In the 2010-2011 equine reproductive season, our Lexington laboratory diagnosed 118 cases of NPA by culture and PCR. Due to the high incidence of NPA that season, a farm-level study was conducted to identify possible risk factors for NPA. A total of 148 horse farms were included in a survey (98 affected, 50 farms unaffected). In total, 8075 mares were at risk on all farms with 429mares diagnosedwith NPA. On average, four mares were affectedper farm.Datawere analysedusing chi-squared,Wilcoxon rank-sum tests, and logistic regression analysis using S-PLUS. Farm acreage,mare numbers, and higher density on affected farmswere significantly and positively associated with NPA. Longer grazing times during January-March administration of progesterone prebreeding, HCG post-breeding, and NSAIDs were significantly and negatively associated with NPA. A study at the University of Kentucky attempted to induce NPA in pregnant and non-pregnant mares via intrauterine, oral, and intranasopharyngeal inoculation of Crosiella equi. However, these attempts did not result in infections or abortions.

Re-emerging Equine Arteritis Virus (EAV) variants

n where p ¼ overall proportion of individual qPCR positive straws and n ¼ number of straws tested (Table 2). 95% Confidence Intervals were adjusted for the clustering of observations in the five ejaculates. The qPCR assay effectively identified T. equigenitalis DNA contaminating multiple straws of different ejaculates processed over four years. Extensive variation in Ct values within and between batches was possibly due to effects of semen collection and processing including addition of antibiotic-containing extender. In conclusion, this data suggested that > three straws per batch should be evaluated for 95% confidence of detecting T. equigenitalis in cryopreserved semen from a potential carrier stallion but the lower range of the 95% CI suggests a larger study is necessary to improve precision of this estimate. Infectivity of the bacterium after cryopreservation warrants further investigation.

Variability of non-structural proteins of equine arteritis virus during persistent infection of the stallion.

The genetic stability of ORF1a encoding non-structural proteins nsp1, nsp2, nsp3 and nsp4 of equine arteritis virus (EAV) has been analysed for nearly seven years in a persistently infected stallion of the Malopolska breed. Between November 2004 and June 2011, 11 semen samples were collected. Viral RNA extracted from semen of this carrier stallion was amplified, sequenced and compared with the sequences of the other known strains of EAV. Sequence analysis of ORF1a showed 84 synonymous and 16 non-synonymous mutations. The most variable part of ORF1a was the region encoding nsp2 protein with 13 non-synonymous substitutions. The degree of amino acid identity between isolates ranged from 98.91 to 100%. Only single non-synonymous mutations were detected in nsp1 (one substitution) and nsp4 (two substitutions). The most stable was nsp3 in which no amino acid substitutions were observed during the whole period of observation.

Abortogenic viruses in horses

SummaryViral 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.

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.

Sequence analysis of ORFs 5, 6 and 7 of equine arteritis virus during persistent infection of the stallion—A 7-year study

Nucleotide and amino acid sequences of ORFs 5, 6 and 7 of EAV during persistent infection in the stallion of the Malopolska breed were analysed in the study. A total of 11 blood and semen samples were collected between 2004 and 2011. The titre of specific EAV antibodies in this carrier stallion was maintained at a high level throughout the study and was equal approximately 1:128. The sequence analysis of ORF5 showed 16 variable sites including 12 with synonymous substitutions and 4 with non-synonymous substitutions. The degree of nucleotide sequence identity among the strains ranged from 98.92% to 100%, whereas amino acid homology ranged from 98.06% to 100%. Ten substitutions were identified including 7 with synonymous mutations and 3 with non-synonymous mutations in ORF6. The degree of similarities among the strains ranged from 94.55 to 100% and from 98.41% to 100% at the level of nucleotide and amino acid sequence, respectively. Only a single point mutation at position 255 of ORF7 (99.6% identity) was found in nucleotide sequences of these strains. Phylogenetic analysis showed that all strains present in the semen of this carrier stallion created a separate cluster of "quasi-species" within the second European subgroup of 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 0h and in stored semen at 24h (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.

Selection and validation of a real-time rt-PCR assay for the detection of equine arteritis virus and preliminary investigations into the prevalence of carrier stallions in the Netherlands

use of a labeled probe, to enable detection of amplified products using a LFD. The assay to identify T. equigenitalis successfully amplified a 121bp fragment of the 23S rDNA genes. The assay detected as few as 46 gene copies in 12 minutes. The assay did not cross-hybridise with any of the 38 different bacterial organisms tested. The RPA assay was tested against a panel of 60 clinical equine genital swabs. All of the culture negative DNA extracts were correctly identified. Of the 30 culture positive DNA extracts, 24 were correctly identified. Culture positive, RPA negative DNA extracts had high real-time PCR CT values, indicating low levels of target DNA present. The introduction of a second probe into the assay enabled the simultaneous detection and differentiation of T. equigenitalis and T. asinigenitalis. The T. equigenitalis assaywas successfully adapted to enable detection of amplification products LFDs. We have successfully developed a rapid and sensitive isothermal assay to identify T. equigenitalis. Additionally, the inclusion of a second probe enables the detection of T. asinigenitalis. The amplification products can be detected in real time or via LFD. This test provides a fast (under 15 minutes) and accurate means of identification. The low energy requirements, lack of expensive equipment and LFD readout makes this test a suitable candidate for rapid diagnosis in low technology laboratories or near to the point of sampling.

Evidence thatIn VitroSusceptibility of CD3+T Lymphocytes to Equine Arteritis Virus Infection Reflects Genetic Predisposition of Naturally Infected Stallions To Become Carriers of the Virus

We investigated the correlation between in vitro susceptibility of CD3(+) T lymphocytes to equine arteritis virus (EAV) infection and establishment of persistent infection among 14 stallions following natural infections. The data showed that carrier stallions with a CD3(+) T lymphocyte susceptibility phenotype to in vitro EAV infection may be at higher risk of becoming carriers than those that lack this phenotype (P = 0.0002).