Disease transmission in horses

Bacterial, viral and protozoal infections may cause severe reproductive losses. The present paper reviews the risk factors, clinical signs and preventive measures for the most important venereal or potential sexually transmitted diseases in horses. The stallion and use of semen for artificial insemination represent major risk factors for the transmission of bacterial contaminants of the penis, including Streptococcus equi subspecies zooepidemicus, Pseudomonas aeruginosa and Klebsiella pneumoniae, known to cause endometritis and infertility in the mare. The role of the stallion in disease transmission is also due to the non-clinical manifestation of diseases such as contagious equine metritis and equine viral arteritis. Dourine has been eradicated from many countries, but continues to be a problem in other areas of the globe. Strategies for the prevention of introduction and transmission of diseases in breeding operation are discussed.     

Equine arteritis virus

Equine arteritis virus (EAV) is a small, enveloped, positive-stranded RNA virus, in the family Arteriviridae , W.H.ich can infect both horses and donkeys. While the majority of EAV infections are asymptomatic, acutely infected animals may develop a wide range of clinical signs, including pyrexia, limb and ventral edema, depression, rhinitis, and conjunctivitis. The virus may cause abortion and has caused mortality in neonates. After natural EAV infection, most horses develop a solid, long-term immunity to the disease. Marzz and geldings eliminate the virus within 60 days, but 30 to 60% of acutely infected stallions will become persistently infected. These persistently infected animals maintain EAV within the reproductive tract, shed virus continuously in the semen, and can transmit the virus venereally. Mares infected venereally may not have clinical signs, but they shed large amounts of virus in nasopharyngeal secretions and in urine, which may result in lateral spread of the infection by an aerosol route. The consequences of venereally acquired infection are minimal, with no known effects on conception rate, but mares infected at a late stages of gestation may abort. Identification of carrier stallions is crucial to control the dissemination of EAV. The stallions can be identified by serological screening using a virus neutralization (VN) test. If positive at a titer of >/= 1:4, the stallion should be tested for persistent infection by virus isolation from the sperm-rich fraction of the ejaculate, or by test mating Shedding stallions should not be used for breeding, or should be bred only to mares seropositive from a natural infection or from vaccination, the mares should be subsequently isolated from seronegative horses for three weeks after natural or artificial insemination. A live attenuated (ARVAC) and a formalin-inactivated (ARTERVAC) vaccine are available. Both vaccines induce virus-neutralizing antibodies, the presence of which correlates with protection from disease, abortion, and the development of a persistent infection. Serological investigations indicate that EAV has a worldwide distribution and that its prevalence is increasing. As a consequence, an increasing number of equine viral arteritis (EVA) outbreaks is being reported. This trend is likely to continue unless action is taken to slow or halt the transmission of this agent through semen.     

Contagious equine metritis: a review

Contagious Equine Metritis (CEM) is a highly contagious venereal disease of horses caused by a fastidious, Gram-negative coccobacillus which grows best on chocolate agar under microaerophilic conditions (5-10% CO2). Clinically, the disease is characterized by a copious watery-to-mucopurulent, vaginal discharge two to ten days after breeding by an infected stallion (11, 13). Shortened estrous cycle lengths are common and may be the only indication of endometritis in some instances (7). Inapparent carriers of the disease in both the mare and stallion make control of the disease more difficult. Outbreaks of CEM have been reported in England, Ireland, France, Australia and the United States. The current information concerning CEM is reviewed.     

Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro

In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates were influenced by factors associated with the freezing process (Experiment 2), a single ejaculate from a second stallion was frozen using eight variations in timing of steps in the freezing protocol. There were no differences among treatments in fertilization rates (range, 0-3%). In Experiment 3, fertilization rates of semen frozen in an extender containing 21.5% egg yolk were lower than fertilization rates of semen from the same ejaculate but frozen with a 3% egg-yolk extender (0% versus 15%, respectively; P<0.01). We inferred that rates of equine IVF with frozen-thawed sperm were influenced by ejaculate, the composition and age of the media used, and freezing extender. To our knowledge, this is the first report of ejaculate or extender differences affecting in vitro fertilization in this species. These factors may help to explain the great variability in fertilization rates reported with equine IVF, both among and within laboratories.     

Genetic divergence with emergence of novel phenotypic variants of equine arteritis virus during persistent infection of stallions

The persistently infected carrier stallion is the critical natural reservoir of equine arteritis virus (EAV), as venereal infection of mares frequently occurs after breeding to such stallions. Two Thoroughbred stallions that were infected during the 1984 outbreak of equine viral arteritis in central Kentucky subsequently became long-term EAV carriers. EAV genomes amplified from the semen of these two stallions were compared by sequence analysis of the six 3' open reading frames (ORFs 2 through 7), which encode the four known structural proteins and two uncharacterized glycoproteins. The major variants of the EAV population that sequentially arose within the reproductive tract of each carrier stallion varied by approximately 1% per year, and the heterogeneity of the viral quasispecies increased during the course of long-term persistent infection. The various ORFs of the dominant EAV variants evolved independently, and there was apparently strong selective pressure on the uncharacterized GP3 protein during persistent infection. Amino acid changes also occurred in the V1 variable region of the GL protein. This region has been previously identified as a crucial neutralization domain, and selective pressures exerted on the V1 region during persistent EAV infection led to the emergence of virus variants with distinct neutralization properties. Thus, evolution of the EAV quasispecies that occurs during persistent infection of the stallion clearly can influence viral phenotypic properties such as neutralization and perhaps virulence.     

Venereal and vertical transmission of deformed wing virus in honeybees (Apis mellifera L.)

Deformed wing virus (DWV) infected semen was used for artificial insemination of DWV-free virgin queens. High titres of DWV could subsequently be detected not only in the spermatheca, but also in the ovaries, demonstrating venereal transmission of DWV in honey bees. Subsequent vertical transmission of the virus to the progeny of DWV infected queens was also demonstrated. Neither transmission route is 100% effective. Whether venereal transmission of DWV occurs during natural mating remains to be determined. The implications for the use, sale and transport of semen samples for artificial insemination are discussed.     

Semen quality of stallions challenged with the Kentucky 84 strain of equine arteritis virus

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days postinoculation (dpi). Semen was collected every other day for the first 15 days and 2 times a week up to 79 dpi. Additional samples were collected at 147, 149, and 151 dpi. Semen from each stallion was evaluated on the basis of motion characteristics, total number of spermatozoa, membrane integrity, and morphology. Virus infectivity titers were determined in RK-13 cells. Significant decreases in sperm quality were observed between 9 and 76 dpi. LOWESS (locally weighted scatterplot smoothing) curves for each horse were fit and integrated to quantify spermatozoa exposure to fever, virus, and edema over a period of 67 days before each ejaculation. Linear mixed models were then fit to isolate the effects of each factor on semen quality. Scrotal edema and fever were found to exert independent effects on all the semen quality parameters (P ≤ 0.002), whereas virus seems to exert little to no direct effect, as virus titers remained high long after semen quality returned to baseline.     

Diagnosis and treatment of four stallions, carriers of the contagious metritis organism--case report

Contagious Equine Metritis (CEM), a venereal disease of horses caused by the bacterium Taylorella equigenitalis, was first diagnosed in 1977 and subsequently spread to many nations [Proc 24th AM Assoc Equine Pract (1979) 287]. The disease was confirmed in the United States in 1978 [Proc Am Assoc Equine Pract (1983) 295]. Specific regulatory procedures for this disease have been established in the United States and 37 other countries. From 1999 through 2001, four of 120 imported European stallions tested positive for CEM at a quarantine facility in Darlington, MD, USA. Two stallions were identified by positive bacterial cultures for T. equigenitalis on arrival. The other two positive stallions were negative on initial bacterial cultures, but were identified as CEM carriers when test mares (that they had mated) were culture-positive for T. equigenitalis. Since T. equigenitalis, is a fastidious slow-growing coccobacillus, additional sets of samples taken over a interval might be required to ensure positive stallions are detected before mating test mares. Likewise, additional sets of samples taken over a long interval after treatment of a stallion for CEM might be required to ensure that positive stallions treated for CEM are detected before mating test mares. Aggressive systemic antibiotic therapy accompanied by routine topical therapy might be required to treat some CEM-positive stallions.     

Cases of Equine Coital Exanthema in Denmark

A venereal disease usually designated equine coital exanthema (ECE) has been observed in horses all over the world. In a very few oases a virus, claimed to be the causative agent of the disease, has been isolated.     

Selection of peptides for serological detection of equine infectious anemia

Equine infectious anemia caused by equine infectious anemia virus is an important disease due to its high severity and incidence in animals. We used a phage display library to isolate peptides that can be considered potential markers for equine infectious anemia diagnosis. We selected peptides using IgG purified from a pool comprised of 20 sera from animals naturally infected with equine infectious anemia virus. The diagnostic potential of these peptides was investigated by ELISA, Western blot and dot blot with purified IgG and serum samples. Based on the results, we chose a peptide mimetic for glycoprotein gp45 epitopes of equine infectious anemia virus, with potential for use as an antigen in indirect diagnostic assays. Synthesis of this peptide has possible applications for the development of new diagnostic tools for this disease.     

Risk of equine infectious anemia virus disease transmission through in vitro embryo production using somatic cell nuclear transfer

Prevention and regulation of equine infectious anemia virus (EIAV) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. Embryo production via the somatic cell nuclear transfer (SCNT) technology uses oocytes collected mainly from untested animals, which creates a potential risk of EIAV transmission through infected embryos. The current review examines the risk of EIAV disease transmission through SCNT embryo production and transfer. Equine infectious anemia virus is a lentivirus from the family Retroviridae. Because of a lack of direct reports on this subject, relevant information gathered from close relatives of EIAV, such as human immunodeficiency virus (HIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), and small ruminant lentiviruses (SRLVs), is summarized and used to predict the biological plausibility of EIAV disease transmission through transfers of the equine SCNT embryos. Based on published information regarding interaction of oocytes with lentiviruses and the sufficiency of oocyte and embryo washing procedures to prevent lentivirus transmission from in vitro-produced embryos of various species, we predicted the risk of EIAV transmission through SCNT embryo production and transfer to be very small or absent.     

Diagnostic aids for the detection of urine in the equine ejaculate

An experiment was conducted to evaluate three commercially available test kits, the Azostix, Multistix and Uric-acid test, for the detection of urine in the equine ejaculate. Azostix, which tests for urea nitrogen, consistently detected urine in the equine ejaculate. Urine contamination was evident when a color change occurred in the reagent pad, going from yellow to green after 10 sec of exposure. The sensitivity of Azostix to urea nitrogen in contaminated samples was 39 mg/dl. The Multistix test kit also successfully detected urine in semen. In the Multistix nitrite pad the color changed from yellow to organge after 3.5 min of exposure to urine-contaminated semen. The Uricacid test kit did not accurately detect urine-contaminated samples. It constantly elicited false positive results in all the control trials. The results of this study show that Azostix and Multistix are cost effective ($1.25 per analysis) and accurate diagnostic aids for detecting urine in the stallion ejaculate.     

Disease Induction by Virus Derived from Molecular Clones of Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.     

Serological survey of equine viral diseases in Mongolia

Three hundred sera were collected from horses in various parts of Mongolia in 2007 and seroepidemiological surveys for several equine viruses performed on them. Equid herpesvirus 1 and equine rhinitis A virus were prevalent, and equine arteritis virus and equid herpesvirus 3 were detected over a wide area though their rates of antibody-positivity were not high. Equine infectious anemia was distributed locally. The rates of horses antibody-positive for Japanese encephalitis virus and equine influenza virus were low, but these were detected. Bovine coronavirus antibodies were detected at a high rate, but it was not clear whether they were due to horse coronavirus.     

Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR)

In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility.Samples of stallion semen (n = 390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test.EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p < 0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs.There was a significant difference (p < 0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing.In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.     

Prostaglandins in stallion semen

The purpose of the experiment was to obtain preparatory information about the presence of prostaglandins in semen collected from various types of horses after different periods of sexual rest. Semen was collected with an artificial vagina. Prostaglandin-like activity was estimated by the bioassay procedure described by Vane (1). Results are expressed in ng/ml PGE(2) of seminal plasma. The total concentration of prostaglandins in the full ejaculate averaged 43.73 +/- 4.93 ng/ml of plasma while the total amount of prostaglandins in the ejaculate was 1076 ng. Taking into consideration the period of sexual rest in the stallion, statistically significant differences were found in the prostaglandin level in the semen of all the stallions.     

Genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus infection

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3+ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3+ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3+ T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3+ T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.     

The efficient use of equine cryopreserved semen

In order to optimize the efficient use of cryopreserved stallion semen, recent research has focused on the minimum insemination dose of frozen-thawed spermatozoa required for maximum fertility rate. The results appear to be highly stallion-dependent. Factors such as the timing of AI with respect to ovulation, as well as the site of insemination within the mare's reproductive tract, also affect success in breeding with frozen-thawed semen. Since acceptable pregnancy rates can be achieved from insemination of mares with very low numbers of spermatozoa, increasing the number of insemination doses processed from a single ejaculate may prove more cost-effective to stallion owners.     

Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen

This scientific review was prompted by recent legislation to curtail the use of semen from potentially virus-infected bulls to produce embryos for import into the European Union. From studies in laboratory animals, humans and horses, it is apparent that viruses may sometimes attach to, or be integrated into, spermatozoa, although in domestic livestock, including cattle, this seems to be a rare phenomenon, and carriage of virus through the zona pellucida into the oocyte by fertilising sperm has never been described in these species. Four specific viruses; enzootic bovine leukosis (EBLV), bovine herpesvirus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV), all of which tend to cause subclinical infections in cattle, but which can occur in bovine semen, are examined with regard to the risks that use of infected semen might lead to production of infected embryos. With regard to in vivo-derived embryos, when internationally approved embryo processing protocols are used, the risks from EBLV- and BTV-infected semen are negligible, and the same is almost certainly true for semen infected with BoHV-1 if the embryos are also treated with trypsin. For BVDV, there is insufficient data on how the virus is carried in semen and how different BVDV strains can interact with sperm, oocytes and embryos. There is a potential, at least, that in vivo-derived embryos resulting from infected semen might carry BVDV, although field studies so far suggest that this is very unlikely. With regard to in vitro-produced embryos, use of semen infected with any of the four viruses, with the probable exception of EBLV, will often lead to contaminated embryos, and virus removal from these embryos is difficult even when the internationally approved embryo processing protocols are used. However, it has never been demonstrated that such embryos have resulted in transmission of infection to recipients or offspring.     

Major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes from horses with equine infectious anemia virus recognize Env and Gag/PR proteins.

Cytotoxic T lymphocytes (CTL) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. Since there is limited evidence for an in vivo role of CTL in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. Horses infected with equine infectious anemia virus (EIAV) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. Viremic episodes may recur, but most horses ultimately control infection and become asymptomatic carriers. To begin dissection of the immune mechanisms involved in EIAV control, peripheral blood mononuclear cells (PBMC) from infected horses were evaluated for CTL to EIAV-infected cells. By using noninfected and EIAV-infected autologous equine kidney (EK) cells in 51Cr-release assays, EIAV-specific cytotoxic activity was detected in unstimulated PBMC from three infected horses. The EIAV-specific cytotoxic activity was major histocompatibility complex (MHC) restricted, as determined by assaying EIAV-infected heterologous EK targets, and was mediated by CD8+ T lymphocytes, as determined by depleting these cells by a panning procedure with an anti-CD8 monoclonal antibody. MHC-restricted CD8+ CTL in unstimulated PBMC from infected horses caused significant specific lysis of autologous EK cells infected with recombinant vaccinia viruses expressing EIAV genes, either env or gag plus 5' pol. The EIAV-specific MHC-restricted CD8+ CTL were detected in two EIAV-infected horses within a few days after plasma viremia occurred and were present after viremia was terminated. The detection of these immune effector cells in EIAV-infected horses permits further studies to determine their in vivo role.