Equine coital exanthema (ECE), caused by equid herpesvirus 3 (EHV-3), is a contagious venereal disease characterised by the formation of painful papules, vesicles, pustules and ulcers on the external genitalia of both mares and stallions. EHV-3 is an alphaherpesvirus, distinct from the other equine herpesviruses, endemic in most horse breeding populations worldwide. EHV-3 is primarily transmitted through coitus, although there is also evidence supporting the possibility of non-coital spreading through infected fomites and contacts other than coitus. The infection does not usually result in systemic illness. Epidemiological observations and serological monitoring suggest the existence of latently infected animals from which EHV-3 is periodically reactivated and transmitted to cohorts but latency of EHV-3 has not been formerly demonstrated.
The negative impacts of ECE on equine breeding enterprises are the forced, temporary disruption of the mating activities of mares and stallions, the additional care and supportive treatment in affected horses, and the risk of virus spread by either fresh or frozen semen as well as by artificial insemination and embryo transfer practices. In intensively managed stud operations, which have heavily-scheduled breeding dates for thoroughbred stallions, breeding disruptions may translate into significant end-of-season decreases in the number of entries into the mare book of affected stallions. Also, delayed foaling dates and/or reduced pregnancy rates may occur in mares that miss breeding opportunities due to the disease. Similarly, in the face of an ECE outbreak in artificial insemination and embryo transfer centres, both donor and recipient affected mares show such discomfort that they are reluctant to be inspected, inseminated or transferred, with the consequent loss of opportunity to become pregnant. The additional time and necessary precautions required to manage the donor and receptor mares due to the presence of the disease also have a substantial negative impact.
Because ECE is a not a notifiable disease and the diagnosis is made on the basis of typical clinical signs, most cases and outbreaks of ECE remain unnoticed and its true prevalence and economic impact is difficult to assess and is probably underestimated.
Therefore, as several aspects of EHV-3 infection are largely unknown and it has severe economic consequences to the horse industry, the general aim of this doctoral study was to increase the knowledge about the biology of EHV-3 infection. The specific objectives were to investigate the iatrogenic transmission of infection and to set up a protocol for experimental reproduction of the disease, to study the reactivation and re-excretion patterns from latency, to evaluate the epidemiological importance of subclinical infections, and to hypothesise about the ECE economic consequences in the current context of the equine industry.
During the occurrence of an outbreak of ECE in an embryo transfer centre, approximately 32% (n=35) of the donor mares and 25% (n=125) of the recipient mares showed typical ECE lesions around the anus and on the perineal skin, discomfort, and anorectal lymphadenopathy. EHV-3 was detected in 7 (58%) of the affected mares and specific antibodies in 23 (88%) of the convalescent mares. Since no natural breeding had taken place on the affected mares, it could be hypothesised that the virus spread was a consequence of contamination by means of the gloves or the ultrasonography scanner used. Lymphadenopathy provides a new concern associated with ECE.
EHV-3 was isolated from nasal swabs obtained during an outbreak of unilateral rhinitis affecting approximately 40 out of 2000 thoroughbred horses. The fact that an endoscopic examination had been performed in the week previous to the onset of the lesions to evaluate the respiratory tract function was a common finding in all the horses affected. EHV-3 was demonstrated as the etiologic agent of the unilateral rhinitis observed in those 40 thoroughbred horses. The endoscope used for respiratory tract examination was identified as the most likely cause of the spread of the infection. This case is an example of non-genital iatrogenic transmission and reinforces the importance of strict application of hygienic measures in order to reduce the risk of spread of infectious diseases.
The virus isolated from this field outbreak as well as that isolated from others were characterized by means of restriction endonuclease (RE) fragment patterns, plaque size and gG gene partial nucleotide sequencing. In the 25 isolates included in the study, different RE patterns were found: two with BamHI (one of them identical to the one of the reference strain), two with Hind III (both different from the one of the reference strain) and one with Eco RI (different from the one of the reference strain). The plaque size was homogeneous between the isolates, and 1.64 and 2.88 times larger than that of the reference strain. Three base substitutions in the gG gene were found at positions 904, 1103 and 1264, which resulted in strains CAT (Australia), AAT (the United States and Brazil), CAG (Argentina) and ACT (Argentina). The RE pattern and the nucleotide sequence of the gG gene obtained revealed that there are genetically distinguishable EHV-3 strains in circulation. Not only the RE patterns, as previously described, but also the nucleotide sequence of the gG gene, could be useful tools for epidemiological studies. The biological implications of these changes are still unknown.
Two sets of experimental infection with EHV-3 were carried out under controlled conditions. In the first experiment, two seronegative mares were topically inoculated in the vagina and perineal area with EHV-3. The same protocol was followed in the second experiment in two seronegative and two seropositive mares (the mares which had been included in the first experiment were used six months later). Clinical samples consisted of swabs from the vagina and perineal area, and blood samples were obtained for virological, serological and haematological studies. A scoring system was designed and used for daily clinical evaluation and rectal temperature records from each mare. Neither hyperthermia nor haematological changes were recorded in the mares analyzed. Typical ECE lesions were observed in seronegative animals: the clinical score was 172 and 90 (average score: 131) for the mares included in the first experiment and 160 and 92 (average score: 124) for the mares included in the second experiment. Only slight lesions were observed in the seropositive mares, being the clinical score 53 and 41 (average score: 47). Also, differences were detected in the duration and intensity of virus shedding, being 15 and 9 days (duration) and 105 versus 104 (the highest virus load detected) in the seropositive and seronegative mares, respectively.
In one study designed to demonstrate EHV-3 latency and to study reactivation and re-excretion patterns, virus shedding, seroconversion and the presence of a small ECE lesion were observed in one out of two previously naturally infected mares after corticosteroid treatment. EHV-3 was isolated from perineal vaginal swabs of one of the mares, both on day 14 after corticosteroid treatment and along the following 10 days. A small and rounded area of erosion was observed on the left labia of the vulva of the same mare on day 19 after corticosteroid treatment and 5 days after the virus shedding was detected. A significant (four-fold) increase in the antibody titre was found in the mare which shed the virus 28 days after corticosteroid treatment and 14 days after the beginning of virus re-excretion. In concordance with epidemiological observation and serological studies, and in common with other members of the subfamily Alphaherpesvirinae, this study indicates that a state of latency is established after natural infection of EHV-3.
A study was carried out to estimate the prevalence of excretion of EHV-3 under field conditions. The virus was detected in perineal-vaginal swabs by real time PCR and specific antibodies were identified by seroneutralization in 14 (6%) and 105 (48%) respectively of 220 thoroughbred mares without clinical signs at the time of breeding. In order to assess the re-excretion patterns of spontaneous reactivation, two seropositive (presumably latently infected) polo mares were kept in isolation for 11 months. Virological investigations on perineal vaginal swabs obtained on a daily basis revealed re-excretion of EHV-3 on two occasions, 3 months apart (each for a 3-day interval) in one of the mares, and on only 1 day in the other mare. Antibodies against EHV-3 were detected with only slight variation during the entire period in both mares. Clear evidence of the existence of EHV-3 shedders in a healthy mare population under both field and isolation conditions is provided. Furthermore, despite the small number of animals included (only two), the study in mares kept in isolation demonstrated that at least two periods of EHV-3 spontaneous reactivation and re-excretion in the presence of serum antibodies were possible in the same animal in an 11-month interval.
In conclusion, EHV-3 infections and ECE are still a threat for the equine industry. In the present study, EHV-3 was found in several field outbreaks of ECE in thoroughbred breeding farms; the disease was reported by the veterinarians as a true sanitary problem and thus demands additional preventive measures. In addition, EHV-3 was detected as a not rare event in clinically healthy mares, which constitutes the most relevant finding from an epidemiological perspective. ECE is also a sanitary problem of concern for embryo transfer and routine veterinary practices.
The population of EHV-3 latently infected mares which reach up to 50% at the time of breeding deserves special attention. Reactivation of the latent virus is not preventable and those mares can spontaneously reactivate the virus and become a source of infection for highly valuable horses like « shuttle stallions », with the consequent economically negative impact on the equine enterprises.
Finally, there is an important need for finding out additional preventive measures including « pen side » diagnostic tools that allow the detection of subclinically EHV-3 shedding mares in order to segregate them from natural breeding and give them an appropriate antiviral treatment before being covered by stallions.