Epidemiology and diagnosis of Equid herpesviruses 1 and 4 in horses in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
Equid herpesvirus 1 (EHV-1) and Equid herpesvirus 4 (EHV-4) are ubiquitous viral pathogens of horses in all major horse-rearing countries in the world. These viruses are associated with four clinical syndromes, respiratory disease, abortion, perinatal disease and neurological disease. Traditional serological tests, such as virus neutralisation and complement fixation, are unable to discriminate between antibodies to EHV-1 and EHV-4. A blocking ELISA test has been developed which showed the potential to be used to screen horses for the presence of specific antibodies to EHV-1. The blocking ELISA test was shown to be specific, sensitive and repeatable for detecting antibodies to EHV-1 even in the presence of antibodies to EHV-4 when tested with polyclonal monospecific antisera to the two viruses raised in equine foetuses and in sheep. Vaccinal antibodies produced with the subunit vaccine Pneumequine® cannot however be reliably distinguished, by the blocking ELISA, from antibodies produced in natural infections or following the use of whole virus vaccines. Possibly, future genetically engineered vaccines which incorporate only the surface glycoproteins will stimulate the production of antibodies which will not be detected and allow the use of the test to differentiate horses naturally infected from those vaccinated with engineered vaccines. In a structured serological survey of Thoroughbred horses in New Zealand it was found that about 70% of adult (>24 months old) horses have specific EHV-1 antibodies and are therefore assumed to be latently infected with the virus. The prevalence increased with age, with 29% of 6-12 month old and 48% of 13-24 month old horses having specific EHV-1 antibodies. Gender was found to have a minor effect on the prevalence of specific antibodies, with female horses having slightly higher prevalence than males. In the survey, samples from two different years were tested. There was a slightly higher prevalence in 1995 than 1993, which is believed to be due to an increase in infection particularly in young horses in that year. In a group of young horses sampled monthly from birth, no clinical signs of respiratory disease were seen but four of the nine foals showed seroconversion to EHV-1 around the time of weaning. In an investigation of an outbreak of EHV-1 abortion on a Thoroughbred stud, high levels of specific EHV-1 antibody were found in sera from four of the six mares that aborted. The blocking ELISA test would have had considerable diagnostic value following the first abortions however, high levels of specific antibody were still present in some of the mares a year later. It was not possible to determine whether this was due to the persistence of these antibodies or whether it was due to reinfection or reactivation of latent virus. With tissue from the aborted foetuses it was possible to evaluate the ability of the EHV-1 specific monoclonal antibody, which forms the basis of the blocking ELISA test, to detect viral antigen in formalin-fixed tissue. After finding a suitable pretreatment involving microwave irradiation and trypsinisation it was possible to 'unmask' and visualise viral antigen in formalin-fixed tissue using the monoclonal antibody and an immunoperoxidase detection system. This provides a useful tool for the direct diagnosis of infection due to EHV-1 in tissue without the need for virus isolation and subsequent typing. The specific blocking ELISA and its associated monoclonal antibody, has proven useful both in the diagnosis of infection due to EHV-1 and in epidemiological studies of the virus. Use of the test in other countries, particularly where the incidence of abortion and neurological disease are higher than in New Zealand, would yield valuable information on the prevalence of the virus in different situations. In addition, the test has application as a management tool on a horse stud for the segregation of horses latently infected with EHV-1 and those naïve to the virus as a control measure for the serious sequelae of abortion and neurological disease.