An outbreak of paresis in mares and geldings associated with equid herpesvirus 1

An outbreak of paresis occurred on a small isolated stud farm in July 1980. Of the 42 horses on the stud, infection was confined to a group of nine in-foal mares and their foals and eight other horses which were either housed together at night or grazed adjacent pastures. Eight mares and two geldings developed ataxia or paresis and one mare died. Equid herpesvirus 1 was isolated from 17 animals and serological studies confirmed that 24 of 26 animals sampled had experienced infection.     

Trials of an inactivated equid herpesvirus 1 vaccine: challenge with a subtype 1 virus

Vaccinated yearlings , two-year-old and in-foal pony mares with appropriate controls were exposed to aerosols of a subtype 1 virus one to two months after two or three vaccinations; all became infected. No obvious differences in the febrile responses, clinical signs and subsequent abortions were found between vaccinated and control mares. All vaccinated yearlings and two-year-old ponies developed a febrile respiratory disease but this was less severe than that suffered by the controls and the amounts and duration of virus shedding were reduced.     

Pathogenicity of equine herpesvirus 1 subtype 2 for foals and adult pony mares

Three pony mares and 4 pony foals were inoculated with a subtype 2 strain of equine herpesvirus 1. Foals had periods of fever 12 h and 2.5 days after inoculation and leukopenia, involving both neutrophils and lymphocytes, followed by leukocytosis. Mares had transient fever and leukopenia 24 hours after inoculation that were less severe than in foals. An increase in circulating virus-neutralizing antibody was seen in 2 of 3 inoculated mares, but not in foals. Attempts to isolate virus from blood were unsuccessful. These studies show that equine herpesvirus 1 subtype 2 is a mild pathogen for ponies and infection may result in inapparent clinical disease.     

Response of pregnant mares to equine herpesvirus 1 (EHV1)

Twenty-one pregnant mares were inoculated with EHV1. Nineteen became infected as evidenced by clinical signs and/or viremia but only one mare aborted a virus-infected fetus. The viremias were leukocyte-associated and appeared to be non-productive, latent infections of these cells. Infectivity, detectable by cocultivation, persisted in the circulating leukocytes for as long as 9 days without resulting in abortion. The data suggest that it is extremely difficult to evaluate the efficacy of vaccines in preventing EHV1 (Rhinopneumonitis) abortion due to the paucity of non-exposed mares, lack of tests which measure residual protection, and an incomplete understanding of the pathogenic mechanisms involved in abortion due to this virus.     

Experimental reactivation of equid herpesvirus 1 (EHV 1) following the administration of corticosteroids

Eight ponies were experimentally infected with equid herpesvirus 1 (EHV 1) (subtype 1). All animals showed clinical and serological evidence of infection and virus was isolated from nasal swabs and leucocytes. These ponies were kept in isolation for a further three months during which time complement fixing antibody decreased at least four-fold. Following immunosuppression with dexamethasone and prednisolone subtype 1 virus was recovered from six of the eight animals within 14 days. Five of these six ponies were viraemic and three of them shed virus in nasal secretions; only four displayed significant rises in complement fixing antibody and only two in neutralising antibody. Clinical abnormalities were not detected during reactivation.     

Serologic responses of pregnant thoroughbred mares to vaccination with an inactivated equine herpesvirus 1 vaccine

The immunogenic potency and safety of a chemically inactivated equine herpesvirus 1 vaccine with added adjuvant was evaluated by testing serum-neutralizing and complement-fixation antibody responses of pregnant Thoroughbred mares. The vaccinated population comprised 321 pregnant mares on 7 farms; 3 in Normandy, France; 1 in Kildare, Ireland; and 3 in central Kentucky. The pattern of antibody response to vaccination was found qualitatively and quantitatively similar to that of pregnant mares previously vaccinated and determined by challenge exposure to be immune to abortigenic infection under experimentally controlled conditions. The safety of the vaccine was demonstrated by the occurrence of only 2 local untoward reactions to the administration of 941 IM injections.     

Serological detection of equid herpesvirus 1 infections of the respiratory tract.

An investigation was made of 3 serological tests (virus neutralization, complement fixation and indirect immunofluorescence), which are applicable to epidemiological studies of infections by Equid herpesvirus 1 (EHV-1). Sera from gnotobiotic foals inoculated intranasally with various strains of EHV-1 were unable in some cases to neutralize heterologous strains and these results were not consistent with the existence of clearly-defined subtypes of EHV-1, as previously proposed. The cross-reactions in complement-fixation tests paralleled those with neutralization but immunofluorescence tests were found to be both more sensitive and more broadly reactive than the other two. Complement-fixing antibodies declined more rapidly following experimental infection than did those measured by neutralization or immunofluorescence. The results are discussed in relation to the diagnosis of EHV-1 infection and the significance they may have for the epidemiology of this disease.     

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Serology of experimental toxoplasmosis in pregnant ewes and their foetuses

Ewes were inoculated orally with 1500 Toxoplasma gondii oocysts at 6 to 14 weeks of pregnancy. Abortions occurred at 26 to 55 days after inoculation, and maternal serum T. gondii antibody titres did not peak/plateau until 20 to 103 days after inoculation, usually after abortion occurred. Serums or body fluids from unautolysed foetuses aborted at 35 days or more after maternal inoculation, contained significant levels of Toxoplasma antibodies as determined by the indirect fluorescent-antibody test. It was considered that foetal serology was a quick and efficient method of diagnosing congenital toxoplasmosis in sheep on a flock basis.     

Serological responses of mares and weanlings following vaccination with an inactivated whole virus equine herpesvirus 1 and equine herpesvirus 4 vaccine

Equine herpesvirus 1 (EHV-1) is a major cause of respiratory disease and abortion in horses worldwide. Although some vaccines have been shown experimentally to reduce disease, there are few reports of the responses to vaccination in the field. This study measured antibody responses to vaccination of 159 mares (aged 4-17 years) and 101 foals (aged 3-6 months) on a large stud farm with a killed whole virus EHV-1/4 vaccine used as per the manufacturer's recommendations. Using an EHV glycoprotein D (gD)-specific ELISA and a type-specific glycoprotein G (gG) ELISA, respectively 13.8 and 28.9% of mares, and 42.6 and 46.6% of foals were classed as responding to vaccination. Additionally, 16.4 and 17.6% of mares were classified as persistently seropositive mares. Using both assays, responder mares and foals had lower week 0 mean ELISA absorbances than non-responder mares and foals. Responder mares were ten times more likely to have responder foals, and non-responder mares were six times more likely to have non-responder foals than other mares using the gG ELISA. Mares aged 7 years or less and foals aged 4 months or more were more likely to respond to vaccination than animals in other age groups. There was no association between response of mares and the number of previous vaccinations received and persistently seropositive mares did not respond to vaccination. This study documents the responses of mares and foals to vaccination in a large scale commercial environment in 2000, and suggests that knowledge of antibody status may allow a more selective vaccination strategy, representing considerable savings to industry.     

Pre-infection frequencies of equine herpesvirus-1 specific, cytotoxic T lymphocytes correlate with protection against abortion following experimental infection of pregnant mares

In general, vaccines containing inactivated equine herpesvirus-1 (EHV-1) fail to prevent abortion in pregnant mares following infection with a virulent strain of EHV-1. We have tested the hypothesis that resistance to EHV-1-induced abortion in pregnant mares is associated with high frequencies of EHV-1 specific, major histocompatibility complex (MHC) class I-restricted, cytotoxic T lymphocytes (CTL) in the circulation. To test this theory, three groups of pregnant mares were assembled with varying backgrounds of infection or vaccination in an attempt to mimic the immune status of the general population. Group 1 mares (n=9) were untreated controls selected at random. Group 2 mares (n=5) were vaccinated three times intramuscularly with inactivated EHV-1. Group 3 mares (n=3) had been infected with EHV-1 on four previous occasions. The frequency of CTL in blood leucocytes was measured by limiting dilution analysis at three time points; at the beginning of pregnancy (approximately 28 weeks before infection) in the Group 2 and Group 3 mares (4-7 weeks of gestation) (Group 1 was unavailable for sampling) and then 2 weeks before (30-40 weeks of gestation) and 3 weeks after experimental infection in all the mares. Serum samples were collected to monitor complement fixing (CF) antibody titres. Mares in all three groups were infected experimentally with EHV-1 strain Ab4/8 by the intranasal route after which they were monitored clinically to determine the outcome of pregnancy and samples were collected to determine the duration of nasopharyngeal shedding and cell-associated viraemia. The untreated control mares showed low pre-infection CTL. After experimental infection, they all seroconverted, aborted and demonstrated expected clinical and virological signs. Some vaccinated mares (3/5) had elevated titres of CF antibody prior to their first vaccination. All the vaccinated mares seroconverted after vaccination and exhibited higher CTL frequencies than controls before infection. Four of the five foaled normally. The multiply infected mares had low CF antibody titres prior to infection and showed neither seroconversion nor clinical or virological signs after infection. All multiply infected mares exhibited high frequencies of CTL before infection and they all foaled normally. The CTL frequencies observed differed significantly from the expected frequencies in the control and multiply infected groups at 2 weeks pre-infection (P=0.034) and between the foaling and aborting mares at 2 weeks pre-infection (P=0.005) and 3 weeks post-infection (P=0.015). The results show a positive correlation between the number of virus-specific CTL in the peripheral blood of pregnant mares and their protection against abortion induced by EHV-1 infection. Therefore, as indicated by this study, rational approaches to the development of new vaccines for EHV-1 should stimulate cytotoxic immune responses and develop virus-specific CTL as pre-requisites for protection against abortion.     

Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals

The envelope glycoprotein D of equine herpesvirus 1 (EHV-1 gD) has been shown in laboratory animal models to elicit protective immune responses against EHV-1 challenge, and hence is a potential vaccine antigen. Here we report that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant Iscomatrix elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses. The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/4 whole virus vaccine. Intramuscular inoculation of EHV-1 gD DNA encoded in a mammalian expression vector was less effective in inducing antibody responses when administered as the sole immunogen, but inoculation with EHV-1 gD DNA followed by recombinant EHV-1 gD induced increased gD ELISA and virus-neutralizing antibody titres in six out of seven horses. However, these titres were not higher than those induced by either EHV-1 gD or the whole virus vaccine. Isotype analysis revealed elevated gD-specific equine IgGa and IgGb relative to IgGc, IgG(T) and IgA in horses inoculated with EHV-1 gD or with the whole virus vaccine. Following inoculation of pregnant mares with EHV-1 gD, their foals had significantly higher levels of colostrally derived anti-gD antibody than foals out of uninoculated mares. The EHV-1 gD preparation did not induce a significant mean antibody response in neonatal foals following inoculation at 12 h post-partum and at 30 days of age, irrespective of the antibody status of the mare. The ability of EHV-1 gD to evoke comparable neutralizing antibody responses in horses to those of a whole virus vaccine confirms EHV-1 gD as a promising candidate for inclusion in subunit vaccines against EHV-1.     

Stimulation of airway neutrophils following dexamethasone administration and equid herpesvirus-2 challenge in horses

The aim of this study was to investigate neutrophil stimulation following experimentally-induced airway inflammation in healthy horses. Six horses received dexamethasone and four were then inoculated with equid herpesvirus-2 (EHV-2). Significant neutrophilia was detected in tracheal wash and bronchoalveolar lavage fluid for up to 6 days. Concentrations of neutrophil elastase (NE) and myeloperoxidase (MPO) were significantly increased compared to baseline for up to 14 days in tracheal washes and both markers were significantly correlated with neutrophil counts. Serum levels of surfactant protein D were not significantly modified throughout the study. These results suggest that dexamethasone administration with or without EHV-2 inoculation is associated with a sustainable activation and degranulation of neutrophils in the trachea along with moderate modifications detectable in the lower airways.     

An outbreak of foal perinatal mortality due to equid herpesvirus type 1: pathological observations.

Twenty-nine cases of EHV1 infection occurred on a property, mainly in full term foals. Some foals were stillborn, some were born alive but weak and soon died and others were healthy at birth, became ill and died within 3 days of birth. Apart from voluminous, oedematous and atelectic lungs there were no gross lesions. Microscopically the lungs showed oedema, pneumonitis and bronchiolitis with intranuclear inclusions and, in many of the foals that survived over 6 hours, there was also hyaline membrane formation. Microscopic lesions were also seen in the liver, adrenal, thymus and spleen of some of these foals.