Detection of EHV-1 and EHV-4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm

REASONS FOR PERFORMING STUDY: A silent cycle of equine herpesvirus 1 infection has been described following epidemiological studies in unvaccinated mares and foals. In 1997, an inactivated whole virus EHV-1 and EHV-4 vaccine was released commercially in Australia and used on many stud farms. However, it was not known what effect vaccination might have on the cycle of infection of EHV-1. OBJECTIVE: To investigate whether EHV-1 and EHV-4 could be detected in young foals from vaccinated mares. METHODS: Nasal and blood samples were tested by PCR and ELISA after collection from 237 unvaccinated, unweaned foals and vaccinated and nonvaccinated mares during the breeding season of 2000. RESULTS: EHV-1 and EHV-4 DNA was detected in nasal swab samples from foals as young as age 11 days. CONCLUSIONS: These results confirm that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned, unvaccinated foals. POTENTIAL RELEVANCE: The evidence that the cycle of EHV-1 and EHV-4 infection is continuing and that very young foals are becoming infected should assist stud farms in their management of the threat posed by these viruses.     

Epidemiology of EHV-1 and EHV-4 in the mare and foal populations on a Hunter Valley stud farm: are mares the source of EHV-1 for unweaned foals.

The prevalence of EHV-1 and EHV-4 antibody-positive horses was determined using a type specific ELISA on serum samples collected from 229 mares and their foals resident on a large Thoroughbred stud farm in the Hunter Valley of New South Wales in February 1995. More than 99% of all mares and foals tested were EHV-4 antibody positive, while the prevalence of EHV-1 antibody positive mares and foals were 26.2 and 11.4%, respectively. Examination of the ELISA absorbance data for the individual mares and foals suggested that the EHV-1 antibody positive foals had been infected recently with EHV-1 and that a sub-group of the mare population was the likely source of infectious virus for the unweaned foals.     

Seroprevalence of equine herpesvirus 1 in Thoroughbred foals before and after weaning

Objective To investigate the seroprevalence of equine herpesvirus 1 in foals around weaning and after weaning on two large Thoroughbred farms using a type-specific enzyme-linked immunosorbent assay to determine exposure to infection.
Design A longitudinal population study in groups of Thoroughbred weanling foals.
Study population Two hundred weanling Thoroughbred foals from a population of about 380 foals were enrolled on two adjacent stud farms in the Hunter Valley of New South Wales. Foals on both farms were weaned from February to May 1995 into randomly selected groups of 10 to 15 foals. Farms were selected because of their willingness to cooperate in the survey and because their detailed records of foals and their movements. They were representative of well-managed large Thoroughbred stud farms in New South Wales. Both studs had upper respiratory tract disease among weanling foals around weaning each year although the sero-prevalence of viral respiratory disease on either farm was not known before the study.
Procedure Serum was collected from foals within each group at fortnightly intervals from 9th February until 1st June 1995, and at a single follow-up period in August 1995. Each sample was tested in triplicate using an antibody-detection ELISA which is type-specific for EHV-1 and EHV-4 antibodies.
Results and conclusions There was serological evidence of EHV-1 infection both before and after weaning. The prevalence of EHV-1 antibody in the sample population increased during the study and individual cases of EHV-1 infection were identified. The increase was caused both by the seroconversion of foals within the groups and by the recruitment into the study of foals with pre-existing EHV-1 antibody. Evidence of EHV-1 infection in Thoroughbred foals after weaning has not been reported previously in Australia and this has implications for vaccination regimens.     

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.     

Temporal detection of equine herpesvirus infections of a cohort of mares and their foals

The objectives of this study were to estimate the prevalence of equine herpesviruses (EHV) 1-5 in the nasal secretions (NS) of a cohort of 12 mares and their foals from birth to 6 months of age, estimate the prevalence of EHV-1-5 infection of peripheral blood mononuclear cells (PBMC) of selected foals, and investigate phylogenetic relationships amongst the various strains of EHV-2 and 5. Virus-specific PCR assays were used to detect EHV-1-5 in NS and PBMC. A homologous portion of the glycoprotein B (gB) gene of the various strains of EHV-2 and 5 was sequenced and compared. EHV-2, 4, and 5 were all detected in NS from the horses, but only EHV-4 was associated with respiratory disease (P=0.005). EHV-2 and 5 infections were both common, but foals shed EHV-2 in their NS earlier in life than EHV-5 (P=0.01). Latent EHV-2 and 5 infections were detected in the PBMC of 75 and 88%, respectively, of the foals at approximately 6 months of age. The strains of EHV-2 shed in the NS of individual horses were more genetically heterogeneous than the strains of EHV-5 (95.5-99.3% versus 98.8-99.3% nucleotide identity, respectively). One-month-old foals typically shed strains of EHV-2 that were identical to those infecting their dams whereas older foals often shed virus strains that were different from those of their dams. Although herpesvirus infections were ubiquitous in this cohort of horses, there were distinct clinical consequences and clear epidemiological differences between infections with the different viruses.     

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.     

Epidemiological studies of equine herpesvirus 1 (EHV-1) in Thoroughbred foals: a review of studies conducted in the Hunter Valley of New South Wales between 1995 and 1997.

Sero-epidemiological studies conducted between 1995 and 1997 on two large Thoroughbred stud farms in the Hunter Valley of NSW showed clear evidence of EHV-1 infection in foals as young as 30 days of age. Similarly, serological evidence suggested that these foals were infected with EHV-1 from their dams or from other lactating mares in the group, with subsequent foal to foal spread of infection prior to weaning. These studies also provided evidence of EHV-1 infection of foals at and subsequent to weaning, with foal to foal spread of EHV-1 amongst the weanlings. These data indicated that the mare and foal population was a reservoir of EHV-1, from which new cases of infection propagated through the foal population both before and after weaning. The results of these studies support the long standing management practices of separating pregnant mares from other groups of horses to reduce the incidence of EHV-1 abortion. Also, these results have important implications for currently recommended vaccination regimens, as the efficacy of vaccination in already latently infected horses is unknown.     

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.     

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.     

Serological responses and clinical outcome after vaccination of mares and foals with equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) vaccines

Equine herpesvirus type 1 and type 4 (EHV-1 and EHV-4) cause infections of horses worldwide. While both EHV-1 and EHV-4 cause respiratory disease, abortion and myeloencephalopathy are observed after infection with EHV-1 in the vast majority of cases. Disease control is achieved by hygiene measures that include immunization with either inactivated or modified live virus (MLV) vaccine preparations. We here compared the efficacy of commercially available vaccines, an EHV-1/EHV-4 inactivated combination and an MLV vaccine, with respect to induction of humoral responses and protection of clinical disease (abortion) in pregnant mares and foals on a large stud with a total of approximately 3500 horses. The MLV vaccine was administered twice during pregnancy (months 5 and 8 of gestation) to 383 mares (49.4%), while the inactivated vaccine was administered three times (months 5, 7, and 9) to 392 mares (50.6%). From the vaccinated mares, 192 (MLV) and 150 (inactivated) were randomly selected for serological analyses. There was no significant difference between the groups with respect to magnitude or duration of the humoral responses as assessed by serum neutralization assays (median range from 1:42 to 1:130) and probing for EHV-1-specific IgG isotypes, although neutralizing responses were higher in animals vaccinated with the MLV preparation at all time points sampled. The total number of abortions in the study population was 55/775 (7.1%), 9 of which were attributed to EHV-1. Seven of the abortions were in the inactivated and two in the MLV vaccine group (p=0.16). When foals of vaccinated mares were followed up, a dramatic drop of serum neutralizing titers (median below 1:8) was observed in all groups, indicating that the half-life of maternally derived antibody is less than 4 weeks.     

Nasal bacterial microbiota during an outbreak of equine herpesvirus 1 at a farm in southern Ontario

The objective of this study was to investigate the nasal bacterial microbiota of healthy horses and horses infected with equine herpesvirus 1 (EHV-1). The nasal bacterial microbiota of 10 horses infected with EHV-1 and 11 control horses from a farm experiencing an outbreak was characterized using the Illumina MiSeq platform targeting the V4 region of the 16S ribosomal RNA gene. The nasal bacterial microbiota of healthy horses and EHV-1 horses was significantly different in community membership and structure. Horses shedding EHV-1 had lower bacterial richness (P = 0.002), evenness (P = 0.008), and diversity (P = 0.026) than healthy horses. Healthy horses had a higher relative abundance of Firmicutes and Bacteroidetes, but lower Proteobacteria than horses with EHV-1 (P < 0.05). This study provides the basis for generating hypotheses and investigations on the role of bacterial-viral interactions in the health and diseases of adult horses.     

Relevance of infection with equine herpesvirus 1 (EHV-1) in a German thoroughbred stud: vaccination, abortion and diagnosis

The aim of the present study was to clarify whether an EHV-1 induced abortion can be prognosticated by an increase of antibody titres, virus shedding and/or viraemia and whether the current abortion diagnostic is suitable. In this context the immune response post immunization and a possible reactivation were of great interest. For this purpose blood samples of 32 mares between the ages of 5-21 years were regularly investigated during a period of two years before and after vaccination and pregnancy. Neutralization tests, indirect immunofluorescence tests as well as PCR and virus isolation were used for EHV-1 diagnostics. It could be shown that the horses reacted individually to vaccination. In 14 cases a EHV-1-reactivation was suggested. An abortion prognosis was not possible even using serological, virological and molecular biological parameters. In addition, virus shedding and antibody titres were individual. An acute infection was detectable by a significant rise of antibodies and viraemia as well as virus shedding in the secretions. For the abortion diagnostics the antigen detection in combination with virus isolation and PCR from fetal lungs gave reliable results. In addition, the virological and serological investigation of the mare is recommendable. For prophylaxis we would advise a regular vaccination and strict hygiene.     

Parascaris equorum in foals and in their environment on a Swedish stud farm, with notes on treatment failure of ivermectin

Environmental contamination and the egg excretion pattern of the ascarid Parascaris equorum (Nematoda) was investigated in relation to anthelmintic treatment on a Swedish stud farm. Faecal samples from 15 foals, dewormed every 8th-week with a paste formulation of ivermectin at the standard dose rate of 0.2 mg/kg bodyweight, were collected at five sampling occasions between August and November 2006. In addition, soil samples were obtained from four paddocks used by these foals in November 2006. The number of eggs per gram (epg) was counted in both faeces and soil. Egg excretion started when the foals were 3-4 months, and reached the highest levels when they were approximately 5-month-old, and was then followed by a decline. Egg excretion seemed to be unaffected by ivermectin despite these foals were dewormed at regular intervals. In four out of five foals examined 10 days after treatment, epg actually increased. In contrast, when either fenbendazol or pyrantel embonate were used instead of ivermectin, treatments were effective. The number of eggs in soil was significantly higher in the permanent paddock compared to in the temporarily used soil paddock and in the summer paddocks.     

Functional and ultrastructural changes in neutrophils from mares and foals experimentally inoculated with a respiratory tract strain of equine herpesvirus-1

Neutrophils isolated from venous blood of adult and foal ponies inoculated with equine herpesvirus-1 were evaluated by in vitro function tests and by electron microscopy. Foals had fever and severe neutropenia 24 hours after inoculation; increased neutrophil random migration under agarose and decreased antibody-dependent cell-mediated cytotoxicity were significant at 24 hours, but values had returned to preinoculation levels by 72 hours. Mares had fever and leukopenia of less severity, increases in neutrophil migration, and longer persistence of primary granule release than were seen in foals. Reduced migration and degranulation, and a decrease in antibody-dependent cell-mediated cytotoxicity seen with neutrophils from foals, as compared with mares, may relate to the high susceptibility of foals to equine herpesvirus-1 infection.     

Respiratory disease in foals and the epizootiology of equine herpesvirus type 2 infection

The epizootiology of equine herpesvirus type 2 (EHV-2) infection was investigated in Thoroughbred foals on a stud farm which in previous years had suffered economic loss due to respiratory disease. Sixteen pairs of foals and their dams were selected for this study and all of the foals became infected with EHV-2 by two to four months of age. These animals responded serologically to the virus infection as detected by an enzyme-linked immunosorbent assay (ELISA). EHV-2 infection persisted in these foals for two to six months with constant or intermittent virus recovery. This persistent infection stimulated continuous production of antibodies against EHV-2. As soon as the antibody levels reached their peak at five to six months, the isolation rate of EHV-2 from the nasal cavity of these animals decreased, and eventually by nine months of age virus could no longer be recovered. Respiratory disease was observed in ten of the 16 foals; and two severely affected animals died at two months of age. EHV-2 was isolated from both foals at ante and/or post mortem examination. It is postulated that EHV-2, either as an initiating agent or by means of immnunosuppression, caused the respiratory disease observed in these foals.     

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.     

Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus.

Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.