Study of the duration and distribution of equine influenza virus subtype 2 (H3N8) antigens in experimentally infected ponies in vivo.

The purpose of this experiment was to study the duration and distribution of equine influenza virus in actively infected ponies over a 3 wk period. Pony foals (6-8 mo old) were infected experimentally by nebulizing equine influenza subtype-2 virus ultrasonically through a face mask. Successful infection was clinically apparent as each of the foals (n = 6) had a febrile response, a deep hacking cough and mucopurulent nasal discharge for 7 to 10 d. The virus was isolated from nasopharyngeal swabs of all the ponies 3 and 5 d after infection and all the ponies seroconverted to the virus. Samples were taken from the nasopharynx, mid-trachea and the mainstem bronchus with cytology brushes through an endoscope as well as from bronchoalveolar lavage fluid. On days 3 to 7 post-infection, ciliacytophtorea (the presence of cilia and ciliated plates separated from columnar epithelial cells) was recognized on routine cytological stain. Indirect immunoperoxidase staining utilizing polyclonal antibodies demonstrated viral antigen in intact and fragmented ciliated epithelial cells and in fragments of ciliated plates. The infected cells and cell fragments were particularly evident on days 3 and 5 post-infection in the nasopharynx, mid-trachea and mainstem bronchus and on days 3 to 7 post-infection in the bronchoalveolar lavage samples. On days 7 and 21 post-infection, viral antigen was identified in vacuoles of alveolar macrophage-like cells collected by bronchoalveolar lavage. It can be concluded from this study that equine influenza virus can infect not only the upper airways but also the bronchial epithelium and that viral antigen can persist up to 21 d post-infection.     

Cytokine responses to EHV-1 infection in immune and non-immune ponies

Protecting equids against equine herpesvirus-1 (EHV-1) infection remains an elusive goal. Repeated infection with EHV-1 leads to protective immunity against clinical respiratory disease, and a study was conducted to measure the regulatory cytokine response (IFN-gamma and IL-4) in repeatedly infected immune ponies compared to non-immune ponies. Two groups of four ponies were established. Group 1 ponies had previously been infected on two occasions, and most recently 7 months before this study. Group 2 ponies had no history no vaccination or challenge infection prior to this study. Both groups were subjected to an intranasal challenge infection with EHV-1, and blood samples were collected pre-infection, and at 7 and 21 days post-infection for preparation of PBMCs. At each time point, the in vitro responses of PBMCs to stimulation with EHV-1 were measured, including IFN-gamma and IL-4 mRNA production, and lymphoproliferation. Group 1 ponies showed no signs of clinical disease or viral shedding after challenge infection. Group 2 ponies experienced a biphasic pyrexia, mucopurulent nasal discharge, and nasal shedding of virus after infection. Group 1 ponies had an immune response characterized both before and subsequent to challenge infection by an IFN-gamma response to EHV-1 in the absence of an IL-4 response, and demonstrated increased EHV-1-specific lymphoproliferation post-infection. Group 2 ponies had limited cytokine or lymphoproliferative responses to EHV-1 pre-challenge, and demonstrated increases in both IFN-gamma and IL-4 responses post-challenge, but without any lymphoproliferative response. Protective immunity to EHV-1 infection was therefore characterized by a polarized IFN-gamma dependent immunoregulatory cytokine response.     

Equine respiratory viruses in foals in New Zealand

AIMS: To identify the respiratory viruses that are present among foals in New Zealand and to establish the age at which foals first become infected with these viruses. METHODS: Foals were recruited to the study in October/ November 1995 at the age of 1 month (Group A) or in March/ April 1996 at the age of 4-6 months (Groups B and C). Nasal swabs and blood samples were collected at monthly intervals. Nasal swabs and peripheral blood leucocytes (PBL) harvested from heparinised blood samples were used for virus isolation; serum harvested from whole-blood samples was used for serological testing for the presence of antibodies against equine herpesvirus (EHV)-1 or -4, equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). Twelve foals were sampled until December 1996; the remaining 19 foals were lost from the study at various times prior to this date. RESULTS: The only viruses isolated were EHV-2 and EHV-5. EHV-2 was isolated from 155/157 PBL samples collected during the period of study and from 40/172 nasal swabs collected from 18 foals. All isolations from nasal swabs, except one, were made over a period of 2-4 months from January to April (Group A), March to April (Group B) or May to July (Group C). EHV-5 was isolated from either PBL, nasal swabs, or both, from 15 foals on 32 occasions. All foals were positive for antibodies to EHV-1 or EHV-4, as tested by serum neutralisation (SN), on at least one sampling occasion and all but one were positive for EHV-1 antibodies measured by enzyme-linked immunosorbent assay (ELISA) on at least one sampling occasion. Recent EHV-1 infection was evident at least once during the period of study in 18/23 (78%) foals for which at least two samples were collected. SN antibodies to ERBV were evident in 19/23 (83%) foals on at least one sampling occasion and 15/23 foals showed evidence of seroconversion to ERBV. Antibodies to ERAV were only detected in serum samples collected from foals in Group A and probably represented maternally-derived antibodies. Haemagglutination inhibition (HI) antibody titres 1:10 to EAdV-1were evident in 21/23 (91%) foals on at least one sampling occasion and 16/23 foals showed serological evidence of recent EAdV-1 infection. None of the 67 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. There was no clear association between infection with any of the viruses isolated or tested for and the presence of overt clinical signs of respiratory disease. CONCLUSIONS: There was serological and/or virological evidence that EHV-1, EHV-2, EHV-5, EAdV-1 and ERBV infections were present among foals in New Zealand. EHV-2 infection was first detected in foals as young as 3 months of age. The isolation of EHV-2 from nasal swabs preceded serological evidence of infection with other respiratory viruses, suggesting that EHV-2 may predispose foals to other viral infections.     

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Isolation of equine herpesvirus type 5 in New Zealand

AIM: To report the first isolation of equine herpesvirus 5 (EHV-5) in New Zealand as part of a study of equine respiratory viruses in New Zealand. METHODS: Nasal swabs and peripheral blood leukocytes were collected from 114 foals and adult horses, inoculated on to equine fetal kidney, rabbit kidney and Vero cell lines and observed for cytopathic effect. EHV-5 isolates were identified using an EHV-5 specific polymerase chain reaction. All samples positive for EHV-5 were also checked for the presence of EHV-2, EHV-1 or EHV-4 DNA using published type-specific primers. The polymerase chain reaction results were further confirmed by dot blot and Southern hybridisation with specific DIG-labelled probes. RESULTS: EHV-5 was isolated from nasal swabs or peripheral blood leukocytes of 38 out of 114 horses sampled. From horses sampled more than once, EHV-5 was often isolated on more than one occasion. Most of the horses were infected with both EHV-2 and EHV-5 viruses. It was not possible to make an association between EHV-5 isolation and the presence of respiratory disease. CONCLUSION: EHV-5 is present in the New Zealand horse population. The exact role it plays in causing, or predisposing to, respiratory disease remains to be elucidated.     

Prevalence of equine herpesvirus type 2 (EHV-2) DNA in ocular swabs and its cell tropism in equine conjunctiva

Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased horses were positive. To define the main virus reservoir ocular tissue from 13 randomly selected horses without pathological evidence of ocular disease were analysed by nested PCR. In two horses optic nerve, lacrimal gland and conjunctiva, in further two cases lacrimal gland and conjunctiva and in four horses the conjunctiva only were EHV-2 PCR positive. For specifying the target cell we focused on conjunctivae and selected 3 out of 15 clinically healthy slaughterhouse horses positive for EHV-2 by PCR. In situ hybridisation on sections of these paraffin embedded conjunctivae localized viral genome in histiocyte-like cells of the submucosa. Immunohistochemical staining with an EHV-2 or S100 specific polyclonal antiserum demonstrated that Langerhans cells were co-localized in the same region of the sample section where virus positive cells were detected. Furthermore, we concluded that detection of viral antigen revealed a productive virus infection.     

Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection

Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.     

Clinical, serological and molecular investigations of EHV-1 and EHV-4 in 15 unweaned thoroughbred foals

Fifteen unweaned thoroughbred foals, born on a stud farm to vaccinated mares, were clinically monitored during their first six months of life and repeatedly tested for equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4). Nasopharyngeal swabs and blood samples were collected and screened respectively by PCR and seroneutralisation to detect the presence of the virus, explore its role as a possible cause of respiratory disease, and to assess the efficiency of the pcr for the diagnosis of this disease. The foals were divided into three groups on the basis of their clinical signs and whether they had seroconverted to EHV-1 and/or EHV-4: first, foals with no clinical signs of disease that had not seroconverted; secondly, foals with clinical signs that had seroconverted, and thirdly, foals with clinical signs that had not seroconverted. The results indicated that the viruses circulated on the stud farm despite stringent vaccination regimens against them, and confirmed their association with respiratory disease. The absence of significantly different pcr results among the three groups of foals showed that the pcr was effective in confirming the circulation of the viruses on the premises without being particularly helpful as a diagnostic tool.     

Isolation of equine herpesvirus type 5 in New Zealand

Aim. To report the first isolation of equine herpesvirus 5 (EHV-5) in New Zealand as part of a study of equine respiratory viruses in New Zealand.

Methods. Nasal swabs and peripheral blood leukocytes were collected from 114 foals and adult horses, inoculated on to equine fetal kidney, rabbit kidney and Vero cell lines and observed for cytopathic effect. EHV-5 isolates were identified using an EHV-5 specific polymerase chain reaction. All samples positive for EHV-5 were also checked for the presence of EHV-2, EHV-1 or EHV-4 DNA using published type-specific primers. The polymerase chain reaction results were further confirmed by dot blot and Southern hybridisation with specific DIG-labelled probes.

Results. EHV-5 was isolated from nasal swabs or peripheral blood leukocytes of 38 out of 114 horses sampled. From horses sampled more than once, EHV-5 was often isolated on more than one occasion. Most of the horses were infected with both EHV-2 and EHV-5 viruses. It was not possible to make an association between EHV-5 isolation and the presence of respiratory disease.

Conclusion. EHV-5 is present in the New Zealand horse population. The exact role it plays in causing, or predisposing to, respiratory disease remains to be elucidated.     

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.     

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.     

Prevalence of equine herpesvirus-1 infection among Thoroughbreds residing on a farm on which the virus was endemic

OBJECTIVE: To determine the incidence of equine herpesvirus-1 (EHV-1) infection among Thoroughbreds residing on a farm on which the virus was known to be endemic. DESIGN: Prospective cohort study. ANIMALS: 10 nonpregnant mares, 8 stallions, 16 weanlings, 11 racehorses, and 30 pregnant mares and their foals born during the 2006 foaling season. PROCEDURES: Blood and nasopharygeal swab samples were collected every 3 to 5 weeks for 9 months, and placenta and colostrum samples were collected at foaling. All samples were submitted for testing for EHV-1 DNA with a PCR assay. A type-specific EHV-1 ELISA was used to determine antibody titers in mares and foals at birth, 12 to 24 hours after birth, and every 3 to 5 weeks thereafter. RESULTS: Results of the PCR assay were positive for only 4 of the 1,330 samples collected (590 blood samples, 590 nasopharyngeal swab samples, 30 placentas, and 30 colostrum samples), with EHV-1 DNA detected in nasal secretions from 3 horses (pregnant mare, stallion, and racehorse) and in the placenta from 1 mare. Seroconversion was detected in 3 of 27 foals during the first month of life. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that there was a low prevalence of EHV-1 infection among this population of Thoroughbreds even though the virus was known to be endemic on the farm and that pregnant mares could become infected without aborting. Analysis of nasopharyngeal swab samples appeared to be more sensitive than analysis of blood samples for detection of EHV-1 DNA.     

Absence of viral envelope proteins in equine herpesvirus 1-infected blood mononuclear cells during cell-associated viremia

In vitro studies demonstrated that most equine herpesvirus 1 (EHV-1)-infected peripheral blood mononuclear cells (PBMC) do not expose viral envelope proteins on their surface. This protects them against antibody-dependent lysis. We examined whether viral envelope proteins are also undetectable on infected PBMC during cell-associated viremia. Further, surface expression of major histocompatibility complex (MHC)-I was examined, since MHC-I assists in making infected cells recognizable for cytotoxic T-lymphocytes (CTL). Four ponies, previously exposed to EHV, and two ponies that had no contact with EHV before, were inoculated with EHV-1. PBMC were collected at different time points up to 28 days post inoculation. Ninety-eight percent of the infected PBMC did not show viral envelope proteins on their surface. Moreover, infected PBMC without surface expression only produced immediate early and, at least, one early protein, ICP22, but not late envelope proteins gB and gM. This indicates that surface expression of viral envelope proteins is absent, simply because the PBMC are in an early phase of infection. The percentage of infected PBMC showing surface expression of MHC-I was similar as observed in non-infected PBMC from the same ponies (80-100%). Therefore, inefficient recognition of EHV-1-infected PBMC by CTLs does not arise from absent surface expression of MHC-I.     

Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination

In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. Two to 3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system.     

Clinical, haematological and biochemical findings in foals with neonatal Equine herpesvirus-1 infection compared with septic and premature foals.

A retrospective multicentre study comparing historical, clinical, haematological, acid-base and biochemical findings of foals with Equine herpesvirus-1 (EHV-1) infection, septicaemia or prematurity was performed to determine if early diagnosis of EHV-1 foals was possible. Fifty-three foals were studied and were assigned to one of 2 groups: herpes positive (n = 14) or herpes negative (n = 39). The latter group included 20 septic, 11 premature, and 8 premature and septic foals. The presence of herpes antigen was confirmed by immunoperoxidase histochemical staining of tissues from necropsied foals. A nonparametric statistical analysis followed by a backwards elimination logistic regression was performed to establish a model at a P value of <0.05. All herpes positive foals died, while 47% (9/19) of the septic foals survived. Based upon our analysis, herpes positive foals were more likely to have total white blood cell counts less than 3 x 10(9)/l and to be icteric as compared to the septic and premature foals. Despite profound hepatic necrosis in the herpes positive foals, liver enzymes were not elevated and were not significantly different from the controls.     

Pathological findings in horses dying during an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection.

In 1988 an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection occurred on a stud farm and several animals died. This provided an opportunity to perform detailed pathological investigations to gain insights into the pathogenesis of this spontaneous disease. Two paretic mares, three foals, an aborted foetus and its non-paretic dam were examined. The endotheliotropism of the virus was clearly demonstrated by the use of an indirect immunoperoxidase (IP) stain. At autopsy, evidence of viral infection was widespread in the foetus and foals, but limited or absent in the mares, probably reflecting differences in their immune status. Vascular lesions were present in the central nervous system (CNS) of the foals as well as the adults; they resulted in minimal neural lesions in the foals. Severe changes in the upper and lower respiratory tracts were a particular feature in the foals, two of which exhibited extensive vasculitis and thrombosis in the lungs. The IP technique was of great value in locating antigen-containing cells in the CNS of one mare when virus isolation was negative. It also revealed the presence of virus in less well documented sites such as the pancreas, gut, thyroid, uveal tract and the skin of the nares.     

Antibody and cellular immune responses following DNA vaccination and EHV-1 infection of ponies

Equine herpesvirus-1 (EHV-1) is the cause of serious disease with high economic impact on the horse industry, as outbreaks of EHV-1 disease occur every year despite the frequent use of vaccines. Cytotoxic T-lymphocytes (CTLs) are important for protection from primary and reactivating latent EHV-1 infection. DNA vaccination is a powerful technique for stimulating CTLs, and the aim of this study was to assess antibody and cellular immune responses and protection resulting from DNA vaccination of ponies with combinations of EHV-1 genes. Fifteen ponies were divided into three groups of five ponies each. Two vaccination groups were DNA vaccinated on four different occasions with combinations of plasmids encoding the gB, gC, and gD glycoproteins or plasmids encoding the immediate early (IE) and early proteins (UL5) of EHV-1, using the PowderJect XR research device. Total dose of DNA/plasmid/vaccination were 25 microg. A third group comprised unvaccinated control ponies. All ponies were challenge infected with EHV-1 6 weeks after the last vaccination, and protection from clinical disease, viral shedding, and viremia was determined. Virus neutralizing antibodies and isotype specific antibody responses against whole EHV-1 did not increase in either vaccination group in response to vaccination. However, glycoprotein gene vaccinated ponies showed gD and gC specific antibody responses. Vaccination did not affect EHV-1 specific lymphoproliferative or CTL responses. Following challenge infection with EHV-1, ponies in all three groups showed clinical signs of disease. EHV-1 specific CTLs, proliferative responses, and antibody responses increased significantly in all three groups following challenge infection. In summary, particle-mediated EHV-1 DNA vaccination induced limited immune responses and protection. Future vaccination strategies must focus on generating stronger CTL responses.