Nasal shedding of equine herpesvirus-1 from horses in an outbreak of equine herpes myeloencephalopathy in Western Canada.

BACKGROUND: There is little information on the duration of nasal shedding of EHV-1 from horses with naturally occurring equine herpesvirus myeloencephalopathy (EHM). OBJECTIVES: To evaluate the duration of nasal shedding of EHV-1 in horses affected by EHM. ANIMALS: One hundred and four horses naturally exposed to EHV-1, 20 of which had clinical signs of EHM. METHODS: All horses on affected premises were monitored. Those horses developing EHM were sampled in a longitudinal outbreak investigation. Nasal swabs were collected daily from 16 of 20 horses affected by EHM. A qPCR was performed on 98 of 246 nasal swab samples to determine nasal shedding duration. Historical and clinical information was analyzed to evaluate potential risk factors for developing EHM and duration of shedding during this outbreak. RESULTS: The last day shedding was detected in any horse was Disease Day 9. EHV-1 was detected in two-thirds of horses tested on Disease Days 0-3. The amount of EHV-1 DNA found in nasal swabs varied markedly and was not associated with disease severity or age. The odds of developing EHM were greater for febrile horses (OR = 20.3; 95% CI 3.4-390.3; P = .01) as well as for horses attending the riding clinic (OR = 4.1; 95% CI 0.84-21.65; P = .08). CONCLUSIONS AND CLINICAL IMPORTANCE: Biosecurity measures should be implemented for a minimum of 14 days beyond the onset of clinical signs of EHM. Animal managers cannot rely on the severity of clinical signs to predict the duration of EHV-1 shedding.

Cardiac troponin I concentrations in ponies challenged with equine influenza virus.

BACKGROUND: Myocarditis is thought to occur secondary to equine influenza virus (EIV) infections in horses, but there is a lack of published evidence. HYPOTHESIS/OBJECTIVES: We proposed that EIV challenge infection in ponies would cause myocardial damage, detectable by increases in plasma cardiac troponin I (cTnI) concentrations. ANIMALS: Twenty-nine influenza-naïve yearling ponies: 23 were part of an influenza vaccine study (11 unvaccinated and 12 vaccinated), and were challenged with 108 EID50 EIV A/eq/Kentucky/91 6 months after vaccination. Six age-matched healthy and unvaccinated ponies concurrently housed in a separate facility not exposed to influenza served as controls. METHODS: Heparinized blood was collected before and over 28 days after infection and cTnI determined. Repeated measures analysis of variance, chi-square, or clustered regression analyses were used to identify relationships between each group and cTnI. RESULTS: All EIV-infected ponies developed clinical signs and viral shedding, with the unvaccinated group displaying severe signs. One vaccinated pony and 2 unvaccinated ponies had cTnI greater than the reference range at 1 time point. At all other times, cTnI was < 0.05 ng/mL. All control ponies had normal cTnI. There were no significant associations between cTnI and either clinical signs or experimental groups. When separated into abnormal versus normal cTnI, there were no significant differences among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrated no evidence of severe myocardial necrosis secondary to EIV challenge with 108 EID50 EIV A/eq/Kentucky/91 in these sedentary ponies, but transient increases in cTnI suggest that mild myocardial damage may occur.

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia.

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for ß-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.

Molecular investigation of the viral kinetics of equine herpesvirus-1 in blood and nasal secretions of horses after corticosteroid-induced recrudescence of latent infection.

BACKGROUND: Recrudescence of latent equine herpesvirus 1 (EHV-1) with subsequent viral shedding via nasal secretions is a potential source of infection for susceptible horses and has been implicated in outbreaks occurring in closed populations. OBJECTIVES: To describe the viral kinetics of reactivated EHV-1 in blood and nasal secretions from latently infected horses after administration of corticosteroids, and to study the infectious nature of reactivated EHV-1 to sentinel horses. ANIMALS: Eight healthy horses. METHODS: Four horses infected 4 months previously with EHV-1 received dexamethasone on 5 consecutive days. Four seronegative horses served as sentinels and had direct contact with the latently infected horses. All horses were monitored daily for development of clinical signs. Whole blood and nasal secretions were collected daily for molecular detection and cell culture of EHV-1. Serum was collected weekly for the detection of antibodies against EHV-1. RESULTS: All horses in the latently infected group showed transient molecular detection of EHV-1 in blood and nasal secretions, but only 1 horse developed fever. Three latently infected horses developed an increase in antibody concentrations against EHV-l. Viral cultures remained negative for all latently infected horses after corticosteroid administration. None of the sentinel horses developed clinical signs, viremia, viral shedding, or seroconversion. CONCLUSIONS AND CLINICAL IMPORTANCE: EHV-1 was successfully reactivated after corticosteroid administration in latently infected horses. However, transmission of reactivated virus to sentinel horses was unsuccessful. Failure to effectively transmit EHV-1 to susceptible horses may have resulted from the low level and short period of viral shedding in latently infected horses.

Control of EHV-1 viremia and nasal shedding by commercial vaccines.

Equine herpesvirus-1 is a cause of outbreaks of abortion and neurological disease. The pathogenesis of both these diseases depends on establishment of viremia. An experiment was performed to determine the protective efficacy of two commercially available vaccines used with an optimized 3-dose vaccination regime: a modified-live viral (MLV) and a high antigen load killed vaccine licensed for abortion control. The study design was a blinded, randomized challenge trial. Three groups of 8 yearling ponies received one of three treatments: MLV vaccine (Rhinomune, Boehringer Ingelheim Vetmedica, Inc.); killed vaccine (Pneumabort-K, Pfizer Animal Health); or a placebo (control group). Three vaccinations were administered at intervals of 27 and 70 days followed by challenge infection 24 days later. Clinical disease after challenge was significantly reduced in both vaccine groups; the reduction was greater in the MLV vaccine group. Nasal shedding was reduced by at least 1-2 logs in both vaccine groups. The number of days of viremia was significantly reduced in the killed vaccine group only. This study demonstrated that both commercial vaccines significantly suppressed EHV-1 disease and nasal viral shedding, and one vaccine suppressed days of viremia.

Vaccination of ponies with the IE gene of EHV-1 in a recombinant modified live vaccinia vector protects against clinical and virological disease.

The control of EHV-1 infection by cytotoxic T-cell responses (CTL) via a reduction in cell associated viremia remains an important goal in horses. Unfortunately, current vaccines are inefficient at inducing these responses. We have identified the immediate early (IE) gene of EHV-1 as a potent stimulator of virus-specific CTL responses in ponies expressing a specific MHC class I serological haplotype (A3/B2). This study was designed to determine if vaccination of A3/B2 MHC I positive ponies with the IE gene could induce protection and immune responses associated with cell mediated immunity. Ponies expressing the MHC-I A3/B2 haplotype (A3/B2 vaccinates) and ponies with a different MHC I haplotype (either non-A3 vaccinates or A3-non-B2 vaccinates) were vaccinated with a recombinant modified vaccinia Ankara (rMVA) vector expressing the IE gene on 3 occasions and vaccinates and unvaccinated controls were challenge infected 8 weeks after the last vaccination. Interferon gamma (IFN-gamma) mRNA and antibody titers were determined throughout the study and clinical signs, nasal virus shedding and viremia were determined following challenge infection. Vaccination of A3/B2 vaccinates conferred significant clinical protection and a significant reduction in EHV-1 viremia. IFN-gamma mRNA increased significantly following vaccination in the A3/B2 vaccinates. Antibody titers remained low until after challenge infection, indicating that no accidental field acquired or recrudescent EHV-1 infection had occurred. In summary, this is an important study showing that vaccination of ponies with the EHV-1 IE protein provides not only reduction in clinical disease but also reduction of cell associated viremia, which is a prerequisite for the prevention of abortion and neurological disease.

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.