Stephen B. Hussey

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.

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.

Evaluation of immune responses following infection of ponies with an EHV-1 ORF1/2 deletion mutant

Equine herpesvirus-1 (EHV-1) infection remains a significant problem despite the widespread use of vaccines. The inability to generate a protective immune response to EHV-1 vaccination or infection is thought to be due to immunomodulatory properties of the virus, and the ORF1 and ORF2 gene products have been hypothesized as potential candidates with immunoregulatory properties. A pony infection study was performed to define immune responses to EHV-1, and to determine if an EHV-1 ORF1/2 deletion mutant (ΔORF1/2) would have different disease and immunoregulatory effects compared to wild type EHV-1 (WT). Infection with either virus led to cytokine responses that coincided with the course of clinical disease, particularly the biphasic pyrexia, which correlates with respiratory disease and viremia, respectively. Similarly, both viruses caused suppression of proliferative T-cell responses on day 7 post infection (pi). The ΔORF1/ORF2 virus caused significantly shorter primary pyrexia and significantly reduced nasal shedding, and an attenuated decrease in PBMC IL-8 as well as increased Tbet responses compared to WT-infected ponies. In conclusion, our findings are (i) that infection of ponies with EHV-1 leads to modulation of immune responses, which are correlated with disease pathogenesis, and (ii) that the ORF1/2 genes are of importance for disease outcome and modulation of cytokine responses.

Onset and duration of immunity to equine influenza virus resulting from canarypox-vectored (ALVAC) vaccination.

Equine influenza virus remains an important problem in horses despite extensive use of vaccination. Efficacy of equine influenza vaccination depends on the onset and duration of protective immunity, and appropriate strain specificity of the immune response. This study was designed to test the protective immunity resulting from vaccination with the North American commercial ALVAC equine influenza vaccine (RECOMBITEK Influenza, Merial, USA)(1) against challenge with American lineage influenza viruses. In experiment 1, 12 ponies were vaccinated twice, at a 35 day interval, using the ALVAC-influenza vaccine expressing the HA genes of influenza A/eq/Newmarket/2/93 and A/eq/Kentucky/94 (H3N8), and 11 ponies served as unvaccinated controls. Six months after the second vaccination, all ponies were challenged with A/eq/Kentucky/91. In experiment 2, 10 ponies received one dose of the ALVAC-influenza vaccine, 10 ponies served as unvaccinated controls, and all ponies were challenge infected with A/equine/Ohio/03, 14 days after vaccination. Parameters studied included serological responses, and clinical disease and nasal viral shedding following challenge infection. In experiment 1, following the two-dose regimen, vaccinated ponies generated high titered anti-influenza virus IgGa and IgGb antibody responses to vaccination and demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. Infection with A/eq/Kentucky/91 produced unusually severe signs in ponies in the control group, requiring therapy with NSAIDs and antibiotics, and leading to the euthanasia of one pony. In experiment 2 following the one-dose regimen, vaccinates generated IgGa responses pre-challenge, and anamnestic IgGa and IgGb responses after challenge. Vaccinates demonstrated statistically significant clinical and virological protection to challenge infection compared to controls. The results of this study clearly demonstrate the early onset, and 6-month duration of protective immunity resulting from ALVAC-influenza vaccination against challenge with American lineage equine influenza viruses.

Comparison of four methods to quantify Equid herpesvirus 1 load by real-time polymerase chain reaction in nasal secretions of experimentally and naturally infected horses

The objective of the current study was to compare the performance of 4 methods to quantify Equid herpesvirus 1 (EHV-1) by real-time polymerase chain reaction (PCR) in nasal secretions from experimentally and naturally infected horses. Nasal secretions were collected on the challenge day and daily thereafter for 13 days from 4 experimentally infected horses. Additional nasal swabs were collected from 30 horses with clinical signs consistent with natural EHV-1 infection. Absolute quantitation of EHV-1 target molecules was performed using standard curves for EHV-1 and equine glyceraldehyde-3-phosphate dehydrogenase, and DNA yield, and was expressed as EHV-1 glycoprotein B (gB) gene copies per million nucleated nasal cells, EHV-1 gB gene copies per entire swab, EHV-1 gB gene copies per 1 μl of purified DNA, and EHV-1 gB gene copies per 1 ng of template DNA. The study results showed that all 4 calculation methods yielded comparable results between experimentally and naturally infected horses, and that the different methods were significantly correlated with each other. Reporting of quantitative results for EHV-1 viral load in nasal swabs collected from infected horses constitutes an important advance in both the research and diagnostic fields, allowing one to determine the infectious risk of affected horses, disease stage, or response to antiviral therapy. However, protocols that normalize the PCR results against a preselected volume of DNA or nasal secretions are likely to be more prone to variations than protocols that calculate the load for the entire swab, incorporate a housekeeping gene, or use a constant amount of extracted DNA.

Strain impact on equine herpesvirus type 1 (EHV-1) abortion models: viral loads in fetal and placental tissues and foals.

Equine herpesvirus-1 (EHV-1) continues to cause both sporadic and epidemic abortions despite extensive vaccination. Lack of progress in the development of protective vaccines may be hindered by the lack of equine abortion models that employ contemporary EHV-1 strains. The objective of our experiments was to compare a contemporary EHV-1 strain with a previously described challenge strain, and to quantify EHV-1 loads in various maternal and fetal tissues. Infection experiments were performed in two groups of 7 pregnant pony mares at 270-290 days of gestation with a contemporary EHV-1 strain (University of Findlay 2003 isolate - OH03) or an EHV-1 strain isolated over 30 years ago, and previously described in abortion models (Ab4). All mares in both groups exhibited nasal viral shedding and viremia. Infection with OH03 resulted in 1/7 abortion and infection with Ab4 resulted in 5/7 abortions. In the OH03 challenge, placentas of foals delivered at term showed little detectable virus, while the aborted fetus expressed high levels of virus infection in the spleen and liver, lower levels in the lung and thymus, and lowest levels in the chorioallantois. After Ab4 challenge, high viral loads were detected in fetal and placental tissues in abortions. In the two normal deliveries, the chorioallantois contained virus levels comparable with the chorioallantois of aborted foals and both foals shed EHV-1 starting on day 4 of life, but were clinically healthy. Our results demonstrate the continued importance of strain selection for abortion models, and this study is the first report of viral load quantification using contemporary methods. Extremely high EHV-1 loads in decidua from abortions illustrate the infection risk posed to other horses.

Evaluation of infectivity of a canine lineage H3N8 influenza A virus in ponies and in primary equine respiratory epithelial cells

OBJECTIVE To evaluate whether an equine-derived canine H3N8 influenza A virus was capable of infecting and transmitting disease to ponies. ANIMALS 20 influenza virus-seronegative 12- to 24-month-old ponies. PROCEDURES 5 ponies were inoculated via aerosol exposure with 10(7) TCID(50) of A/Canine/Wyoming/86033/07 virus (Ca/WY)/pony. A second group of 5 ponies (positive control group) was inoculated via aerosol exposure with a contemporary A/Eq/Colorado/10/07 virus (Eq/CO), and 4 sham-inoculated ponies served as a negative control group. To evaluate the potential for virus transmission, ponies (3/inoculation group) were introduced 2 days after aerosol exposure and housed with Ca/WY- and Eq/CO-inoculated ponies to serve as sentinel animals. Clinical signs, nasal virus shedding, and serologic responses to inoculation were monitored in all ponies for up to 21 days after viral inoculation. Growth and infection characteristics of viruses were examined by use of Madin-Darby canine kidney cells and primary equine and canine respiratory epithelial cells. RESULTS Ponies inoculated with Ca/WY had mild changes in clinical appearance, compared with results for Eq/CO-inoculated ponies. Additionally, Ca/WY inoculation induced significantly lower numbers for copies of the matrix gene in nasal secretions and lower systemic antibody responses in ponies than did Eq/CO inoculation. The Ca/WY isolate was not transmitted to sentinel ponies. CONCLUSIONS AND CLINICAL RELEVANCE Inoculation of ponies with the canine H3N8 isolate resulted in mild clinical disease, minimal nasal virus shedding, and weak systemic antibody responses, compared with responses after inoculation with the equine H3N8 influenza isolate. These results suggested that Ca/WY has not maintained infectivity for ponies.

Comparison of temperature readings from a percutaneous thermal sensing microchip with temperature readings from a digital rectal thermometer in equids

OBJECTIVE To compare temperature readings from an implantable percutaneous thermal sensing microchip with temperature readings from a digital rectal thermometer, to identify factors that affect microchip readings, and to estimate the sensitivity and specificity of the microchip for fever detection. DESIGN Prospective study. ANIMALS 52 Welsh pony foals that were 6 to 10 months old and 30 Quarter Horses that were 2 years old. PROCEDURES Data were collected in summer, winter, and fall in groups 1 (n = 23 ponies), 2 (29 ponies), and 3 (30 Quarter Horses), respectively. Temperature readings from a digital rectal thermometer and a percutaneous thermal sensing microchip as well as ambient temperature were recorded daily for 17, 23, and 19 days in groups 1 through 3, respectively. Effects of ambient temperature and rectal temperature on thermal sensor readings were estimated. Sensitivity and specificity of the thermal sensor for detection of fever (rectal temperature, >or= 38.9 degrees C [102 degrees F]) were estimated separately for data collection at ambient temperatures <or= 15.6 degrees C (60 degrees F) and > 15.6 degrees C. RESULTS Mean ambient temperatures were 29.0 degrees C (84.2 degrees F), -2.7 degrees C (27.1 degrees F), and 10.4 degrees C (50.8 degrees F) for groups 1 through 3, respectively. Thermal sensor readings varied with ambient temperature and rectal temperature. Rectal temperatures ranged from 36.2 degrees to 41.7 degrees C (97.2 degrees to 107 degrees F), whereas thermal sensor temperature readings ranged from 23.9 degrees (75 degrees F) to 42.2 degrees C (75 degrees to 108 degrees F). Sensitivity for fever detection was 87.4%, 53.3%, and 58.3% in groups 1 to 3, respectively. CONCLUSIONS AND CLINICAL RELEVANCE The thermal sensor appeared to have potential use for initial screening of body temperature in equids at ambient temperatures > 15.6 degrees C.

Low-dose DNA vaccination into the submandibular lymph nodes in ponies

THE ability to elicit an antigen-specific immune response in horses is desirable, either for protecting them against a pathogen, or for immunomodulation as a means of treating hypersensitivity diseases. While intramuscular administration of an antigen-adjuvant combination often elicits an adequate