Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR.

Abstract

BACKGROUND: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples. OBJECTIVES: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses. ANIMALS: Fifteen horses experimentally infected with EHV-1. METHODS: Experimental study: Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed. RESULTS: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI (P< .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86-100) and 27% (95% CI: 20-35). CONCLUSIONS AND CLINICAL IMPORTANCE: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C(T) values are provided as well as justification of a minimum 10-day quarantine period.