Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR.
ABSTRACT 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.
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.
Fifteen horses experimentally infected with EHV-1.
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.
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).
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.
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ABSTRACT: The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15 pg/μl for EHV-1 and 5 copies/μl or 2.5 fg/μl for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.Applied Microbiology and Biotechnology 03/2014; · 3.69 Impact Factor
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ABSTRACT: Abstract Equid herpesvirus (EHV) type 1 is a common pathogen of horses with worldwide distribution. Infection with EHV-1 can be subclinical, or can result in sociologically and economically important outcomes such as abortion, neonatal death or neurological disease. The perceived recent increase in the reported cases of EHV-1 neurological disease in the USA and Europe over the past decade has caused concerns amongst veterinarians and horse owners worldwide. Despite evidence of EHV-1 circulation among horses and foals in New Zealand, EHV-1 neurological disease has not yet been reported here. This review provides an update on the recent developments in our understanding of the pathogenesis and epidemiology of EHV-1 and associated diseases, with an emphasis on epidemiological data from Australasia. Many aspects of the pathogenesis and epidemiology of equine herpesvirus myeloencephalopathy still remain to be elucidated. This is an active area of current research worldwide.New Zealand veterinary journal 03/2014; · 1.06 Impact Factor