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: There is little information on the duration of nasal shedding of EHV-1 from horses with naturally occurring equine herpesvirus myeloencephalopathy (EHM). To evaluate the duration of nasal shedding of EHV-1 in horses affected by EHM. One hundred and four horses naturally exposed to EHV-1, 20 of which had clinical signs of EHM. 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. 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). 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.Journal of Veterinary Internal Medicine 02/2012; 26(2):384-92. · 2.06 Impact Factor
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ABSTRACT: A single-nucleotide polymorphism (A(2254) or G(2254)) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E(2)) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69-72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E(1)) was developed by redesigning primers and probes specific to ORF30. The E(1) and E(2) rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A(2254) or G(2254)) in all archived isolates plus 168 of the clinical samples. The E(1) assay was 10 times more sensitive than E(2), with a lower detection limit of 10 infectious virus particles. Furthermore, all A(2254) and G(2254) genotypes along with samples from three cases of dual infection (A(2254)+G(2254)) were correctly identified by E(1), whereas E(2) produced 20 false dual positive results with only one actual mixed A(2254)+G(2254) genotype confirmed. Based on these findings, E(1) offers greater sensitivity and accuracy for the detection and A/G(2254) genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.Journal of clinical microbiology 04/2012; 50(6):1981-8. · 4.16 Impact Factor