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. Objective: 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. Methods: 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: 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.
Publication Date: 2008-08-06 PubMed ID: 18691363DOI: 10.1111/j.1939-1676.2008.0172.xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article focuses on the efficiency of using fast quantitative PCR testing on nasal swabs from horses to detect equine herpesvirus-1 (EHV-1) as compared to traditional virus isolation methods. The study concludes that fast qPCR testing is more effective for diagnosing EHV-1 and suggests a minimum of 10 days of quarantine.
Introduction
- This study is centered around the detection and diagnosis of equine herpesvirus type 1 (EHV-1) in horses.
- The primary comparison made was between the conventional virus isolation (VI) methods and the real-time quantitative PCR (qPCR) approach.
- A crucial requirement for direct comparison was a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Methodology
- 15 horses were experimentally infected with EHV-1 for this research.
- Nasal swab samples were acquired daily from the subjects for up to 21 days following experimental infection.
- The DNA was prepared for qPCR with the inclusion of a known DNA quantity of Marek’s disease virus acting as an internal control.
- The researchers employed the conventional methods for performing VI.
Results
- The qPCR method showed an ability to detect the virus for a more extended duration, up to day 21 after the challenge was initiated. In comparison, VI was only able to detect the virus until day 5.
- The median Kaplan-Meier estimation for the detection of EHV-1 was significantly longer for the qPCR method at 12 days, compared to only 2 days with the VI method.
- Furthermore, the sensitivity and specificity of qPCR when compared with VI were 97% and 27% respectively.
Conclusion
- The results suggest that qPCR testing on nasal swab samples from horses is a more effective method for diagnosing and monitoring EHV-1, leading to the recommendation that fast qPCR should be selected over VI methods.
- The study additionally provides recommended reference ranges of C(T) values.
- Given the extended detection window for EHV-1 with qPCR, the researchers also offer justification for a minimum 10-day quarantine period for horses suspected of or confirmed to have EHV-1.
Cite This Article
APA
Perkins GA, Goodman LB, Dubovi EJ, Kim SG, Osterrieder N.
(2008).
Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR.
J Vet Intern Med, 22(5), 1234-1238.
https://doi.org/10.1111/j.1939-1676.2008.0172.x Publication
Researcher Affiliations
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. gap7@cornell.edu
MeSH Terms
- Animals
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / isolation & purification
- Horse Diseases / virology
- Horses
- Nose / virology
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / veterinary
- Sensitivity and Specificity
- Time Factors
- Virus Shedding
Citations
This article has been cited 13 times.- Eady NA, Holmes C, Schnabel C, Babasyan S, Wagner B. Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies. J Virol 2024 Jun 13;98(6):e0025024.
- Minuto J, Bedenice D, Ceresia M, Zaghloul I, Böhlke M, Mazan MR. Clinical effects and pharmacokinetics of nebulized lidocaine in healthy horses. Front Vet Sci 2022;9:984108.
- Thieulent CJ, Sutton G, Toquet MP, Fremaux S, Hue E, Fortier C, Pléau A, Deslis A, Abrioux S, Guitton E, Pronost S, Paillot R. Oral Administration of Valganciclovir Reduces Clinical Signs, Virus Shedding and Cell-Associated Viremia in Ponies Experimentally Infected with the Equid Herpesvirus-1 C(2254) Variant. Pathogens 2022 May 4;11(5).
- Carvelli A, Nielsen SS, Paillot R, Broglia A, Kohnle L. Clinical impact, diagnosis and control of Equine Herpesvirus-1 infection in Europe. EFSA J 2022 Apr;20(4):e07230.
- Nielsen SS, Alvarez J, Bicout DJ, Calistri P, Canali E, Drewe JA, Garin-Bastuji B, Gonzales Rojas JL, Gortázar C, Herskin M, Michel V, Miranda Chueca MÁ, Roberts HC, Padalino B, Pasquali P, Spoolder H, Ståhl K, Calvo AV, Viltrop A, Winckler C, Carvelli A, Paillot R, Broglia A, Kohnle L, Baldinelli F, Van der Stede Y. Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infection with Equine Herpesvirus-1. EFSA J 2022 Jan;20(1):e07036.
- Stasiak K, Dunowska M, Rola J. Outbreak of equid herpesvirus 1 abortions at the Arabian stud in Poland. BMC Vet Res 2020 Oct 6;16(1):374.
- Yan L, Toohey-Kurth KL, Crossley BM, Bai J, Glaser AL, Tallmadge RL, Goodman LB. Inhibition monitoring in veterinary molecular testing. J Vet Diagn Invest 2020 Nov;32(6):758-766.
- Kishimoto M, Tsuchiaka S, Rahpaya SS, Hasebe A, Otsu K, Sugimura S, Kobayashi S, Komatsu N, Nagai M, Omatsu T, Naoi Y, Sano K, Okazaki-Terashima S, Oba M, Katayama Y, Sato R, Asai T, Mizutani T. Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex. J Vet Med Sci 2017 Mar 18;79(3):517-523.
- Taktaz Hafshejani T, Nekoei S, Vazirian B, Doosti A, Khamesipour F, Anyanwu MU. Molecular Detection of Equine Herpesvirus Types 1 and 4 Infection in Healthy Horses in Isfahan Central and Shahrekord Southwest Regions, Iran. Biomed Res Int 2015;2015:917854.
- Harman RM, Bussche L, Ledbetter EC, Van de Walle GR. Establishment and characterization of an air-liquid canine corneal organ culture model to study acute herpes keratitis. J Virol 2014 Dec;88(23):13669-77.
- Smith KL, Li Y, Breheny P, Cook RF, Henney PJ, Sells S, Pronost S, Lu Z, Crossley BM, Timoney PJ, Balasuriya UB. New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G2254 polymorphisms. J Clin Microbiol 2012 Jun;50(6):1981-8.
- Goodman LB, Wimer C, Dubovi EJ, Gold C, Wagner B. Immunological correlates of vaccination and infection for equine herpesvirus 1. Clin Vaccine Immunol 2012 Feb;19(2):235-41.
- Brosnahan MM, Damiani A, van de Walle G, Erb H, Perkins GA, Osterrieder N. The effect of siRNA treatment on experimental equine herpesvirus type 1 (EHV-1) infection in horses. Virus Res 2010 Feb;147(2):176-81.
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