Diagnosis of equine influenza by the polymerase chain reaction.
Abstract: Influenza A is a common respiratory infection of horses, and rapid diagnosis is important for its detection and control. Sensitive detection of influenza currently requires viral culture and is not always feasible. The polymerase chain reaction (PCR) was used to detect DNA produced by reverse transcription of equine influenza in stored nasal secretions, vaccines, and allantoic fluids. Primers directed at a target of 212 bp on conserved segment 7 (matrix gene) of human influenza A/Bangkok/1/79(H3N2) produced amplification products of appropriate size with influenza A/Equine/Prague/1/56 (H7N7), A/Equine/Miami/63 (H3N8), A/Equine/Kentucky/79 (H3N8), and A/Equine/Kentucky/2/91 (H3N8) in infected frozen allantoic fluids and in frozen extracts of nasal swabs of 2 horses with naturally acquired influenza. The products bound a 32P-labeled hybridization probe to an inner region of the target. Control samples, including nasal secretions from a horse infected with herpesvirus, were negative. In a prospective study, 2 ponies inhaled aerosols of influenza A/Equine/Kentucky/2/91 (H3N8), and thereafter supernatants of nasal swabs in transport medium were obtained daily for 10 days for culture and PCR. Amplification products were evaluated by size and binding of a 32P-labeled probe and also by dot-blotting and binding of a biotin-labeled probe. Culture detected influenza more consistently than did PCR in the first 2 days of infection, but PCR detected virus more often later in infection. Gels were the most sensitive, but radiometric and biotin-labeled probes gave specific results and were consistently positive from days 3-6. PCR is suitable for detection of equine influenza in clinical samples.
Publication Date: 1994-01-01 PubMed ID: 8011780DOI: 10.1177/104063879400600108Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease
- Disease control
- Disease Diagnosis
- Disease Treatment
- Epidemiology
- Equine Diseases
- Equine Health
- Horses
- Infectious Disease
- Influenza
- Laboratory Methods
- Molecular biology
- Polymerase Chain Reaction
- Respiratory Disease
- Veterinary Medicine
- Veterinary Research
- Virology
- Virus
Summary
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The research focuses on using the polymerase chain reaction (PCR) method for detecting equine influenza, a common respiratory infection in horses. The study finds the PCR detection method suitable and effective, especially later in the infection period, compared to the traditional viral culture method.
Methodology and Targeting
- The researchers tried detecting the DNA produced by reverse transcription of equine influenza in stored nasal secretions, vaccines, and allantoic fluids using PCR.
- The primers were directed at a target of 212 bp on a conserved segment 7 (also known as the matrix gene) of human influenza.
- Varieties of equine influenza were used in the study, including A/Equine/Prague/1/56 (H7N7), A/Equine/Miami/63 (H3N8), A/Equine/Kentucky/79 (H3N8), and A/Equine/Kentucky/2/91 (H3N8).
PCR Detection in Horses
- For testing, amplification products of appropriate size were observed in infected frozen allantoic fluids and in frozen extracts of nasal swabs in horses with naturally acquired influenza.
- The PCR products were confirmed by the binding of a 32P-labeled hybridization probe to an inner region of the target.
- Control samples, standalone nasal secretions from a horse infected with herpesvirus, did not show any signs of equine influenza, verifying the specificity of the PCR detection method.
Prospective Study on Ponies
- In a more practical, forward-looking experiment, researchers subjected two ponies to aerosols of influenza A/Equine/Kentucky/2/91 (H3N8) and took nasal swabs daily for ten days to monitor the infection via culture and PCR.
- The amplified products were evaluated based on the size, binding of a 32P-labeled probe, and also the binding of a biotin-labeled probe by dot-blotting.
Findings on PCR vs. Culture Detection
- The study found that although culture detection was more consistent than PCR in the early stages (first two days) of infection, PCR performed better later in the infection period.
- Gels showed the highest sensitivity among the different methods used. However, both radiometric and biotin-labeled probes gave specific results and were consistently positive from days 3-6 post infection.
Conclusion
- The study shows that PCR is appropriate and is an effective alternative method for detecting equine influenza in clinical samples, particularly later in infection.
Cite This Article
APA
Donofrio JC, Coonrod JD, Chambers TM.
(1994).
Diagnosis of equine influenza by the polymerase chain reaction.
J Vet Diagn Invest, 6(1), 39-43.
https://doi.org/10.1177/104063879400600108 Publication
Researcher Affiliations
- Veterans Affairs Medical Center, Lexington, KY.
MeSH Terms
- Animals
- Base Sequence
- Body Temperature
- Chick Embryo
- DNA Primers
- Hemagglutination Tests / methods
- Horse Diseases
- Horses
- Influenza A virus / genetics
- Influenza A virus / isolation & purification
- Molecular Sequence Data
- Oligonucleotide Probes
- Orthomyxoviridae Infections / diagnosis
- Orthomyxoviridae Infections / veterinary
- Polymerase Chain Reaction / methods
- RNA, Viral / isolation & purification
Citations
This article has been cited 3 times.- Singh RK, Dhama K, Karthik K, Khandia R, Munjal A, Khurana SK, Chakraborty S, Malik YS, Virmani N, Singh R, Tripathi BN, Munir M, van der Kolk JH. A Comprehensive Review on Equine Influenza Virus: Etiology, Epidemiology, Pathobiology, Advances in Developing Diagnostics, Vaccines, and Control Strategies. Front Microbiol 2018;9:1941.
- Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB. Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus. J Clin Microbiol 2009 Dec;47(12):3907-13.
- Pfeffer M, Wiedmann M, Batt CA. Applications of DNA amplification techniques in veterinary diagnostics. Vet Res Commun 1995;19(5):375-407.
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