Diagnosis of equine influenza by the polymerase chain reaction.

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.