Rapid and sensitive detection of equine arteritis virus in semen and tissue samples by reverse transcription-polymerase chain reaction, dot blot hybridisation and nested polymerase chain reaction.

This research shows that the use of reverse transcription-polymerase chain reaction (RT-PCR), dot blot hybridisation, and nested polymerase chain reaction (PCR) methods together increase the speed and accuracy of detecting equine arteritis virus (EAV) in semen and tissue samples from horses.

Testing Methods

• The research utilized reverse transcription-polymerase chain reaction (RT-PCR) with different primer pairs to detect equine arteritis virus (EAV) RNA. Among the different primers used, the one encoding the leader sequence of the EAV genome showed the most successful amplification.
• The effectiveness of RT-PCR was further validated using various methods including virus isolation in cell culture, restriction analysis, and dot blot hybridisation, and nested PCR.

Sample Analysis

• The samples for testing these techniques consisted of 23 semen samples from seropositive stallions and 11 tissue samples from 4 aborted foals. These specific samples were chosen because they least likely hold a high concentration of EAV, making them a good measure for the sensitivity of the detection methods.

Results

• Compared to virus isolation in cell culture, the molecular methods of RT-PCR, dot blot hybridisation, and nested PCR proved to be more sensitive to EAV detection.
• Semen samples from 11 stallions and tissue samples from all 4 foals were identified as positive in the RT-PCR, dot blot hybridisation, and nested PCR tests.
• The traditional method of virus isolation in cell culture identified only 4 positive semen samples and only 1 positive tissue sample from aborted foals.

Sensitivity of the Techniques

• The dot blot hybridisation test showed superior sensitivity compared to the RT-PCR test.
• The nested PCR test was found to be the most sensitive among all, as 3 semen samples were identified as positive by this method only.

Conclusions

• Considering the overall sensitivity, speed, and reliability of the tests, the research suggests that a combined use of RT-PCR followed by dot blot hybridisation or nested PCR offers the best method for diagnosing EAV.
• The authors recommend inclusion of these methods in the official diagnostic regimes for detecting the EAV in horses.