Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA.

This research paper presents the successful use of a reverse transcriptase polymerase chain reaction (RT-PCR) test to detect the presence of Equine Arteritis Virus (EAV) in the semen of horses and donkeys, suggesting this method is potent, speedy and effective for diagnostic purposes.

Methodology and Sample

• The study deployed the RT-PCR assay to check for EAV in the semen of 90 animals, which included 88 horses and 2 donkeys. These animals had previously demonstrated neutralising antibodies against EAV.
• The test focused specifically on detecting a 279 bp long fragment that is found in the EAV’s polymerase gene.

Test Sensitivity and Comparative Analysis

• The RT-PCR assay was able to detect the presence of the virus when the concentration was as low as 4 TCID50/ml in cell culture. This indicates a high sensitivity as it succeeded in discovering a large percentage (54.4%) of positive cases.
• RT-PCR proved to be more sensitive in comparison to the cell culture isolation method, which had a sensitivity of only 33.3%.
• Most importantly, the assay was successful in identifying the virus in the semen samples of the two donkeys included in the experiment.

Genomic Divergence and Probe Identification

• On sequencing the cDNAs obtained from the semen samples of 14 horses and 2 donkeys, a high level of genomic heterogeneity was noticed among the RNAs extracted.
• However, within the amplified fragment, a consistently well-preserved region of 24 bp was identified, making it an ideal candidate for a probe in future RT-PCR-ELISA testing.
• When compared with the sequence of reference strain Bucyrus, the analysed samples showed a 78-100% identity – further suggesting this particular region’s suitability as a probe.

Feasibility and Diagnostic Application

• The study demonstrated that the RT-PCR-ELISA assay is not only sensitive and specific, but can be directly applied to semen, making it significantly useful for diagnosing EAV.
• Another notable advantage of this testing method is the short duration required for the overall procedure, enhancing its practical application in a diagnostic setting.