#03-052

Complete

Date Full Report Received

07/28/2004

Date Abstract Report Received

07/26/2006

Investigation

Institution:
Primary Investigator:
Co-Investigators: Raymond R. Rowland

The detection of PRRSv in boars is critical for developing and maintaining a biosecure breeding program, however it is complicated by many variables. Correlating the current PRRSv infectious status of a boar using only one diagnostic approach at one point in time from only one type of sample can result in misdiagnoses. As was demonstrated in this study, all boars seroconverted to PRRSv using a commercial ELISA indicating past-exposure to PRRSv antigens, but only one boar on one sampling date (day after infection, [dai-13]) demonstrated PRRSv in semen by traditional tissue culture procedures for recovering the virus. Virus was detectable in all boars in serum samples by virus isolation, however these were only detectable by dai-5 and the viremias were short-lived as only one boar was viremic by dai-13. Comparing different PCR detection procedures on these different boar samples also demonstrated the potential for misdiagnoses.
The original intent of this study was to evaluate the efficiency of a SYBR green “real-time” PCR for detecting PRRSv in boar semen and serum. Unfortunately, when the SYBR green PCR was compared to PCR results detected with electrophoretic gels stained with ethidium bromide (EtBr) or blotted and hybridized with a DNA probe (Southern blots), the SYBR green PCR was unable to detect the presence of PRRSv RNA in all serum and semen samples evaluated. While the gels stained with EtBr were superior to the SYBR green PCR results for detecting PRRSv in serum and semen, the Southern blots on the same PCRs were able to detect PRRSv RNA on samples that were otherwise undetectable.
The need for a DNA hybridization step in any PCR assay for detecting PRRSv RNA in serum or semen cannot be overstated based on these results and the potential for misdiagnoses, particularly false-negative PCR test results. To overcome this, we recommend the development and evaluation of PCR approaches that incorporate automated DNA hybridization steps into the PCR analysis, such as TaqMan or Molecular Beacon detection systems. These fluorgenic-based PCR assays will allow for improvements in detection sensitivity and specificity that is lacking with the SYBR green detection system, but also allow for rapid “real-time” (rt)-PCR to be applied. Also recognized in this study were the importance of effective RNA recovery procedures from semen samples and their importance to any sensitive PRRSv PCR detection procedure. Additional research will be required to identify and optimize the most efficient PRRSv RNA recovery processes from semen and other tissues. The goal of the process should be the development of a straight-forward, user-friendly process that can be totally automated, self-contained PRRSv-RNA detection system.