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Title: Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

Author
item GOODELL, CHRISTA - Iowa State University
item ZHANG, JIANQIANG - Iowa State University
item STRAIT, ERIN - Iowa State University
item HARMON, KAREN - Iowa State University
item PATNAYAK, DEVI - University Of Minnesota
item OTTERSON, TRACY - University Of Minnesota
item GRAMER, MARIE - University Of Minnesota
item CHRISTOPHER-HENNINGS, JANE - South Dakota State University
item Baker, Amy
item Kitikoon, Pravina
item McGill, Jodi

Submitted to: Canadian Journal of Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2015
Publication Date: 1/16/2016
Citation: Goodell, C.K., Zhang, J., Strait, E., Harmon, K., Patnayak, D., Otterson, T., Culhane, M., Christopher-Hennings, J., Clement, T., Leslie-Steen, P., Hesse, R., Anderson, J., Skarbek, K., Vincent, A., Kitikoon, P., Swenson, S., Jenkins-Moore, M., McGill, J., Rauh, R., Nelson, W., O'Connell, C., Shah, R., Wang, C., Main, R., Zimmerman, J.J. 2016. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation. Canadian Journal of Veterinary Research. 80(1):12-20.

Interpretive Summary: Influenza A virus (IAV) is an important respiratory pathogen in pigs with occasional spread of swine IAV to humans. Rapid and reliable detection of IAV infections in pigs is necessary to prevent spread of the disease. Diagnostic samples that can be collected on live animals without restraint are preferred in many situations, and oral fluids (OF) are such a sampling option. This study was conducted to evaluate the performance of routine diagnostic tests (RT-PCR and virus isolation) for IAV among 8 laboratories on OF samples from swine spiked with known amounts of IAV. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the laboratories for detecting IAV in OF, particularly as concentration of virus decreased. Further efforts are required to improve accurate and consistent performance of OF as a diagnostic specimen for IAV among veterinary laboratories.

Technical Abstract: The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold diluted (10-1 to 10-8). Eight participating laboratories received 180 randomized samples (10 replicates x 8 dilutions x 2 IAV subtypes plus 20 IAV-negative oral fluid samples) and performed the IAV rRT-PCR and VI procedure(s) of their choice. Analysis of the results using a mixed-effect logistic regression model identified dilution (p <0.0001) and assay (p <0.0001) as variables significant to IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant to IAV detection by either rRT-PCR (p = 0.4566) or VI (p = 0.1006). For rRT-PCRs, cycle threshold (Ct) values increased consistently with dilution, but exhibited wide variation. Therefore, it was not possible to predict VI success based on Ct values. VI was inversely related to the dilution of the sample and was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated.