#09-193

Complete

Date Full Report Received

05/01/2012

Date Abstract Report Received

05/01/2012

Investigation

Institution:
Primary Investigator:

The objective of this research was to compare the detection of influenza virus A in nasal swabs vs. pen-based oral fluids. In addition, this study evaluated the impact of vaccination on IAV shedding as detected from nasal swabs and oral fluid.

Briefly, 21-day-old weaned pigs (n = 82) were isolated for 30 days and confirmed negative to antibodies against PRRSV, M. hyopneumonia, and influenza A virus (IAV). A subset (n = 28) was vaccinated twice with a commercial multivalent influenza vaccine (FluSure XP™, Pfizer Animal Health, New York, NY) twenty-one days apart. Pigs were then transported to the research facility, re-confirmed serologically negative for PRRSV, IAV and M. hyopneumonia, grouped according to treatment and inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus or left uninoculated (negative controls). Pigs were housed in pens by treatment group and oral fluid (OF) was collected from rope hung daily in each pen (DPI 0-16). Individual flocked nasal swabs (NS) were collected daily DPI 0 through DPI 6, then on DPI 8, 10, 12, 14, and 16 in the inoculated animals and every fourth day in control animals. OF and NS samples were tested for IAV by matrix real-time reverse transcriptase polymerase chain reaction (RT-PCR) (DPI 0-16) screening assay independently at 2 laboratories, virus isolation (VI) (DPI 0-16), and VetScan® Avian Influenza Rapid Test Kit (Abaxis, Union City CA) (DPI 0-10). To report NS at a pen based level, a pen was considered NS positive (+) if at least 1 pig was detected positive in that pen.

Response by all three detection assays were similar between inoculating virus serotype, therefore pens of animals were grouped by vaccination status (unvaccinated or vaccinated) and compared by DPI.

● RT-PCR results demonstrated prolonged detection of IAV in OF compared to NS for the 16 day period, in both vaccinated and unvaccinated animals. In vaccinated pens, influenza detection by OF using RT-PCR was better than NS. There was 100% agreement with NS through DPI 6, but OF also detected 50% of the positive pens through DPI 8, as well as intermittent detection through DPI 14. RT-PCR IAV detection results of both sample types were significantly less than IAV detection in unvaccinated pigs.
● IAV was detected from OF by VI, and in unvaccinated groups DPI 1 through 6, similar positive results were detected from both sample types. After DPI 6, virus was not detected by VI from either sample type in unvaccinated or vaccinated groups. In addition, vaccinated pigs did not appear to shed significant virus in OF (VI routinely negative), however, some nasal shedding was detected in these pigs (VI positive on NS up to DPI 6).
● The VetScan® Rapid Avian Influenza A Antigen Test had a similar sensitivity to VI for both OF and NS samples in unvaccinated groups; detection was limited to, but equivalent, during DPI 2-5. In vaccinates, OF was not a suitable sample for the detection of IAV by the VetScan®, although NS only detected between 80% and 17% of the 6 pens and only through DPI 5.
● False positive RT-PCR results were reported in 2 OF and 4 NS on DPI 0 and 1 OF on DPI 4 (sample from the negative control group). One false positive VI was reported in 1 NS on DPI 0. RT-PCR positives from OF were accurately characterized by H and N subtyping 85.3% of the time, mis-characterized 1.9% and reported untypeable in 13.1% of the samples (9% on DPI 1, and 75% after DPI 7).

The results of this study have significant importance to the swine industry and influenza surveillance programs. From this study, a pen based OF sample is at least equivalent (DPI 1-6) to the traditional sampling technique (NS) and often a superior sample (≤DPI 16) to NS for the detection of IAV using RT-PCR. In addition, IAV can be isolated from OF but vaccination reduces the level and duration of oral shedding of IAV. Because successful VI is unlikely from vaccinated pigs, RT-PCR should be the detection method of choice. In particular, the matrix RT-PCR screening assay using oral fluid is highly suitable for surveillance programs. The Vetscan® Avian Influenza Rapid Test can detect contemporary IAV from DPI 2-5 in both OF and NS samples from unvaccinated pigs, but like VI, sensitivity is low after DPI 6 in unvaccinated animals and shedding in OF is not easily detected in vaccinated animals.