#11-001

Complete

Date Full Report Received

02/14/2013

Date Abstract Report Received

02/14/2013

Investigation

Institution:
Primary Investigator:

With classical swine fever virus (CSFV) commonplace in the world and barely outside the borders of North America, it is reasonable to expect that CSFV will eventually be reintroduced into North America. The primary objective of this project is to optimize and validate technology capable of rapidly identifying premises infected with CSFV following its introduction into North America or other CSFV-free areas using oral fluid samples. In the present study, twenty pigs at the age of 21 days were obtained from a free-CSFV farm and divided into 3 groups. Pigs in group A (n=8) were intramuscularly inoculated with a low virulence CSFV strain (ALD, 105TCID50/pig) at day 0 and re-challenged with a virulence strain (Bangkok-1950, 103TCID50/pig) intramuscularly at 14 days post inoculation (DPI). Pigs in group B (n=8) were intramuscularly vaccinated with a commercial modified live CSFV vaccine (LOM) at day 0 and challenged with the virulence strain similar to pigs in group A at 14 DPI. Group C (n=4) was a negative control group. This modified experimental protocol allowed us to successfully collect the oral fluid samples from subclinically infected pigs and might mimic the CSFV situation in CSFV-endemic countries using modified live CSFV vaccines. Based on the modified real-time RT-PCR results (sensitivity at 101.5 TCID50/ml), a few vaccinated pigs having viremia for a few days after vaccination and after re-challenged with a high virulence strain, a few viremic pigs were found in both vaccinated and low virulence CSFV challenged groups. However, all samples were tested negative using a routine RT-PCR method (sensitivity at 103 TCID50/ml). In addition, none of the oral fluid samples from all groups were tested positive using either the modified real-time RT-PCR or the routine RT-PCR. Possible use of a commercial blocking ELISA of classical swine fever antibody was also evaluated by using oral fluid samples from a CSFV-free herd mixed with serially diluted known CSFV antibody titers as a pilot study and compared to the neutralizing peroxidase-linked assay (NPLA). Interestingly, the low levels of NPLA antibody titers (log2 1- log2 3) could evidently be detected in the oral fluid samples obtained from the in vivo experiment (lower than average serum NPLA titers of pigs in the same group, log2 3- log2 6) but none of the oral fluid samples from this present study were positive by a commercial ELISA (sensitivity at least log2 4 based on the pilot study). In conclusion, the modified real time RT-PCR yielded satisfactory sensitivity than the routine RT-PCR, but the sensitivity was not good enough to detect low virus levels, particularly in the oral fluid samples. Extracting genetic material from the oral fluid samples may be hampered by the mucous component in the saliva or other organic matters. Achievement of this objective could be done by developing a better genetic material extraction from oral fluid samples. However, detecting CSFV antibody in the oral fluid samples is another alternative method and when adjusting a commercial blocking ELISA protocol to increase its sensitivity for improved surveillance in CSFV-free areas, thereby would enhance elimination and control efforts. In addition, testing oral fluid samples using an improved sensitivity blocking ELISA may benefit the North American swine producers to rapidly identify premises infected with CSFV following its introduction into North America much faster than using the molecular diagnostic methods.