Faster, better, cheaper (PRRSV) surveillance using oral fluid-based sampling Jeff Zimmerman DVM PhD Iowa State University Ames, Iowa

Basic assumptions We need better surveillance of pathogens of swine, but current methods provide inadequate detection, are unacceptable to farmers, or are too expensive to implement Good oral fluid antibody and PCR assays can be developed for a variety of pathogens How well would these assays work?

Performance of oral fluids in PRRSV surveillance - a field study C Olsen,1 C Wang,1 J Christopher-Hennings,2 K Doolittle,3 K Harmon,1 S Abate,1 A Kittawornrat,1 S Lizano,5 R Main,1 E Nelson,2 T Otterson,6 Y Panyasing,1 C Rademacher,4 R Rauh,7 R Shah,8 J Zimmerman1   1Iowa State University, 2South Dakota State University, 3Boehringer Ingelheim Vetmedica, Inc., 4Murphy-Brown LLC, 5IDEXX Laboratories Inc., 6University of Minnesota, 7Tetracore®, Inc., 8Life Technologies®, Inc.

Objective - Estimate the probability of detecting PRRSV infection as a function of within-pen prevalence

Samples tested by RT-PCR and ELISA Experimental design 25 pens, 25 pigs per pen Prevalence was established using pigs vaccinated with PRRSV MLV vaccine Prevalence (pigs +)   Samples tested by RT-PCR and ELISA Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 0% (0+) 0% 2% 4% (1+) 12% 16% 8% 20% 12% (3+) 85% 95% 55% 100% 90% 20% (5+) 72% 88% 40% 32% 80% 36% (9+) 96% 76%

PRRSV MLV SITE 1 SITE 2 … 14 DPV - serum antibody and virus positive 25 pigs per pen

Experimental design ● DPV 0 PRRSV-negative pigs (n = 90) in Missouri vaccinated with MLV PRRSV MLV. ● DPV 10 PRRSV-vaccinated pigs (n = 90) in Missouri brought to Iowa farm and placed in isolation ● DPV 12 PRRSV-negative pigs (n = 535) from Oklahoma brought to Iowa farm, placed in 25 pens, oral fluid collected from each pen. ● DPV 13 Morning: blood sample from each of 535 negative pigs. Afternoon: Within pen PRRSV prevalence (0%, 4%, 12%, 20%, or 36%) established by placing 0, 1, 3, 5, or 9 PRRSV-vaccinated pigs in the 25 pens. Each pen held a total of 25 pigs after placement. ● DPV 14 5 successive oral fluid samples were collected from each pen, i.e., a total of 125 samples. ● DPV 14 Serum samples were collected from each of the 90 vaccinated pigs to establish PRRSV status.

Probability of detection as a function of prevalence?

PRRSV RT-PCR results on oral fluids

PRRSV RT-PCR results on oral fluids PREV (pigs +) *n   Percent of RT-PCR positive oral fluid samples p-value Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 0% (0+) 50 0% 2% 0.700 4% (1+) 25 12% 16% 8% 20% 0.443 8% (2+) 5 80% 100% 0.015 12% (3+) 20 85% 95% 55% 90% <0.001 20% (5+) 72% 88% 40% 32% 36% (9+) 96% 76% 0.002 *n = number of oral fluid samples

PRRSV ELISA results on oral fluids

PRRSV ELISA results on oral fluids PREV (pigs +) *n   Percent of ELISA positive oral fluid samples p-value Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 0% (0+) 50 2% 0% 0.416 4% (1+) 25 24% 16% 12% 8% 0.067 8% (2+) 5 1.000 12% (3+) 15 67% 60% 80% 47% 73% 53% 0.048 20% (5+) 68% 88% 92% 0.015 32% (8+) 10 90% 100% 0.722 36% (9+) 87% 93% 0.352 *n = number of oral fluid samples

COMPARISON PREV (pigs +) *n   Percent of RT-PCR positive oral fluid samples Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 0% (0+) 50 0% 2% 4% (1+) 25 12% 16% 8% 20% 8% (2+) 5 80% 100% 12% (3+) 20 85% 95% 55% 90% 20% (5+) 72% 88% 40% 32% 36% (9+) 96% 76% COMPARISON PREV (pigs +) *n   Percent of ELISA positive oral fluid samples Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 0% (0+) 50 2% 0% 4% (1+) 25 24% 16% 12% 8% 8% (2+) 5 12% (3+) 15 67% 60% 80% 47% 73% 53% 20% (5+) 68% 88% 92% 32% (8+) 10 90% 100% 36% (9+) 87% 93%

Within-pen prevalence One oral fluid sample One serum sample X x Within-pen prevalence

Within-pen prevalence Increased probability of PRRSV detection with one oral fluid samples vs. one serum sample X x Within-pen prevalence

Conclusions Oral fluid-based detection of PRRSV infection using either ELISA or RT-PCR is effective, efficient, and easy. The estimates in this study are conservative: 1. Vaccine-induced viremia and antibody response is "weaker" than natural infection (Johnson et al., 2004) 2. Vaccinated pigs were introduced into pens ~16 hours prior to collection. Lack of socialization adversely affects sampling behavior. 3. Results from all laboratories were included in the estimates. 4. Oral fluid-based surveillance could facilitate faster, better, cheaper surveillance of PRRSV and other pathogens

Thank you! jjzimm@iastate.edu