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

2. 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?

3. 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.

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

5. 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%

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

7. 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 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.

8. Probability of detection as a function of prevalence?

9. PRRSV RT-PCR results on oral fluids

10. 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

11. PRRSV ELISA results on oral fluids

12. 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

13. 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%

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

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

16. 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

17. Thank you!