Faster, better, cheaper (PRRSV) surveillance using oral fluid-based sampling

Jeff Zimmerman DVM PhDIowa 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

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% 0% 0% 2% 2% 2%

4% (1+)   12% 16% 12% 8% 20% 20%

12% (3+)   85% 95% 55% 55% 100% 90%

20% (5+)   72% 88% 40% 32% 80% 72%

36% (9+)   96% 96% 76% 76% 100% 96%

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

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% 0% 0% 2% 2% 2% 0.700

4% (1+) 25   12% 16% 12% 8% 20% 20% 0.443

8% (2+) 5   80% 100% 80% 20% 100% 80% 0.015

12% (3+) 20   85% 95% 55% 55% 100% 90% <0.001

20% (5+) 25   72% 88% 40% 32% 80% 72% <0.001

36% (9+) 25   96% 96% 76% 76% 100% 96% 0.002

PRRSV RT-PCR results on oral fluids

*n = number of oral fluid samples

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% 0% 0% 0% 0.416

4% (1+) 25   24% 16% 12% 8% 24% 8% 0.067

8% (2+) 5   0% 0% 0% 0% 0% 0% 1.000

12% (3+) 15   67% 60% 80% 47% 73% 53% 0.048

20% (5+) 25   68% 88% 92% 80% 92% 88% 0.015

32% (8+) 10 90% 100% 90% 90% 100% 90% 0.722

36% (9+) 15   87% 93% 93% 87% 100% 87% 0.352

PRRSV ELISA results on oral fluids

*n = number of oral fluid samples

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% 0% 0% 0% 0%

4% (1+) 25 24% 16% 12% 8% 24% 8%

8% (2+) 5 0% 0% 0% 0% 0% 0%

12% (3+) 15 67% 60% 80% 47% 73% 53%

20% (5+) 25 68% 88% 92% 80% 92% 88%

32% (8+) 10 90% 100% 90% 90% 100% 90%

36% (9+) 15 87% 93% 93% 87% 100% 87%

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% 0% 0% 2% 2% 2%

4% (1+) 25 12% 16% 12% 8% 20% 20%

8% (2+) 5 80% 100% 80% 20% 100% 80%

12% (3+) 20 85% 95% 55% 55% 100% 90%

20% (5+) 25 72% 88% 40% 32% 80% 72%

36% (9+) 25 96% 96% 76% 76% 100% 96%

CO

MP

AR

ISO

N

X

xx

One oral fluid sampleOne serum sample

Within-pen prevalence

X

xx

Within-pen prevalence

Increased probability of PRRSV detection with one oral fluid samples vs. one

serum sample

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

jjzimm@iastate.edu

Thank you!