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Introduction

Diagnostic performance of a commercial PRRS serum antibody ELISA adapted to oral fluid specimens: field samples.

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Introduction

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  1. Diagnostic performance of a commercial PRRS serum antibody ELISA adapted to oral fluid specimens: field samples A. Kittawornrat1, J. Prickett1, C. Wang1,2, C. Olsen1, C. Irwin1, Y. Panyasing1, A.Ballagi3, A. Rice3, R. Main1, C. Rademacher4, M. Hoogland4, J. Lowe5, J. Zimmerman1 1Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA 2Department of Statistics, College of Liberal Arts and Sciences, Iowa State University, Ames, IA 3IDEXX Laboratories, Inc., Westbrook, ME 4Murphy-Brown LLC, Ames, IA 5Carthage Veterinary Service, Ltd., Carthage IL

  2. Introduction • In human diagnostic medicine • 1909. “The agglutinating properties of several body fluids during Malta Fever.” Pollaci and Ceralo • 1986. Oral fluid antibodies used to detect HIV infection. J Clin Microbiol 24(5): 873 • Lots of research in humans (1909-2010) • Primary focus on antibody detection • Widely used in surveillance

  3. Diagnostic monitoring in pigs today is Based on blood testing BOARS: Confirm negative PRRS status of individual “seed stock” pigs GILTS: Confirm PRRS status before entering breeding herds, preferably at the beginning and end of quarantines SOWS and PIGLETS: Monitor vaccine effectiveness or confirm PRRS status Boars Gilts Sows Piglets Simplified sampling would allow more frequent testing and provide more exact epidemiological information

  4. Pictures courtesy of Dr. John Prickett

  5. Indirect ELISA format

  6. Steps in adapting ELISA to oral fluid 1. Optimize antibody isotypes 2. Optimize incubation period 3. Optimize sample dilution 4. Interactions (isotype x sample dilution x sample volume) 5. Optimize conjugate dilution

  7. Antibody Isotype Detection Conclusion from previous results

  8. Modified Oral fluid PRRS ELISA • Serum antibody assays (IDEXX PRRS X3 can be optimized to detect antibodies in oral fluid by • IgGFC isotype (IgM, IgA, IgG can be detected) • 250 μl sample volume • 16 hour incubation • 1:2 sample dilution • Other conjugate

  9. Source of samples • 10 barns: 1,100 head wean-finish • - 1 oral fluid sample / pen; 6 pens /barn • Samples collected every 2 weeks from placement to close-out (10 sample points) • 600 oral fluid samples (PCR, ELISA) Wean-to-finish barns (1100 pigs) 10 sites x 6 pens per site x sampling each 2 weeks. n = 600 total samples

  10. Results – ELISA S/P ratios

  11. Results 5 serum samples per pen (n = 30) at week 0 Avg. S/P = 0.30 Pos. = 23.3% Avg. S/P = 0.04 Pos. = 3.3% Avg. S/P = 0.72 Pos. = 53.3% Avg. S/P = 0.34 Pos. = 30.0%

  12. Results

  13. Results

  14. Results

  15. Results

  16. Results

  17. Results

  18. Results

  19. Results

  20. Results 30 serum samples at week 18 Avg. S/P = 0.93 Pos. = 93.3% Avg. S/P = 0.78 Pos. = 70.0% Avg. S/P = 1.40 Pos. = 96.7% Avg. S/P = 1.06 Pos. = 86.7%

  21. Test performance (Se, Sp) • Source of positive samples • Experimental samples • Field samples • For any pen, all samples after the first PCR positive result were considered positive • Source of negative samples • Experimental (DPI 0) • Field samples from expected PRRSV negative herds submitted to ISU VDL

  22. Test performance (Se, Sp) NEGATIVE SAMPLES Experimental= 84 Field neg(ISU VDL) = 283 TOTAL = 367 POSITIVE SAMPLES Experimental= 251 Field = 241 TOTAL = 492

  23. Test performance (Se, Sp)

  24. Conclusions • Kinetics of anti-PRRSV antibodies in oral fluid is amenable to rapid detection of infection. • The oral fluid ELISA could provide an efficient, cost-effective approach to PRRSV monitoring in commercial herds and surveillance in elimination programs.

  25. Thank you • IDEXX Laboratories, Inc. • National Pork Board (Pork Checkoff funds) • PRRS CAP, USDA NIFA award 2008-55620-1932 • Request reference standard samples contact to Dr. Zimmerman (jjzimm@iastate.edu) • Request SOP contact to Dr. Johnson (jkjohn@iastate.edu)

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