Dr. Korakrit Poonsuk - Effect of circulating antibody on the course of PEDV infection in neonatal pigs

Information about Dr. Korakrit Poonsuk - Effect of circulating antibody on the course of...

Published on January 17, 2016

Author: trufflemedia

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1. Role of passively administered systemic antibodies in protecting piglets from PEDV K Poonsuk, LG Giménez-Lirola, J Zhang, P Arruda, Q Chen, L Correa da Silva Carrion, R Magtoto, P Piñeyro, L Sarmento, C Wang, KJ Yoon,J Zimmerman, R Main Iowa State University

2. PEDV protection? • Maternal immunity important in protection • What is the role of colostral antibodies in protecting piglets from PEDV? – Experimental model based on "passive antibody transfer model", i.e., parenterally administered antibody.

3. Objective of the study • Identify effect of passively administered PEDV-specific Ab on: 1) Body weight 2) Body temperature 3) Survival 4) Fecal shedding 5) Humoral immune response

4. Experimental design (overview) • Source of "passive antibody" – Serum from PEDV Ab+ sows (n = 2) – Salt out/concentrate antibody • Treatments (n = 6) randomized to piglets w/n litters – All litters received all treatments • Piglets inoculated with PEDV – Observed/sampled for 14 days

5. PEDV immune sows (n = 2) Euthanized Preparation of "passive" antibody Blood Serum RBCs Serum Ab purification & concentration Blood

6. PEDV Ab negative sows (n = 6) (~110 days gestation) Piglets (n = 62) 6 Treatment groups IP inoculation -1 DPI Anti-PEDV antibody PEDV CHALLENGE 1x 103 TCID50/ml Challenge 0 DPI Animal study DATA - Clinical outcomes - PEDV shedding in feces - Serum/milk Ab profile WV IgG ELISA: S/P < 0.6 IFA titer < 8

7. Treatments • Treatments randomized to piglets by sow – All litters received all treatments • Randomized block design TREATMENT Dilution Treatment 1 0 Treatment 2 1:80 Treatment 3 1:160 Treatment 4 1:320 Treatment 5 1:640 Treatment 6 1:1280

8. Treatments- all litters received all treatments Litter No. of piglets Treatments (no. piglets within treatments) Piglet age (days) at DPI 0 1 2 3 4 5 6 1 13 2 3 2 2 2 2 5 2 11 2 1 2 2 2 2 4 3 9 1 2 1 2 2 1 4 4 10 2 1 2 1 1 3 4 5 10 2 2 1 2 2 1 4 6 9 2 2 2 1 1 1 4 Σ 62 12 12 13 13 12 12

9. PEDV inoculum • PEDV USA/IN/2013/19338E (Vero cells) – 7th passage of the virus • Inoculation – 1 x 103 TCID50 per ml – Mixed with milk replacer (1:4) – 5 ml given orally

10. Daily clinical assessment • Survival • Piglet weight • Body temperature

11. Sample collection • Serum samples (-4, 0, 14) • Milk samples (daily) – centrifuge 13,000 x g, 15 min, 4 ⁰C • Fecal samples (daily) – 1 g + 1 ml PBS (1X)

12. Testing • Antibody profile – Sow milk and piglet serum samples – PEDV FFN, IgG, and IgA ELISA assays • PEDV shedding – Individual piglet fecal swab samples – PEDV real time RT-PCR

13. Data analysis • Linear mixed models – Body weight – Body temperature – Fecal shedding (PEDV rRT-PCR Cq) • Hazard regression analysis – Compare survival x trt • Kruskal-Wallis test – Compare time to death x litter • Wilcoxon rank test – Antibody responses (WV ELISA IgA, IgG)

14. Data analysis • Linear mixed models – Body weight – Body temperature – Fecal shedding (PEDV rRT-PCR Cq) • Hazard regression analysis – Compare survival x trt • Kruskal-Wallis test – Compare time to death x litter • Wilcoxon rank test – Antibody responses (WV ELISA IgA, IgG)

15. Data analysis 1. Treatment defined as 6 groups • 5 different PEDV Ab levels (trt. 2 to 6) • 1 antibody-negative control (trt. 1) IF NO DIFFERENCE DETECTED … then 2. Treatment defined as 2 groups • Group 1: Antibody-positive (trt. 2+3+4+5+6) • Group 2: Antibody-negative control (trt. 1)

16. Piglet weight (lb)

17. Piglet weight (lb) NO SIGNIFICANT DIFFERENCE AMONG TREATMENTS

18. Piglet body temperature (F)

19. Piglet body temperature (F) NO SIGNIFICANT DIFFERENCE AMONG 6 TREATMENTS

20. Piglet body temperature (F)

21. Feces qRT-PCR (converted CT)

22. Feces qRT-PCR (converted CT) NO SIGNIFICANT DIFFERENCE AMONG TREATMENTS

23. Survival (%)

24. Assay (piglet serum) Treatment Day post inoculation -4 0a 14 FFN arithmetic mean 1 <1:8 <1:8 1:64 2 <1:8 1:5.3 1:19.7 3 <1:8 1:6.1b 1:19.7 4 <1:8 1:8.0b 1:32.0 5 <1:8 1:17.1b 1:11.3 6 <1:8 1:32.0b 1:16.0 PEDV IgA ELISA least square mean S/P 1 0.5 0.2 2.2 2 0.7 0.7 2.0 3 0.6 1.1 1.9 4 0.6 1.8b 1.3 5 0.6 2.8b 1.2 6 0.7 3.3b 1.4 PEDV IgG ELISA least square mean S/P (SE) 1 0.6 0.5 1.7 2 0.7 0.7b 1.0 3 0.7 0.7b 1.5 4 0.7 0.8b 1.3 5 0.7 1.1b 1.0 6 0.7 1.4b 0.9 a 24 hours following intraperitoneal administration of concentrated PEDV antibody

25. a 24 hours following intraperitoneal administration of concentrated PEDV antibody Assay (piglet serum) Treatment Day post inoculation -4 0a 14 FFN arithmetic mean 1 <1:8 <1:8 1:64 2 <1:8 1:5.3 1:19.7 3 <1:8 1:6.1b 1:19.7 4 <1:8 1:8.0b 1:32.0 5 <1:8 1:17.1b 1:11.3 6 <1:8 1:32.0b 1:16.0 PEDV IgA ELISA least square mean S/P 1 0.5 0.2 2.2 2 0.7 0.7 2.0 3 0.6 1.1 1.9 4 0.6 1.8b 1.3 5 0.6 2.8b 1.2 6 0.7 3.3b 1.4 PEDV IgG ELISA least square mean S/P (SE) 1 0.6 0.5 1.7 2 0.7 0.7b 1.0 3 0.7 0.7b 1.5 4 0.7 0.8b 1.3 5 0.7 1.1b 1.0 6 0.7 1.4b 0.9

26. a 24 hours following intraperitoneal administration of concentrated PEDV antibody Assay (piglet serum) Treatment Day post inoculation -4 0a 14 FFN arithmetic mean 1 <1:8 <1:8 1:64 2 <1:8 1:5.3 1:19.7 3 <1:8 1:6.1b 1:19.7 4 <1:8 1:8.0b 1:32.0 5 <1:8 1:17.1b 1:11.3 6 <1:8 1:32.0b 1:16.0 PEDV IgA ELISA least square mean S/P 1 0.5 0.2 2.2 2 0.7 0.7 2.0 3 0.6 1.1 1.9 4 0.6 1.8b 1.3 5 0.6 2.8b 1.2 6 0.7 3.3b 1.4 PEDV IgG ELISA least square mean S/P (SE) 1 0.6 0.5 1.7 2 0.7 0.7b 1.0 3 0.7 0.7b 1.5 4 0.7 0.8b 1.3 5 0.7 1.1b 1.0 6 0.7 1.4b 0.9

27. Assay (piglet serum) Treatment Day post inoculation -4 0a 14 FFN arithmetic mean 1 <1:8 <1:8 1:64 2 <1:8 1:5.3 1:19.7 3 <1:8 1:6.1b 1:19.7 4 <1:8 1:8.0b 1:32.0 5 <1:8 1:17.1b 1:11.3 6 <1:8 1:32.0b 1:16.0 PEDV IgA ELISA least square mean S/P 1 0.5 0.2 2.2 2 0.7 0.7 2.0 3 0.6 1.1 1.9 4 0.6 1.8b 1.3 5 0.6 2.8b 1.2 6 0.7 3.3b 1.4 PEDV IgG ELISA least square mean S/P (SE) 1 0.6 0.5 1.7 2 0.7 0.7b 1.0 3 0.7 0.7b 1.5 4 0.7 0.8b 1.3 5 0.7 1.1b 1.0 6 0.7 1.4b 0.9 b Treatment 2-6 showed Ab levels significantly different from treatment 1 (p < 0.02)

28. a 24 hours following intraperitoneal administration of concentrated PEDV antibody Assay (piglet serum) Treatment Day post inoculation -4 0a 14 FFN arithmetic mean 1 <1:8 <1:8 1:64 2 <1:8 1:5.3 1:19.7 3 <1:8 1:6.1b 1:19.7 4 <1:8 1:8.0b 1:32.0 5 <1:8 1:17.1b 1:11.3 6 <1:8 1:32.0b 1:16.0 PEDV IgA ELISA least square mean S/P 1 0.5 0.2 2.2 2 0.7 0.7 2.0 3 0.6 1.1 1.9 4 0.6 1.8b 1.3 5 0.6 2.8b 1.2 6 0.7 3.3b 1.4 PEDV IgG ELISA least square mean S/P (SE) 1 0.6 0.5 1.7 2 0.7 0.7b 1.0 3 0.7 0.7b 1.5 4 0.7 0.8b 1.3 5 0.7 1.1b 1.0 6 0.7 1.4b 0.9

29. Assay (piglet serum) Treatment Day post inoculation -4 0a 14 FFN arithmetic mean 1 <1:8 <1:8 1:64 2 <1:8 1:5.3 1:19.7 3 <1:8 1:6.1b 1:19.7 4 <1:8 1:8.0b 1:32.0 5 <1:8 1:17.1b 1:11.3 6 <1:8 1:32.0b 1:16.0 PEDV IgA ELISA least square mean S/P 1 0.5 0.2 2.2 2 0.7 0.7 2.0 3 0.6 1.1 1.9 4 0.6 1.8b 1.3 5 0.6 2.8b 1.2 6 0.7 3.3b 1.4 PEDV IgG ELISA least square mean S/P (SE) 1 0.6 0.5 1.7 2 0.7 0.7b 1.0 3 0.7 0.7b 1.5 4 0.7 0.8b 1.3 5 0.7 1.1b 1.0 6 0.7 1.4b 0.9 a 24 hours following intraperitoneal administration of concentrated PEDV antibody

30. Conclusions • Passively administered antibody affected – Survival – Thermoregulation • Did not affect – Body weight – PEDV shedding in feces – Antibody responses

31. IgA response sow milk

32. IgA response sow milk Sows exposed to PEDV contaminated piglet feces Sows responded differently

33. IgA response sow milk Treatments were randomized to sows. This means "sow effect" is accounted for.

34. Milk IgA - % survival

35. Milk IgA - % survival Most of the mortality occured before IgA levels in sow milk increased

36. Conclusion • Passive ab protects piglets – Thermoregulation – Survivability • “Sow effect” was accounted for by experimental design • Results do not contradict mainstream ideas of PEDV immunity

37. How might circulating ab affect PEDV infection ? 1. Neutralized PEDV during viremia? 2. Facilitated systemic humoral and/or CMI responses? 3. Antibody-dependent cell-mediated cytotoxicity (ADCC)? 4. Transported IgG neutralized PEDV in the intestinal lumen and/or assisted the GI humoral and CMI responses?

38. Acknowledgement • Funding from the National Pork Board • Thanks to collaborators – Luis G Giménez-Lirola – Marisa Rotolo – Qi Chen – Lucas Correa da Silva Carrion – Chris Olsen – Ronaldo Magtoto

39. Questions?

40. The 2015 North American PRRS Symposium wishes to thank the following sponsors for their generous support:

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