dr. korakrit poonsuk - effect of circulating antibody on the course of pedv infection in neonatal...

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 MainIowa State University

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.

Objective of the study• Identify effect of passively administered PEDV-

specific Ab on: 1) Body weight2) Body temperature3) Survival4) Fecal shedding5) Humoral immune response

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

PEDV immune sows (n = 2)

Euthanized

Preparation of "passive" antibody

Blood Serum

RBCs

Serum

Ab purification & concentration

Blood

PEDV Ab negative sows (n = 6)

(~110 days gestation)

Piglets (n = 62)

6 Treatment groups

IP inoculation-1 DPI

Anti-PEDVantibody

PEDV CHALLENGE1x 103 TCID50/ml

Challenge0 DPI

Animal study

DATA- Clinical outcomes- PEDV shedding in feces- Serum/milk Ab profile

WV IgG ELISA: S/P < 0.6IFA titer < 8

Treatments• Treatments randomized to piglets by sow– All litters received all treatments

• Randomized block design TREATMENT DilutionTreatment 1 0Treatment 2 1:80Treatment 3 1:160Treatment 4 1:320Treatment 5 1:640Treatment 6 1:1280

Treatments - all litters received all treatments

Litter No. of piglets

Treatments (no. piglets within treatments) Piglet age (days)

at DPI 01 2 3 4 5 6

1 13 2 3 2 2 2 2 52 11 2 1 2 2 2 2 43 9 1 2 1 2 2 1 44 10 2 1 2 1 1 3 45 10 2 2 1 2 2 1 46 9 2 2 2 1 1 1 4Σ 62 12 12 13 13 12 12

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

Daily clinical assessment

• Survival• Piglet weight• Body temperature

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)

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

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)

Data analysis1. 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)

Piglet weight (lb)

Piglet weight (lb)

NO SIGNIFICANT DIFFERENCE AMONG TREATMENTS

Piglet body temperature (F)

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

Piglet body temperature (F)

Feces qRT-PCR (converted CT)

Feces qRT-PCR (converted CT)

NO SIGNIFICANT DIFFERENCE AMONG TREATMENTS

Survival (%)

Assay (piglet serum) TreatmentDay post inoculation

-4 0a 14

FFN arithmetic mean

1 <1:8 <1:8 1:642 <1:8 1:5.3 1:19.73 <1:8 1:6.1b 1:19.74 <1:8 1:8.0b 1:32.05 <1:8 1:17.1b 1:11.36 <1:8 1:32.0b 1:16.0

PEDV IgA ELISA least square mean S/P

1 0.5 0.2 2.22 0.7 0.7 2.03 0.6 1.1 1.94 0.6 1.8b 1.35 0.6 2.8b 1.26 0.7 3.3b 1.4

PEDV IgG ELISA least square mean S/P (SE)

1 0.6 0.5 1.72 0.7 0.7b 1.03 0.7 0.7b 1.54 0.7 0.8b 1.35 0.7 1.1b 1.06 0.7 1.4b 0.9

a 24 hours following intraperitoneal administration of concentrated PEDV antibody



-4 0a 14

FFN arithmetic mean

1 <1:8 <1:8 1:642 <1:8 1:5.3 1:19.73 <1:8 1:6.1b 1:19.74 <1:8 1:8.0b 1:32.05 <1:8 1:17.1b 1:11.36 <1:8 1:32.0b 1:16.0


1 0.5 0.2 2.22 0.7 0.7 2.03 0.6 1.1 1.94 0.6 1.8b 1.35 0.6 2.8b 1.26 0.7 3.3b 1.4


1 0.6 0.5 1.72 0.7 0.7b 1.03 0.7 0.7b 1.54 0.7 0.8b 1.35 0.7 1.1b 1.06 0.7 1.4b 0.9


-4 0a 14

FFN arithmetic mean

1 <1:8 <1:8 1:642 <1:8 1:5.3 1:19.73 <1:8 1:6.1b 1:19.74 <1:8 1:8.0b 1:32.05 <1:8 1:17.1b 1:11.36 <1:8 1:32.0b 1:16.0


1 0.5 0.2 2.22 0.7 0.7 2.03 0.6 1.1 1.94 0.6 1.8b 1.35 0.6 2.8b 1.26 0.7 3.3b 1.4


1 0.6 0.5 1.72 0.7 0.7b 1.03 0.7 0.7b 1.54 0.7 0.8b 1.35 0.7 1.1b 1.06 0.7 1.4b 0.9

b Treatment 2-6 showed

Ab levels significantly

different from treatment 1

(p < 0.02)



-4 0a 14

FFN arithmetic mean

1 <1:8 <1:8 1:642 <1:8 1:5.3 1:19.73 <1:8 1:6.1b 1:19.74 <1:8 1:8.0b 1:32.05 <1:8 1:17.1b 1:11.36 <1:8 1:32.0b 1:16.0


1 0.5 0.2 2.22 0.7 0.7 2.03 0.6 1.1 1.94 0.6 1.8b 1.35 0.6 2.8b 1.26 0.7 3.3b 1.4


1 0.6 0.5 1.72 0.7 0.7b 1.03 0.7 0.7b 1.54 0.7 0.8b 1.35 0.7 1.1b 1.06 0.7 1.4b 0.9

Conclusions• Passively administered antibody affected – Survival– Thermoregulation

• Did not affect– Body weight– PEDV shedding in feces– Antibody responses

IgA response sow milk

IgA response sow milkSows exposed to PEDV

contaminated piglet fecesSows responded differently

IgA response sow milkTreatments were randomized

to sows. This means "sow effect" is accounted for.

Milk IgA - % survival

Milk IgA - % survivalMost of the mortality occured before IgA

levels in sow milk increased

Conclusion• Passive ab protects piglets – Thermoregulation– Survivability

• “Sow effect” was accounted for by experimental design

• Results do not contradict mainstream ideas of PEDV immunity

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?

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

Questions?

The 2015 North American PRRS Symposium wishes to thank the following sponsors for their generous

support:

dr. korakrit poonsuk - effect of circulating antibody on the course of pedv infection in neonatal...

Science