Role of Pre-Farrow Natural Planned Exposure of Gilts in Shaping the Passive Antibody Response to Rotavirus A in Piglets

Abstract

Natural planned exposure (NPE) remains one of the most common methods in swine herds to boost lactogenic immunity against rotaviruses. However, the efficacy of NPE protocols in generating lactogenic immunity has not been investigated before. A longitudinal study was conducted to investigate the dynamics of genotype-specific antibody responses to different doses (3, 2 and 1) of Rotavirus A (RVA) NPE (genotypes G4, G5, P[7] and P[23]) in gilts and the transfer of lactogenic immunity to their piglets. Group 1 gilts received three doses of NPE at 5, 4 and 3 weeks pre-farrow (WPF), group 2 received two doses at 5 and 3 WPF, group 3 received one dose at 5 WPF, and group 4 received no NPE (control group). VP7 (G4 and G5) and truncated VP4* (P[7] and P[23]) antigens of RVA were expressed in mammalian and bacterial expression systems, respectively, and used to optimize indirect ELISAs to determine antibody levels against RVA in gilts and piglets. In day-0 colostrum samples, group 1 had significantly higher IgG titers compared to the control group for all four antigens, and either significantly or numerically higher IgG titers than groups 2 and 3. Group 1 also had significantly higher colostrum IgA levels than the control group for all antigens (except G4), and either significantly or numerically higher IgA levels compared to groups 2 and 3. In piglet serum, group 1 piglets had higher IgG titers for all four antigens at day 0 than the other groups. Importantly, RVA NPE stimulated antibodies in all groups regardless of the treatment doses and prevented G4, G5, P[7] and P[23] RVA fecal shedding prior to weaning in piglets in the absence of viral challenge. The G11 and P[34] RVA genotypes detected from pre-weaning piglets differed at multiple amino acid positions with parent NPE strains. In conclusion, the results of this study suggest that the group 1 NPE regimen (three doses of NPE) resulted in the highest anti-RVA antibody (IgG and IgA) levels in the colostrum/milk, and the highest IgG levels in piglet serum.

Keywords: ELISA; antibody; natural planned exposure; rotavirus A; sequencing; swine.

Figure 1

NPE administration and sample collection (serum, colostrum, and milk) schedule. NPE administration to gilts, farrowing and weaning are indicated by red, blue and green triangles. Gilts (N = 12 per group) and five piglets per gilt were sampled individually. Control group gilts did not receive NPE.

Figure 2

Validation of antigen expression via immunocytometric staining and immunoblotting. (A) HEK293A cells transfected with pcDNA3.1 (+) construct encoding G5 VP7 RVA showing positive anti-His tag staining. (B) HEK293A cells transfected with pcDNA3.1 (+) construct encoding G4 VP7 RVA showing positive anti-His tag staining. (C) Negative control. (D) Purified G5 RVA VP7 (37 kd). (E) Purified G4 RVA VP7 (37 kd). M—protein marker; S—culture supernatant; P—cell pellet; FT—flow through; W—wash fraction; E1, E2, E3 and E4—sequential G4 protein elutes. (F) Purified P[7] and P[23] VP4* (55 kd) protein elutes. (G) Western blot confirmation of the affinity-purified G5, G4, P[7] and P[23] antigens using anti-His monoclonal primary and HRP-tagged anti-mouse IgG secondary antibodies.

Figure 3

Kinetics of gilt serum antibody levels against RVA. Progression of serum IgG and IgA levels over time in gilts receiving three (group 1), two (group 2), one (group 3) or no (group 4, control) doses of NPE. Gilt serum IgG levels against G5 (A), G4 (B), P[7] (C) and P[23] (D) RVA antigens. Gilt serum IgA levels against G5 (E), G4 (F), P[7] (G) and P[23] (H) RVA antigens. The horizontal axis represents sample collection time-points (−5 W = 5 weeks pre-farrow; −3 W = 3 weeks pre-farrow; F = at farrowing; +3 W = 3 weeks post-farrow or at weaning). The vertical axis represents geometric mean antibody titers (GMTs). * p < 0.033, ** p < 0.002, *** p < 0.001, mixed effects model.

Figure 3

Kinetics of gilt serum antibody levels against RVA. Progression of serum IgG and IgA levels over time in gilts receiving three (group 1), two (group 2), one (group 3) or no (group 4, control) doses of NPE. Gilt serum IgG levels against G5 (A), G4 (B), P[7] (C) and P[23] (D) RVA antigens. Gilt serum IgA levels against G5 (E), G4 (F), P[7] (G) and P[23] (H) RVA antigens. The horizontal axis represents sample collection time-points (−5 W = 5 weeks pre-farrow; −3 W = 3 weeks pre-farrow; F = at farrowing; +3 W = 3 weeks post-farrow or at weaning). The vertical axis represents geometric mean antibody titers (GMTs). * p < 0.033, ** p < 0.002, *** p < 0.001, mixed effects model.

Figure 4

Kinetics of colostrum/milk antibody levels against RVA. Progression of colostrum/milk IgG and IgA levels over time in gilts receiving three (group 1), two (group 2), one (group 3) or no (group 4, control) doses of NPE. Colostrum/milk IgG levels against G5 (A), G4 (B), P[7] (C) and P[23] (D) RVA antigens. Colostrum/milk IgA levels against G5 (E), G4 (F), P[7] (G) and P[23] (H) RVA antigens. The horizontal axis represents sample collection time-points (−5 W = 5 weeks pre-farrow; −3 W = 3 weeks pre-farrow; F = at farrowing; +3 W = 3 weeks post-farrow or at weaning). The vertical axis represents geometric mean antibody titers (GMTs). * p < 0.033, ** p < 0.002, *** p < 0.001, mixed effects model.

Figure 4

Kinetics of colostrum/milk antibody levels against RVA. Progression of colostrum/milk IgG and IgA levels over time in gilts receiving three (group 1), two (group 2), one (group 3) or no (group 4, control) doses of NPE. Colostrum/milk IgG levels against G5 (A), G4 (B), P[7] (C) and P[23] (D) RVA antigens. Colostrum/milk IgA levels against G5 (E), G4 (F), P[7] (G) and P[23] (H) RVA antigens. The horizontal axis represents sample collection time-points (−5 W = 5 weeks pre-farrow; −3 W = 3 weeks pre-farrow; F = at farrowing; +3 W = 3 weeks post-farrow or at weaning). The vertical axis represents geometric mean antibody titers (GMTs). * p < 0.033, ** p < 0.002, *** p < 0.001, mixed effects model.

Figure 5

Kinetics of piglet serum antibody response to RVA. Progression of piglet serum IgG and IgA levels over time in piglets born to gilts receiving three (group 1), two (group 2), one (group 3) or no (group 4, control) doses of NPE. Piglet serum IgG levels against G5 (A), G4 (B), P[7] (C) and P[23] (D) RVA antigens. Piglet serum IgA levels against G5 (E), G4 (F), P[7] (G) and P[23] (H) RVA antigens. The horizontal axis represents sample collection time-points (−5 W = 5 weeks pre-farrow; −3 W = 3 weeks pre-farrow; F = at farrowing; +3 W = 3 weeks post-farrow or at weaning). The vertical axis represents geometric mean antibody titers (GMTs). * p < 0.033, ** p < 0.002, *** p < 0.001, mixed effects model.

Figure 5

Kinetics of piglet serum antibody response to RVA. Progression of piglet serum IgG and IgA levels over time in piglets born to gilts receiving three (group 1), two (group 2), one (group 3) or no (group 4, control) doses of NPE. Piglet serum IgG levels against G5 (A), G4 (B), P[7] (C) and P[23] (D) RVA antigens. Piglet serum IgA levels against G5 (E), G4 (F), P[7] (G) and P[23] (H) RVA antigens. The horizontal axis represents sample collection time-points (−5 W = 5 weeks pre-farrow; −3 W = 3 weeks pre-farrow; F = at farrowing; +3 W = 3 weeks post-farrow or at weaning). The vertical axis represents geometric mean antibody titers (GMTs). * p < 0.033, ** p < 0.002, *** p < 0.001, mixed effects model.