Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae

Abstract

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA(+) B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA(+) B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.

Figure 1

The immune response of pigs after immunization with F4 fimbriae. The F4-specific IgG antibody responses (A), the F4-specific IgA antibody responses (B), number of F4-specifc total antibody secreting cells (ASCs) per 3.0 × 10⁸ peripheral blood monomorphonuclear cells (PBMCs) (C), number of F4-specific IgA⁺ ASCs per 3.0 × 10⁸ PBMCs (D). Piglets were immunized with F4 fimbriae at day 0 and at 14 days post primary immunization (dppi). Data are presented as the mean ± SEM. The asterisk indicates a statistical significant difference (p < 0.05) within a group between a time point and day 0, while letters indicate a statistical significance (p < 0.05) between groups at a designated time point; (A) in comparison to the maternal-PBS group, (b) to the seronegative-oral group. ↑, immunization time points.

Figure 2

The efficiency of using ELISpot on enriched IgA ⁺ B-cell fractions to enumerate F4-specific IgA ⁺ antibody secreting cells (ASCs). A In one well, only one F4-specific IgA⁺ ASC was detected from 5 × 10⁵ peripheral blood monomorphonuclear cells (PBMCs) (left) while 18 F4-specific spots were detected from 5 × 10⁵ enriched IgA⁺ B-cells (right) at 21 days post primary immunization (dppi) of an oral immunized pig having maternal antibodies; each blue spot is counted as one F4-specific IgA⁺ ASC. B From eight wells, less than seven F4-specific IgA⁺ ASCs were detected from 4.0 × 10⁶ PBMCs whereas more than 28 F4-specific IgA⁺ ASCs were detected from 4.0 × 10⁶ enriched IgA⁺ B-cells at 21 dppi of the three immunized groups. The asterisk indicates a statistical significant difference (p < 0.05) between groups in comparison to the maternal-PBS group.