Orally-delivered Bacillus subtilis carrying Eimeria profilin gene protects against Eimeria tenella infection in chickens

Protective effect of orally-delivered Bacillus subtilis spores expressing Eimeria profilin (3-1E) antigen against Eimeria tenella (E. tenella) infection in broiler chickens was evaluated. Total 168 male broiler chickens (Ross 708, 24/group) were orally immunized with B. subtilis-3-1E recombinants or B. subtilis-EV spores (control) at a dose of 1 × 10⁹/bird from day 2 of age for three consecutive days per week for two weeks as follows: uninfected control (NC), E. tenella-infected control (PC), chickens immunized with B. subtilis-EV (EV), chickens immunized with B. subtilis-3-1E #147 spores (#147), chickens immunized with B. subtilis-3-1E #241 spores (#241), chickens immunized with B. subtilis-3-1E #285-2 spores (#285-2), and chickens immunized with B. subtilis-3-1E #879 spores (#879). On day 16, all chickens, except NC group, were orally infected with 1.0 × 10⁴ sporulated-E. tenella oocysts per chicken, and the protective effect against E. tenella was assessed on days 4 and 7 post infection (dpi). There was no significant change in body weight (BW) due to immunization with B. subtilis spores prior to E. tenella infection. Following E. tenella infection, a significantly decreased (p<0.05) BW was observed in the PC group of chickens at 4 and 7 dpi compared to the NC group. However, chickens in the #147 and #879 groups showed a significantly higher BW (p<0.01) compared to those in the PC group on 7 dpi. Intestinal lesion scores were significantly lower in the #147 group compared to those in the PC and EV groups. Serum IgY antibody levels were significantly higher (p<0.05) in the #147 and #241 groups, and intestinal IgA antibody levels were significantly increased (p<0.01) in the #147, #285-2 and #879 groups compared to the PC group. Splenocytes collected from #147 and #285-2 groups showed a significantly higher (p<0.05) antigen-specific lymphocyte proliferation response following stimulation with recombinant 3-1E protein. Quantitative RT-PCR analysis revealed that the expression levels of occludin (OCLN) in the ceca of E. tenella-infected chickens were significantly upregulated (p<0.05) in the #147 and #285-2 groups, and the expression of JAM-2 gene was increased in the #147 group compared to the PC group following E. tenella infection. Importantly, most spores collected from the cecal tissues of B. subtilis-3-1E-immunized chickens still retained 3-1E plasmids 12 days post-immunization, demonstrating the high stability and remarkable potential of orally delivered B. subtilis spores to act as an effective vaccine vehicle.