Expression of Eimeria profilin in Bacillus subtilis

Authors: Panebra, Alfredo; LEE, YOUNGSUB - US Department Of Agriculture (USDA); Lillehoj, Hyun

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2025
Publication Date: 6/1/2025
Citation: Panebra, A., Lee, Y., Lillehoj, H.S. 2025. Expression of Eimeria profilin in Bacillus subtilis. Poultry Science. https://doi.org/10.1016/j.psj.2025.105112.
DOI: https://doi.org/10.1016/j.psj.2025.105112

Interpretive Summary: Coccidiosis is caused by several different species of Eimeria parasites which invade the chicken intestinal epithelial cells resulting in serious gut damage. This disease is a major poultry welfare problem, causing significant economic losses estimated at $13 billion annually worldwide. In this paper, ARS scientists in Beltsville describe a novel method of constructing Bacillus spores to carry an immunodominant antigen of Eimeria called profilin to vaccinate chickens orally. Profilin is a major immunodominant antigen of all Eimeria parasites and the authors demonstrate in this work its ability to induce protective host immunity against coccidiosis, a disease caused by several distinct species of Eimeria parasites. Furthermore, Bacillus spores are heat resistant so they can be given orally in the feed at the farm level. Ability to vaccinate chickens orally at the farm level using Bacillus spores carrying a major immunogenic protein of Eimeria parasites will reduce the economic losses due to coccidiosis and enable industry to control coccidiosis in commercial chickens.

Technical Abstract: Coccidiosis is caused by apicomplexan protozoa of the genus Eimeria, which invade chicken intestinal epithelial cells, resulting in gut damage and a major poultry welfare problem worldwide. In this study, we developed a Bacillus subtilis (B. subtilis)-based vaccine delivering E. acervulina profilin (3-1E) antigen to induce protective immunity against coccidiosis in the host. A library of pBE-S-3-1E plasmid was constructed by subcloning a 3-1E open reading frame into the shuttle vector pBE-S. This library comprised approximately 900 recombinants, all expressing and secreting 3-1E, but each recombinant contained a distinct signal peptide. Following three rounds of screening using 3-1E-specific monoclonal antibodies (mAbs), 25 higher expressors were isolated and sequenced to identify the signal peptide driving 3-1E expression. From these 25 candidates, four high-expressing recombinants (#147, #241, #285-2, and #879), along with the empty vector (EV), were selected for further in vitro and in vivo assays. All recombinant clones sporulated, but clone #241 germinated at a higher rate compared to the others. Secretion of 3-1E by all germinated recombinant clones was confirmed by western blot and indirect ELISA, and further visualized by immunofluorescence assay (IFA). The cultured-medium of all recombinant clones induced nitrite release and were neutralized by 3-1E mAb (#320). In addition, the cultured-medium of all recombinant clones induced significant expression of chicken IL-4, IL-6, TNF-a, IL-10 and IFN-' (p<0.05). In vitro, phagocytosis of recombinants by HD11 cells was significantly decreased in order of #147, #241, #285-2 and #879 compared to EV (P<0.0001). A pilot trial (N=30) was conducted to evaluate humoral and cellular immune responses in broiler chickens immunized with recombinant spores, as well as to assess spore persistence in chicken ceca in vivo. Chickens immunized with all recombinant spores exhibited significantly higher serum IgY and cecal IgA levels to recombinant 3-1E protein compared to EV group (P<0.0001). Furthermore, splenocytes from immunized chickens demonstrated significantly increased proliferation when stimulated with recombinant 3-1E protein compared to the EV group. All colonies collected from the ceca of chickens immunized with recombinant spores 10 days post-immunization with B. subtilis-3-1E were identified as positive by colony PCR.