Oral Delivery of Bacillus subtilis expressing chicken NK-2 peptide protects against Eimeria acervulina challenge infection in broiler chickens

Chicken NK-lysin peptide 2 (cNK-2) is a natural lytic peptide with direct cytotoxicity against many apicomplexan parasites, including Eimeria. Developing an effective oral delivery strategy to express cNK-2 in the intestine, where Eimeria parasites interact with the host's gut epithelial cells, may effectively reduce the fecundity of parasites and minimize intestinal damage. Furthermore, cNK-2 modulates gut immune responses to decrease local inflammation elicited by Eimeria infection in the intestine. Therefore, we developed a stable strain of Bacillus subtilis (B. subtilis) that carries chicken NK2 peptide to the gut to determine its effectiveness in ameliorating the negative impacts of coccidiosis and to replace the use of antibiotics in controlling coccidiosis in commercial broiler chicken production. Chickens were allocated into eight treatment groups in a completely randomized design that consisted of two controls (NC: challenged negative control without B. subtilis; CON: unchallenged control), three B. subtilis-treated groups with empty vector (EV6: 106 cfu/day; EV8: 108 cfu/day; EV10: 1010 cfu/day), and three treatment groups with B. subtilis-cNK2 (NK6: 106 cfu/day/bird; NK8: 108 cfu/day/bird; NK10: 1010 cfu/day/bird). All chickens were challenged with 5,000 sporulated E. acervulina oocysts through oral gavage, except the CON group, on day 15. Chickens were given an oral dose of B. subtilis on days 14, 15, and 16, and body weight, weight gain, and fecal oocyst shedding were measured. To investigate the efficacy of B. subtilis-cNK2 against coccidiosis, gene expression of gut health-related biomarkers, including SOD1, CAT, and HMOX1, for oxidative stress in the spleen and intestinal mucosa and tight junction protein (occludin, ZO-1, and JAM2) and mucin gene (MUC2) expression in the gut were measured. The results showed that B. subtilis-cNK2 is a promising and effective alternative strategy to replace antibiotics against coccidiosis based on its ability to reduce parasite survival, to reduce body weight loss caused by coccidiosis and to decrease gut damage based on the enhanced expression of proteins associated with the integrity of the gut.