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

Authors: WICKRAMASURIYA, SAMIRU - US Department Of Agriculture (USDA); PARK, INKYUNG - US Department Of Agriculture (USDA); VAN OOSTERWIJK, JOLIEKE - Us Biologics; Gay, Cyril; PRZYBYSZWEK, CHRIS - Us Biologics; Lillehoj, Hyun

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2021
Publication Date: 6/4/2021
Citation: Wickramasuriya, S.S., Park, I., Van Oosterwijk, J., Gay, C.G., Przybyszwek, C., Lillehoj, H.S. 2021. Bacillus subtilis expressing chicken NK-2 peptide protects against Eimeria acervulina challenge infection in broiler chickens. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2021.684818.
DOI: https://doi.org/10.3389/fvets.2021.684818

Interpretive Summary: Coccidiosis is a major intestinal disease of poultry caused by several different species of Eimeria parasites infecting the different areas of the gut. Coccidiosis primarily damages the intestine decreasing nutrient utilization and causes an annual loss of over $3.2 billion to the poultry industry globally. In this paper, ARS scientists collaborated with scientists at a private company to construct a stable Bacillus strain that can transports a chicken NK lysin gene (Bacllus-cNk2) to the gut. Chicken NK-lysin (cNK-2) is a natural peptide that has been previously reported by ARS scientists to have direct killing and immunity-modulating effects against Eimeria parasites. When chickens were fed with dietary probiotics using Bacillus-cNK2, they showed reduced oocyst production following Eimeria challenge infection. These results support a notion that Bacillus spores can be effectively used to transport an anti-microbial protein to the chicken gut to kill intestinal parasites to reduce gut damage due to coccidiosis. Therefore, dietary treatment of young chickens with Bacillus-cNK2 is a practical antibiotic alternative method to control coccidiosis infection in poultry.

Technical Abstract: 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.