Orally delivered bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens

Authors: WICKRAMASURIYA, SAMIRU - US Department Of Agriculture (USDA); PARK, INKYUNG - US Department Of Agriculture (USDA); LEE, YOUNGSUB - US Department Of Agriculture (USDA); RICHER, LUCIANA - Us Biologics; PRZYBYSZEWSKI, C - Us Biologics; Gay, Cyril; VAN OOSTERWIJK, JOLIEKE - Us Biologics; Lillehoj, Hyun

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2023
Publication Date: 5/1/2023
Citation: Wickramasuriya, S.S., Park, I., Lee, Y., Richer, L.R., Przybyszewski, C., Gay, C.G., Van Oosterwijk, J.G., Lillehoj, H.S. 2023. Orally delivered bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens. Poultry Science. https://doi.org/10.1016/j.psj.2023.102590.
DOI: https://doi.org/10.1016/j.psj.2023.102590

Interpretive Summary: Coccidiosis is caused by several distinct species of Eimeria parasites which costs global poultry industry more than $ 14.5 billion annual losses. Development of a novel antibiotic-alternative strategy to mitigate coccidiosis will have a significant impact on poultry industry since the use of in-feed antibiotics in subtherapeutic levels has been banned since 2014 although therapeutic levels of antibiotics are still used to treat sick chickens. In 2021, we reported the successful construction and delivery of a stable transgenic bacteria (Bacillus subtilis) carrying a chicken anti-microbial peptide, NK-lysin peptide (B. subtilis-cNK-2), as an effective oral therapeutic antibiotic alternative strategy to reduce the negative impact of coccidiosis in commercial poultry production. This new report describes the underlying gut microbiota changes in the Eimeria-uninfected, Eimeria-infected (two control groups), and in chickens which are treated with B. subtilis-cNK-2. Changes in gut microbiota populations in B. subtilis-cNK2-treated group compared to the untreated control group after coccidiosis infection indicates many beneficial changes associated with B. subtilis-cNK-2 treatment that may be practical and effective to mitigate the negative effects of avian coccidiosis in commercial broilers.

Technical Abstract: We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose(1×10^12) of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, one hundred (14-day old) broiler chickens were allocated into four treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts through oral gavage on day 15. Chickens given B. subtilis (EV or NK) were orally gavaged (1×10^12) daily from day 14 to day 18. Growth performances were measured on days 6, 9, and 13 post-infections (dpi). Spleen and duodenal samples were collected from the randomly selected chickens on 6 dpi to assess the gut microbiota, gut integrity, and local inflammation. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3–1E antibody levels. E. acervulina-infected chickens treated with B. subtilis with cNK-2 showed significantly improved (p<0.05) growth performance, gut integrity, and mucosal immunity compared to the NC group. Moreover, NK chickens showed reduced fecal oocyst shedding (p<0.05) compared to the other infected (NC and EV) chickens. Interestingly, there was a distinct shift in the gut microbiota profile in NK group compared to that of NC and EV groups. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In chickens in the NK group, however, the ratio between Firmicutes and Cyanobacteria was not affected, and was similar to that of CON group. Taken together, treatment with B. subtilis-cNK2 restored dysbiosis incurred by E. acervulina infection and provided the general protective effects on coccidiosis infection including reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis.