Effect of orally administered B. subtilis-cNK-2 on Growth performance, immunity, gut health, and gut microbiome in chickens during Eimeria acervulina challenge, and its potential as an alternative to antibiotics

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

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2024
Publication Date: 11/1/2024
Citation: Wickramasuriya, S.S., Park, I., Lee, Y., Richer, L.M., Przybyszewski, C., Gay, C.G., Van Oosterwijk, J.G., Lillehoj, H.S. 2024. Effect of orally administered B. subtilis-cNK-2 on Growth performance, immunity, gut health, and gut microbiome in chickens during Eimeria acervulina challenge, and its potential as an alternative to antibiotics. Poultry Science. 103(11). Article e104156. https://doi.org/10.1016/j.psj.2024.104156.
DOI: https://doi.org/10.1016/j.psj.2024.104156

Interpretive Summary: Since the regulatory restriction of using antibiotics in animal agriculture starting early 2010, there have been increasing research activities in developing antibiotic alternative strategies to improve gut health to mitigate negative consequences of intestinal infections in commercial poultry production. Recently, we reported the clinical success of a novel oral-delivery system using B. subtilis spores expressing chicken NK-lysin peptide (B. subtilis-cNK-2) as an alternative to antibiotics strategy to mitigate avian coccidiosis caused by Eimeria intestinal parasites. In this report, we evaluated the gut bacterial population changes associated with B. subtilis-cNK-2 treatment to better understand the underlying mechanisms associated with enhanced resistance against coccidiosis associated with dietary B. subtilis-cNK-2 treatment. In chickens which have been treated with cNK-2, we found a higher population of certain beneficial bacterial populations such as Lactobacillus in the gut compared to the nontreated control group. Furthermore, cNK-2-treated chickens showed a favorable immune response profile associated with improved host gut health. These results support a notion that an antibiotic alternative strategy using B. subtilis-cNK-2 may be effective in the mitigation of coccidiosis in broiler chickens. Finally, we confirm that B. subtilis-cNK-2 treatment of young chickens led to enhanced growth performance, increased immune modulation, reduced parasite numbers, and favorable alteration of gut microbiome in chickens. These results support the potential use of dietary B. subtilis-cNK-2 spores as an effective alternative to antibiotics to improve gut health in broiler chickens. Further studies will focus on the development of the most effective delivery of the B. subtilis-cNK-2, focusing on scaling up the technology to bring it into commercial poultry operations.

Technical Abstract: This study investigated the effects of oral delivery of B. subtilis expressing the chicken anti-microbial peptide cNK2 (B. subtilis-cNK-2) in comparison to Monensin, in chickens challenged with Eimeria acervulina. Oral B. subtilis-cNK-2 resulted in beneficial effects on broiler chicken growth performance, oocyst shedding, and gut health of broiler chickens. A total of 120 broiler chickens were randomly allocated into five treatment groups: 1) non-challenged chickens fed a basal diet (NC), 2) E. acervulina-challenged chickens fed a basal diet (PC), 3) E. acervulina-challenged chickens fed a basal diet supplemented with 90 mg Monensin/kg feed (MO), 4) E. acervulina-challenged chickens fed a basal diet and orally gavaged with B. subtilis-cNK-2 at 1×10^10 cfu/day (CNK-O), and 5) E. acervulina-challenged chickens fed a basal diet mixed with B. subtilis-cNK-2 at 1×10^10 cfu/kg feed (CNK-F). The challenge consisted of 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts through oral gavage on day 15. Body weights were measured on days 7, 14, 21, and 23. Duodenal tissue and digesta samples were collected at 6-days post-infection (dpi) to assess the gut integrity, oxidative makers, mucosal immunity, and the gut microbiome. To enumerate oocyst shedding, fecal samples were collected from 6 to 8 dpi. Chickens in the CNK-O group showed improved (p < 0.05) growth performance, gut integrity, and mucosal immunity compared to untreated control (PC), comparable to chickens in the MO group. Chickens in the MO, CNK-F, and CNK-O treatment groups all showed lower (p < 0.05) oocyst shedding compared to untreated (PC) chickens. Moreover, distinct cytokine production, oxidative stress measures, tight junction, and shifts in the gut microbiome and associated functional changes were observed in all challenge groups. Here we show that oral administration of B. subtilis-cNK-2 improves growth performance, enhances local protective immunity, and reduces fecal oocyst shedding of broiler chickens, demonstrating potential as an alternative to antibiotics to protect chickens against coccidiosis.