Non Technical Summary
The continuing circulation of multiple Salmonella serovars in poultry flocks, along with increasing reports of human salmonellosis, warrants the necessity of complementary strategies against a broad range of serovars. The goal of this application is to evaluate the safety and efficacy of a live probiotic Bacillus subtilis strain in reducing the Salmonella load in broiler birds. The PI has engineered B. subtilis-FliC to secrete monovalent and bivalent neutralizing nanobodies (Nbs) against a key virulent factor, Flagellin C in Salmonella. Nbs from Camelidae feature small size, high solubility, protease resistance, and high thermal stability compared to conventional antibodies. While natural B. subtilis probiotics have been identified to promote gut health in poultry, a major distinction of this proposal is to rationally design B. subtilis with traits against virulent factors to directly inhibit Salmonella colonization and loads in broilers. The platform technology from this proposal will ultimately open opportunities for targeting other enteric pathogens in poultry and livestock, thereby promoting the well-being of agricultural animals, and ensuring a safe and abundant food supply.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1040	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology;

Goals / Objectives
The objective of this study is to engineer live probiotic Bacillus subtilis (Bs) strains as feed additives and evaluate their efficacy in reducing the Salmonella load in broiler birds. These strains are genetically modified to secrete monovalent (BsFliC) or bivalent (BsbiFliC) neutralizing nanobodies (Nbs) against key virulent factors such as Flagellin C (FliC), which facilitates bacterial motility, colonization, and host cell invasion for Salmonella. Because Bs can form spores that are resistant to various stress conditions, engineered Bs strains are shelf-stable, cost- effective, and feed-stable, and hence will facilitate widespread applications in the poultry industry.

Project Methods
1)Characterize and compare thein vitroefficacy ofBsFliCandBsbiFliCstrains in multiple serovars ofS. enterica2) Characterize thein vivocolonization dynamics and safety ofBsFliCandBsbiFliCsupplementation in chickens3)Quantify the efficacy ofBsFliCandBsbiFliCin decreasingSalmonellaload in broilers challenged withSalmonella