Effective Competitive Exclusion: Increasing Compliance and Widespread Utilization with Cost-Effective Feed-Stable Isolates

Non-Technical Summary: Foodborne illness is a significant worldwide public health problem, and poultry and poultry products, reportedly, continue to be prevailing vehicles for pathogens. This research examines the use of feed-based probiotics as a mechanism for antemortem food safety intervention. <P> Approach: (Objective A) In preliminary screening, three Bacillus isolates were similarly identified (2 species are GRAS listed) with in vitro activity with one showing in vivo activity similar to the previously commercialized LAB culture. Very recent studies have suggested that the mode of action of one effective culture involves initiation of host defense mechanisms, involving very rapid submucosal translocation of live Lactobacilli and rapid activation of innate immune responses (see below). Although an effective LAB combination in vivo was identified and commercialized, this was done without mechanistic understanding of host innate immune activation other than indirect exclusion of Salmonella. Labeling of effective LAB cultures with fluorescein isothiocyanate (FITC), while maintaining viability, has indicated rapid submucosal translocation of effective isolates (36, 44). This characteristic may be essential for activation of host immune defense and may quickly differentiate effective candidate isolates and identify gut regions initially colonized by individual isolates, allowing for increased coverage of the gastrointestinal tract with selected mixed cultures. To further characterize and differentiate effects on innate immune system activation, candidate cultures will be evaluated for effects on goblet cell type and numbers, effects on mucin secretion and type, heterophil activation and submucosal infiltration, and monocyte/macrophage activation according to published techniques ongoing in our laboratories (32, 33). (Objective B) Using a novel thin layer semi-solid culture system recently developed in our laboratories, we have confirmed generation of >1010 spores/gm wet weight, with more than 1011 spores/gm dry weight, numbers much higher than recently published fermentation methods declaring their methods as the best to date (25). Importantly, neither vegetative growth nor sporulation have been completely optimized for this system. Achieving these final numbers of spores, we only observed sporulation efficiency of ~22%, suggesting that optimization could greatly improve yield. Current estimates indicate that this process could be up-scaled for production of very high beneficial spore counts at very low cost, using medium containing only food-grade ingredients. However, preliminary results have been confirmed. (Objective C) Previous research has elucidated an effective in vitro screening technique for identification of candidate probiotic organisms (5). Initial screens will be similarly performed on enteric Bacillus isolates from commercial broiler sources. As these isolates are subjected to in vivo screening as described in Objective A, retained frozen isolates will be sorted according to species (3) and highly effective combinations will be further evaluated in neonatal chicks for exclusion efficacy for Salmonella and Campylobacter as previously described (8). Safety testing will be performed as recently described for initial successful isolates (5). Selected isolates will be submitted to American Type Culture Collection (Manassas, VA) patent depository for reference and storage safety purposes.