Our long-term goal is to develop drinking water deliverable safe and effective Salmonella and Campylobacter vaccines for poultry, which could confer an increased depth and breadth of immunity to reduce food poisoning from Salmonella and Campylobacter. Our overall objective is to develop an efficacious subunit bivalentSalmonellaandCampylobactervaccines using chitosan- nanoparticles (NPs) and potent mucosal adjuvant/s, containing immunogenic antigens outer membrane proteins(OMP)and flagella (FLA). BothSalmonellaandCampylobacterantigens will be entrapped in chitosan NPs and surface labeled withSalmonellaFLA and mannose to specifically target NPs to immune cells in the intestines to elicit mucosal immunity.It was estimated that poultry is associated with 25% of the foodborne outbreaks in the US (Chai, Cole et al. 2017). The presence of multiple serotypes of Salmonella and Campylobacter species (primarily C. jejuni) in poultry flocks with increased reports of human Salmonellosis and Campylobacteriosis warrants the necessity for developing a broadly protective and easily deliverable vaccine to use in poultry. Poultry is known to be a predominant reservoir of C. jejuni, where it can be detected in high abundance (up to 109 CFU/g of cecum) and for an extended period (until slaughter) in the intestinal track and chicken fecal shedding, and thus, can be rapidly disseminated across the whole flock (up to 100%). Similar observations were made with Salmonella in addition to its capacity to become systemic. By consequence, poultry products contaminated with Campylobacter and Salmonella are frequent post-slaughter. However, no effective control strategies and vaccines are available to control these two dreaded enteric foodborne pathogens in broilers.We have an optimized method to prepare mucoadhesive chitosan NPs vaccine entrapped with immunodominant antigens of S. Enteritidis, OMP and FLA, with surface labeled FLA and mannose (Sal-NPs). In our pilot studies in broilers, oral inoculated Sal-NPs was found localized in intestinal immune sites and induced specific IgA and IgG antibodies, activated (IFNγ+) lymphocytes and reduced the challenge bacterial load by over 1 log10 in the cecal content. This study is aimed at improving the efficacy of Sal-NPs vaccine by including strong mucosal vaccine adjuvants (Mycobacterium smegmatis whole cell lysate and c-di-GMP) and optimize its drinking water delivery method to reduce the risk of food poisoning in broilers. Further, by adapting similar platform develop a Campylobacter-NPs (Campy-NPs) subunit vaccine to reduce the risk of Campylobacteriosis in humans caused by the consumption of contaminated poultry meat.Soluble subunit antigens are poorly immunogenic, but they can be made highly immunogenic by entrapping in NPs (Bacon, Makin et al. 2000, Bertram, Bernard et al. 2010). Nanoscale materials (<500 nm) have favorable physicochemical properties for vaccine delivery, because of their flexibility in preparing NPs of any size, shape, charge and composition (Magenheim and Benita 1991, Panyam and Labhasetwar 2003, Duncan 2005, Nel, Xia et al. 2006). Several biodegradable and biocompatible natural and synthetic polymers including chitosan are approved by FDA for drug and vaccine delivery (Panyam and Labhasetwar 2003, Duncan 2005, McNeil 2005, Rytting, Nguyen et al. 2008, Lu, Wang et al. 2009, Thomas, Rawat et al. 2011). We earlier developed a biodegradable NPs based Porcine Reproductive and Respiratory virus intranasal vaccine for pigs, and the technology is licensed to a company and the vaccine is in use by the pork industry. In this study, we will focus on development of a potent drinking water deliverable vaccine for broilers to control Salmonellosis and Campylobacteriosis in humans. Ourhypothesisis that drinking water delivery of Sal-NPs and Campy-NPs vaccines delivered with a potent mucosal adjuvant/s elicit robust antibody and cell-mediated immune responses in intestines and systemic and reduce colonization and shedding of Salmonella and Campylobacter in broilers. We have three Objectives to test our hypothesis.Objective 1:To develop an innovative immune targeted chitosan nanoparticle based bivalentSalmonellasubunit vaccine and analyze its efficacy against challenge infections in broilers by delivering the vaccine orally with a potent adjuvant.Rationale: Our goal in this study is to elicit robust mucosal immunity against Salmonella enterica serotype Enteritidis and Heidelberg by developing and testing an immune targeted bivalent Sal-NPs vaccine containing OMP and FLA co-delivered with a potent mucosal adjuvant/s. Because these two Salmonella serotypes have been implicated in majority of the foodborne Salmonellosis from poultry meat in the U.S. during the last decade (Antunes, Mourao et al. 2016). The outcome of this objective will provide an optimized Sal-NPs vaccine formulation which is effective and safe to use in broilers against most common two serotypes. Further, provides a scope for incorporating other Salmonella serotype and other bacterial antigens in NPs vaccine in future.Objective 2:To determine the efficiency of adjuvanted bivalentSalmonellaNPs vaccine delivered through drinking water in broilers.Rationale: The route of vaccination should be easy and convenient for mass application in large commercial poultry settings. In poultry, both drinking water and spray delivery of live Salmonella and several live viral vaccines have been in practice. Though drinking water delivery of killed or subunit vaccines are preferred, currently we do not have any due to lack of a proven vaccine delivery vehicle which protects the vaccine cargo and target it to intestinal immune cells in birds. In a pilot study, Sal-NPs vaccine was administered through drinking water and feed in layers and in vaccinates detected reduced challenge bacterial load in the cecal content by over 1 log10, associated with induction of specific enhanced mucosal IgA and IgG antibody responses (Renu, Han et al. 2020). Due to rapid growth and short lifespan of broilers until slaughter (~42 days), we need to elicit robust mucosal immunity in the intestines to reduce the colonization of Salmonella. Thus, in this Objective, we will optimize the dose and number of boosters of Sal+Adj-NPs vaccine delivered through drinking water to achieve robust immunity required to reduce bacterial load and shedding and assess any changes in birds in terms of the growth performance after vaccination.Objective 3:To develop a chitosan nanoparticle-basedCampylobactersubunit vaccine and determine its efficiency in broilers co-delivered with an adjuvant orally. Rationale: Poultry is a predominate reservoir of Campylobacter. A major concern is Campylobacter-contaminated carcasses are often associated with multistate outbreaks in the US (Newell and Fearnley 2003, Luangtongkum, Morishita et al. 2006, Jorgensen, Ellis-Iversen et al. 2011, Sibanda, McKenna et al. 2018, Hazards, Koutsoumanis et al. 2020, Kuhn, Nygard et al. 2020). Currently, we do not have any vaccines to mitigate the colonization of chickens by Campylobacter, and thus prevention of contamination of poultry product post-slaughter is important. Therefore, in this Objective, we will design a Campylobacter-NPs vaccine using the similar procedure followed for Sal-NPs vaccine presented in Objective 1 and evaluate its efficacy in broilers vaccinated orally. Specifically, our study will focus on C. jejuni, which is the most represented Campylobacter species associated with human infections and poultry (Facciola, Riso et al. 2017).