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Sankar Renu, 1 Ashley D Markazi, 2 Santosh Dhakal, 1 Yashavanth S Lakshmanappa, 1 Revathi Shanmugasundaram, 2 Ramesh K Selvaraj, 3 Gourapura J Renukaradhya 1 1Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA and Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA; 2Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Columbus, OH, USA; 3Department of Poultry Science, University of Georgia, Athens, GA 30602, USACorrespondence: Gourapura J RenukaradhyaFood Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USATel +1 330-263-3748Fax +1 330-263-3677Email gourapura.1@osu.eduRamesh K SelvarajDepartment of Poultry Science, University of Georgia, Athens, GA 30602, USAEmail selvaraj@uga.eduPurpose: Salmonellosis in poultry is a serious economic burden. A major concern is the public health hazard caused by consumption of Salmonella-contaminated poultry products. Currently used Salmonella vaccines are ineffective in combating poultry Salmonellosis warranting the need of a potent vaccine, especially an oral vaccine that can elicit robust local intestinal immunity.Materials and Methods: A Salmonella subunit chitosan nanoparticles (NPs)-based vaccine was prepared that contained immunogenic outer membrane proteins (OMPs) and -flagellin (F) protein (OMPs-F-CS NPs). OMPs-F-CS NPs were administered as an oral vaccine in layer chickens and the resultant humoral and cell-mediated immune responses and localization of NPs were examined using standard detection methods.Results: We demonstrated targeting of surface F-protein coated chitosan NPs to immune cells when delivered orally to layer chickens, the particles were localized in ileal Peyer’s patches. The OMPs-F-CS NPs vaccinated layer chickens had significantly higher OMPs-specific mucosal IgA production and lymphocyte proliferation response. The candidate vaccine increased the expression of toll-like receptor (TLR)-2, TLR-4, IFN-γ, TGF-ß and IL-4 mRNA expression in chicken cecal tonsils.Conclusion: Our study demonstrated that the chitosan-based oral Salmonella nanovaccine targets immune cells of chickens and induced antigen-specific B and T cell responses. This candidate oral Salmonella nanovaccine has the potential to mitigate Salmonellosis in poultry.Keywords: chickens, chitosan nanoparticle, Salmonella antigens, oral delivery, mucosal immune response

Sankar Renu,1,2,* Ashley D Markazi,3,* Santosh Dhakal,1,2 Yashavanth S Lakshmanappa,1,2 Suren R Gourapura,1,2 Revathi Shanmugasundaram,3 Sujata Senapati,4 Balaji Narasimhan,4 Ramesh K Selvaraj,5 Gourapura J Renukaradhya1,2 1Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA; 2Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA; 3Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA; 4Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA; 5Department of Poultry Science, University of Georgia, Athens, GA 30602, USA *These authors contributed equally to this work Purpose: Salmonellosis is a severe economic threat in poultry and a public health concern. Currently available vaccines are ineffective, and thus, developing effective oral Salmonella vaccine is warranted. Especially, a potent oral vaccine such as the mucoadhesive polyanhydride nanoparticle (PNP) protects the vaccine cargo and delivers to intestinal immune sites to elicit robust mucosal immunity and mitigate Salmonella colonization and shedding.Materials and methods: We designed a Salmonella subunit vaccine using PNP containing immunogenic Salmonella outer membrane proteins (OMPs) and flagellar (F) protein-entrapped and surface F-protein-coated PNPs (OMPs-F-PNPs) using a solvent displacement method. Using high-throughput techniques, we characterized the OMPs-F-PNPs physicochemical properties and analyzed its efficacy in layer birds vaccinated orally.Results: The candidate vaccine was resistant in acidic microenvironment and had ideal physicochemical properties for oral delivery in terms of particle size, charge, morphology, biocompatibility, and pH stability. In vitro, in vivo, and ex vivo studies showed that F-protein surface-anchored nanoparticles were better targeted to chicken immune cells in peripheral blood and splenocytes and intestinal Peyer’s patch sites. In layer chickens inoculated orally with OMPs-F-PNPs, substantially higher OMPs-specific IgG response and secretion of Th1 cytokine IFN-γ in the serum, enhanced CD8+/CD4+ cell ratio in spleen, and increased OMPs-specific lymphocyte proliferation were observed. OMPs-F-PNPs vaccination also upregulated the expression of toll-like receptor (TLR)-2 and -4, TGF-β, and IL-4 cytokines’ genes in chicken cecal tonsils (lymphoid tissues). Importantly, OMPs-F-PNPs vaccine cleared Salmonella cecal colonization in 33% of vaccinated birds.Conclusion: This pilot in vivo study demonstrated the targeted delivery of OMPs-F-PNPs to ileum mucosal immune sites of chickens and induced specific immune response to mitigate Salmonella colonization in intestines. Keywords: Salmonella antigens, polyanhydride nanoparticles, oral delivery, ileum, chickens

Three separate experiments were conducted to study the effects of whole yeast cell product supplementation in pullets and layer hens. Body weight gain, fecal and intestinal coccidial oocyst counts, cecal microflora species, cytokine mRNA amounts, and CD4+ and CD8+ T-cell populations in the cecal tonsils were analyzed following an experimental coccidial infection. In Experiment I, day-old Leghorn layer chicks were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product (CitriStim®, ADM, Decatur, IL). At 21 d of age, birds were challenged with 1 × 105 live coccidial oocysts. Supplementation with whole yeast cell product decreased the fecal coccidial oocyst count at 7 (P = 0.05) and 8 (P < 0.01) d post-challenge. In Experiment II, 27-week old Leghorn layer hens were fed 3 experimental diets with 0, 0.05 or 0.1% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 25 of whole yeast cell product feeding. Supplementation with whole yeast cell product decreased the coccidial oocyst count in the intestinal content (P < 0.01) at 5, 13, and 38 d post-coccidial challenge. Supplementation with whole yeast cell product increased relative proportion of Lactobacillus (P < 0.01) in the cecal tonsils 13 d post-coccidial challenge. Supplementation with whole yeast cell product decreased CD8+ T cell percentages (P < 0.05) in the cecal tonsils at 5 d post-coccidial challenge. In Experiment III, 32-week-old Leghorn layer hens were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 66 of whole yeast cell product feeding. At 5 d post-coccidial challenge, whole yeast cell product supplementation down-regulated (P = 0.01) IL-10 mRNA amount. It could be concluded that supplementing whole yeast cell product can help minimize coccidial infection in both growing pullets and layer chickens., (© 2017 Poultry Science Association Inc.)