Utilization of Synbiotics, Acidifiers, and a Polyanhydride Nanoparticle Vaccine in Enhancing the Anti-Salmonella Immune Response in Laying Hens Post-Salmonella Challenge Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ashley D. Markazi, M.S. Graduate Program in Animal Sciences The Ohio State University 2018 Dissertation Committee: Ramesh K. Selvaraj, Advisor Michael S. Lilburn, Co-Advisor Renukaradhya Gourapura Lisa Bielke Copyright by Ashley D. Markazi 2018 ABSTRACT Salmonellosis, a zoonotic disease caused by the bacterium Salmonella, is most commonly attributed to the consumption of poultry eggs and meat. The current project examined the effects of drinking water synbiotics, in-feed acidifiers, and a polyanhydride nanoparticle Salmonella vaccine in enhancing the anti-Salmonella immune response and decreasing Salmonella infection in laying hens. The synbiotic experiment was conducted to study the effects of drinking water supplementation of synbiotic product in laying hens with and without a Salmonella challenge. A total of 384 one-day-old layer chicks were randomly distributed to the drinking water synbiotic supplementation or control groups. At 14 wk of age, the birds were vaccinated with a Salmonella vaccine, resulting in a 2 (control and synbiotic) X 2 (non-vaccinated and vaccinated) factorial arrangement of treatments. At 24 wk of age, half of the birds in the vaccinated groups and all the birds that were not vaccinated were challenged with Salmonella Enterica serotype Enteritidis, resulting in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial arrangement. At 8 d post-Salmonella challenge, synbiotic supplementation decreased (P = 0.04) cecal S. Enteritidis in the challenge group compared to the un-supplemented challenge group. At 17 d post-Salmonella challenge, synbiotic supplementation increased bile anti- Salmonella IgA in the challenge group compared to the birds in the challenge group without synbiotic supplementation. The acidifier experiment was conducted to study the effects of ii acidifier supplementation in laying hens with and without a Salmonella challenge. A total of 384 one-day-old layer chicks were randomly distributed to the acidifier supplementation or control groups. At 14 wk of age, the birds were vaccinated with a Salmonella vaccine, resulting in a 2 (control and acidifier) X 2 (unvaccinated and vaccinated) factorial arrangement. At 24 wk of age, half of the birds in the vaccinated groups and all the birds that were not vaccinated were challenged with Salmonella Enterica serotype Enteritidis, resulting in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and acidifier) factorial arrangement. At 8 d post-Salmonella challenge, birds supplemented with acidifier in the challenge group had decreased (P = 0.04) cecal S. Enteritidis percentage compared to the challenge group without acidifier supplementation. At 22, 24, and 30 d post-Salmonella challenge, acidifier supplementation had higher (P < 0.01) plasma anti-Salmonella IgA titers in the challenge group compared to the challenge group without acidifier supplementation. The polyanhydride nanoparticle experiment analyzed the immunological effects of an oral Salmonella nanoparticle vaccine (OMPs-F-PNPs) loaded with Salmonella outer membrane proteins (OMPs) and flagellin proteins. At 6 wk of age, chickens were either orally vaccinated with PBS (Mock), soluble OMPs (0.05 mg) + flagellin (0.05 mg) in PBS (Antigen), or OMPs (0.05 mg) + flagellin (0.05 mg) loaded OMPs-F-PNPs suspension in 1 mL of sterile PBS (OMPs-F-PNPs). The same dose and route of delivery were repeated at 9 and 12 wk of age. At 15 wk of age, chickens were challenged with 1 x 109 CFU of live S. Enteritidis. Birds that were vaccinated with OMPs-F- PNPs had significantly (P < 0.01) higher serum and bile OMPs-specific IgG titers compared to that from the mock + challenge and the mock treatment groups. The OMPs-F-PNPs vaccinated iii birds had significantly fewer positive samples for cecal S. Enteritidis compared to that of the mock + challenge group. In conclusion, drinking water synbiotic supplementation, in-feed acidifier supplementation, and the polyanhydride Salmonella nanoparticle vaccine each resulted in significant increases in the anti-Salmonella immune response as measured by Salmonella- specific antibody titers and decreased Salmonella colonization in layer chickens. iv Dedication I dedicate my dissertation to my father, mother, and sister, who gave me constant encouragement in pursuing my goals in education. v Acknowledgements I would like to give my sincere thank you to my advisor, Dr. Ramesh Selvaraj, for providing me with the opportunity to receive my doctorate degree. The knowledge and growth that I have gained working with him these last five years have been invaluable. I also would like to thank Dr. Mike Lilburn for his mentorship and guidance. I greatly appreciate all the help he provided me with going over my dissertation, organizing my results, and organizing my thoughts. Thank you also to Revathi for her technical help in the laboratory. She helped me immensely with my projects and I cannot be thankful enough for her. Thank you also to Dr. Lisa Bielke for always being friendly, helpful, and welcoming any questions I had. I would like to thank Dr. Aradhya Gourapura for his encouragement and help with the nanoparticle vaccine project. I am very grateful to have worked on such an impactful and novel project. I would also like to give my sincere thank you to Sankar, who was a patient mentor who taught me a lot of valuable laboratory and research techniques. Thank you to Theros, Yi, Keila, and Amanda for their help with bird trials and lab. I would also like to thank Keith B. Patterson, Jarrod Snell, Jack E.Sidle, and Jordan L.Welsh for all of their help with my bird trials and sample collections. Additionally, I would like to thank my loving family and friends who helped support me during my academic journey. My mom, dad, and sister were all extremely kind and encouraging of my pursuits. Thank you also to Jaron Caudill for his kindness, love, and encouragement. Also, I would like to thank Sherry Zhang for always encouraging me and connecting with me about the vi challenges of graduate school. Lastly, thank you to all the friends I met at OARDC (graduate students, faculty and staff), for your guidance, kindness, and support. vii Vita 2008…………………………………………………Barrington High School Barrington, Illinois 2012…………………………………………………B.S. Natural Resources and Environmental Sciences, University of Illinois at Urbana- Champaign 2013-2018…………………………………………...Graduate Research Associate, Graduate program in Animal Sciences, The Ohio State University Fields of Study Major Field: Animal Sciences Specialization: Poultry Nutrition Immunology viii Table of Contents ABSTRACT ......................................................................................................................... ii Dedication ............................................................................................................................ v Acknowledgements .............................................................................................................. vi List of Tables ....................................................................................................................... xi List of Figures .................................................................................................................... xii Chapter 1: Introduction ....................................................................................................... 1 Chapter 2: Review of Literature......................................................................................... 10 Chapter 3: Effects of Drinking Water Synbiotic Supplementation in Laying Hens Challenged with Salmonella ................................................................................................................... 45 ABSTRACT................................................................................................................................ 45 INTRODUCTION ...................................................................................................................... 47 MATERIALS AND METHODS ................................................................................................. 49 RESULTS ................................................................................................................................... 55 DISCUSSION AND CONCLUSION .......................................................................................... 58 Chapter 4: Effects of Acidifier Product Supplementation in Laying Hens Challenged with Salmonella .......................................................................................................................... 63 ABSTRACT................................................................................................................................ 63 INTRODUCTION ...................................................................................................................... 65 MATERIALS AND METHODS ................................................................................................. 67 RESULTS ................................................................................................................................... 73 DISCUSSION AND CONCLUSION .........................................................................................