ABSTRACT BALLOU, ANNE LAEL. Probiotic Modulation of the Avian Microbiome Releases Systemic Signals that Alter the Lymphocyte Transcriptome. (Under the direction of Dr. Matthew Koci). The gastrointestinal (GI) microbiome plays an important role in animal development, health, and performance. Previous research in our laboratory has demonstrated probiotic supplementation alters host intestinal development, mucosal and systemic immune responses, and increases ATP production in circulating leukocytes. The ability to influence these diverse systems could be a powerful tool for promoting health and maximizing performance of food animals. However, to achieve this level of control I need to identify the specific members of the microbiome involved, understand where they reside in the host, and understand what governs their colonization. The goal of this dissertation research was to address this knowledge gap. Over the course of several distinct, yet interconnected studies I investigated the kinetics of microbial colonization of the chicken cecum from hatch to maturity, examined the impact of early exposure to distinct bacteria on long-term population dynamics, compared the microbial differences along the entire length of the gastrointestinal tract, studied role different diets have on the microbial populations in different regions of the gut, and developed an in vitro system to characterize probiotic-induced signals disseminated in the blood that regulate host immunity. From these studies I learned the microbiome of growing chicks starts off with only a few members (Enterobacteriaceae), but develops rapidly, adding complexity and diversity. First increasing the numbers of Firmicutes around day 7 until it reaches maturity by day 28. As the microbiome develops, the influence of the treatments becomes more apparent; however, treatments that influence colonization early in life before the microbiome is established appear to have lost lasting effects. Additionally, taxonomic differences may not be the best predictor of biologically relevant changes in the microbiomes as predicted metagenomic content suggest fewer functional differences despite the strong taxonomic differences. Evaluation of the crop, gizzard, duodenum, jejunum, ileum, and cecum of chickens demonstrated GI location was the strongest predictor of similarity among samples, influencing the microbiome more than diet or probiotic treatment. Of the locations evaluated, the cecum had the highest phylogenetic diversity, but the ileal microbiome had more treatment-related changes, and more predicted metagenomic differences. Further, the ileum of probiotic-fed animals had reductions in bacteria associated with inflammation and increased potential for production of anti-inflammatory butyrate. This suggests changes in ileal microbiota are more closely related to changes in host physiology, highlighting the need for a better understanding of how treatments influence changes in the microbiome along the GI. Finally, I hypothesized that previously observed changes in immune energy metabolism were regulated by a probiotic-stimulated factor in the serum. To test this I developed an in vitro cell culture assay and demonstrated serum isolated from probiotic-fed animals can augment the amount of ATP/cell in vitro. The specific serum factor still has not been identified; however, the results of this study suggest it is a compound under 30 kDa in size, lipid or associated with lipids, and based on the response of the evaluated cell lines, its effect is specific to immune cells. Transcriptomic analysis of an avian T-cell line (CU205) stimulated with serum from probiotic-treated animals demonstrates gene expression changes consistent with an increase in cell survival and T-helper cell differentiation as compared to the control. This research has added to our understanding of how probiotics alter the GI microbiome to affect host immune function. It indicates that probiotic-directed alteration of the ileal microbiome during development could result in the upregulation of a serum compound that stimulates circulating immune cells in a variety of ways. Continued study of this system will reveal how these ileal changes lead to the lymphocyte-stimulating serum signal, as well as other immune functions impacted by this probiotic. © Copyright 2017 Anne Lael Ballou All Rights Reserved Probiotic Modulation of the Avian Microbiome Releases Systemic Signals that Alter the Lymphocyte Transcriptome by Anne Lael Ballou A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Functional Genomics Raleigh, North Carolina 2017 APPROVED BY: _______________________________ _______________________________ Matthew Koci Hsiao-Ching Liu Committee Chair _______________________________ _______________________________ Hosni Hassan Jack Odle BIOGRAPHY Anne Ballou is a native of Louisville, Kentucky. She attended the University of Kentucky, where she received a B.S. in Animal Science, followed by an M.S. in Animal Science. After a brief internship with the Mote Marine Laboratory, she decided to pursue a Ph.D. in Functional Genomics at North Carolina State University. This has allowed Anne to apply modern genomics principals to solving animal health challenges facing the food animal industry. She intends to continue in her support of food animal agriculture in her professional career and through service focused on agriculture and science policy. ii ACKNOWLEDGMENTS I would like to thank the many supporters, mentors, and collaborators who contributed to my growth and to the success of this research project. Dr. Matthew Koci guided and fostered my training, providing an example of tireless and dedicated mentorship. None of this would have been possible without him. Drs. W. James Croom, Hosni Hassan, Hsiao-Ching Liu, and Jack Odle provided valuable feedback, encouragement, and assistance during my research and writing. Rebecca-Ayme Hughes, Rizwana Ali, Mary Mendoza, Brian Troxell, Amy Savage, Chris Ellis, and Aaron Oxendine assisted with the collection, analysis, and interpretation of data. I was supported in part by NIFA National Needs Fellowship 2009-38420-05026, NIFA Predoctoral Fellowship 2016-67011-25168, and a generous gift from Star- Labs/Forage Research. Countless others not mentioned also contributed their time and energy to support me during this process. Many thanks to all. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................. vii LIST OF FIGURES ............................................................................................................... viii CHAPTER 1: LITERATURE REVIEW ................................................................................ 1 INTRODUCTION ............................................................................................................... 2 ROLE OF MICROBES IN DEVELOPMENT OF THE GI ............................................... 3 Kinetics of gut colonization ............................................................................................. 3 Impact of bacteria on GI morphology and growth. ......................................................... 5 Mucosal immune development ........................................................................................ 7 Microbial niches with in the gastrointestinal tract ........................................................... 8 ROLE OF MICROBES IN FUNCTION AND HEALTH .................................................. 10 Interactions between diet, the microbiome, and digestive function ................................ 10 Metabolism ...................................................................................................................... 14 Mucosal immune activity ................................................................................................ 15 Systemic immune activity ............................................................................................... 17 PRIMALAC USE AS A MODEL FOR THE STUDY OF THE AVIAN MICROBIOME IN IMMUNE FUNCTION .................................................................................................. 19 REFERENCES .................................................................................................................... 22 CHAPTER 2: DEVELOPMENT OF THE CHICK MICROBIOME: HOW EARLY EXPOSURE INFLUENCES FUTURE MICROBIAL DIVERSITY .................................... 32 ABSTRACT ........................................................................................................................ 33 INTRODUCTION ............................................................................................................... 34 MATERIAL AND METHODS .......................................................................................... 36 Animals and Treatments .................................................................................................. 36 Sample Collection............................................................................................................ 37 DNA isolation and 16S sequencing ................................................................................. 37 Sequence data analysis .................................................................................................... 38 RESULTS...........................................................................................................................