An Integrated Study on Chicken Gut Microbiome Associated with Diets and Feed Utilization Using Microarray and Illumina Sequencing DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Deng Pan Graduate Program in Animal Sciences The Ohio State University 2014 Dissertation Committee: Dr. Zhongtang Yu, Advisor Dr. Hua Wang Dr. Michael Lilburn Dr. Thaddeus Ezeji Copyrighted by Deng Pan 2014 Abstract The gastrointestinal (GI) tract of chicken harbors a complex and dynamic microbiome that is almost exclusively composed of bacteria. This bacterial community is considered as an essential component which contributes to the wellbeing of animal host in a wide range of aspects, especially nutrition and disease resistance. The overall objective of the studies presented here was to better understand the role gut microbiome plays in host growth performance and disease resistance by investigating the diversity of gut microbiome and identifying bacteria with different relative abundance in chickens with different feed utilization efficiencies and fed with different diets. In the first study (Chapter 3), the relationship between gut microbiome and host feed utilization efficiency was investigated. Poultry Intestinal Tract Chip version 2 (PITChip2), a poultry specific phylogenetic microarray was used to characterize gut microbiome structures in 24 male Cobb 500 broiler chickens with low feed conversion ratio (FCR) and 24 chickens with high FCR. No clear separation in the gut microbiome structure was observed between good- and bad-performance chickens as indicated by hierarchical clustering (HCL) and principal component analysis (PCA). As indicated by significant difference (p≤0.05) in relative abundance between high FCR and low FCR chickens, 100 phylotypes were found to correlate with bird performance, with 48 of them showing greater than 3-fold differences. Five phylotypes were more abundant in good- performance low FCR chickens. Three of them represent species of Bacteroides, ii Peptococcus, and Ruminococcaceae. The other two can only be classified as Bacteria. These five phylotypes may serve as indicators of feed utilization efficiency in chicken and also as potential probiotic candidates or targets of enhancement by prebiotics to improve growth performance of broilers. In the second study (Chapter 4), PITChip3, the latest version of PITChip, was developed. It has 1,204 customized oligonucleotide probes designed based on the V1-V3 region of bacterial 16S rRNA and are able to detect 62 genus-level phylotypes, 662 species-level phylotypes, and 34 pathogens. Each PITChip3 slide has six microarrays and every probe has 4 replicates randomly located on each microarray. The optimal hybridization temperature of PITChip3 was determined to be 48℃. At the optimal hybridization temperature, PITChip3 has a linear detection range from 3×107 to 3×1010 copies of a target per hybridization reaction. The utility of PITChip3 was tested using metagenomic DNA samples extracted from the cecal content samples of broilers fed either corn-based or wheat-based diet. Clear separations between chickens fed with different diets and between chickens at two different ages were observed on PCA plots. Eighteen species- level phylotypes, including Akkermansia muciniphila, Clostridium perfringens, and Corynebacterium variabile, along with 2 genus-level phylotypes Bacteroides and Escherichia/Shigella/Salmonella, were further identified with different relative abundance in chickens fed different diets. Such difference in gut microbiome revealed by PITChip3 may help to better understand the interactions between gut microbiome and diet and the benefits and risk associated with the two common diets. iii In the last study (Chapter 5), the difference in gut microbiome between chickens fed with corn- or wheat-based diet was further investigated using Illumina sequencing. Two hundred 1-day-old straight run Cobb 500 broiler chicks were randomly assigned to one of two dietary treatments. Each dietary treatment had 20 replicate pens with 5 birds per pen. At 14 and 35 days of age, ileal mucosa and cecal content were collected from one bird per pen. Metagenomic DNA was extracted from those samples and Illumina MiSeq platform was used to sequence the V1-V3 hypervariable region (2×300bp paired-end) of bacterial 16S rRNA gene. A phylotype-based analysis was performed and no clear difference in microbial diversity between the two dietary treatments was observed. Yet we were able to identify phylotypes that have different relative abundance in 14-day-old chickens fed with the two different diets. One phylotype representing unclassified members in the family Ruminococcaceae and one representing unclassified Bacteria had higher relative abundance in the cecal content of chickens fed with corn-based diet, whereas a phylotype classified as Escherichia/Shigella had higher relative abundance in chickens fed with wheat-based diet. It is known that chickens fed with corn-based diet perform better and are less likely to have Clostridium perfringens induced necrotic enteritis (NE). Further studies on the phylotypes identified may help to find non- antibiotic alternatives to enhance growth and protect chickens from disease such as NE. Taken together, the series of studies provided a comprehensive characterization of gut microbiome in chickens fed with corn- and wheat-based diets and also in chickens with different feed utilization efficiencies. These studies expanded our knowledge on the potential role gut microbiome plays in host growth performance and disease resistance, iv and established a poultry specific phylogenetic microarray which can serve as a powerful tool for the study of poultry gut microbiome. v Acknowledgements Foremost, I would like to express my sincere gratitude to my advisor Dr. Zhongtang Yu for his support and the guidance during my Ph.D. study at OSU. I appreciate him giving me the opportunity to advance my education. His immense knowledge and inspiration helped me in all the time of my research and dissertation writing. Besides my advisor, I would also like to thank Drs. Hua Wang, Michael Lilburn, and Thaddeus Ezeji for their service on both my candidacy and dissertation committee. The insightful suggestions from my committee were of great value to my research. I also extend my gratitude to the former and present members of Dr. Yu’s lab: Jill Stiverson, Lingling Wang, Yueh-Fen Li, Shan Wei, Dr. Paul, Minseok Kim, Tansol Park, Amlan Patra, Wen Lv, Hao Wu, Elmerson Ferreira de Jesus, Jackie Gano, Bethany Denton, Gabriella Cobellis, and anyone else I may have missed, for their willingness to share knowledge and experience with me, as well as their support and friendship for the past three years. In particular, I am grateful to Lingling Wang, Yueh-Fen Li, and Shan Wei for their helps in troubleshooting and discussion. Last but not the least I would like to thank my beloved wife Fangfei for her unconditional love and support. I would also like to thank my family for being a constant source of support and this dissertation would certainly not have existed without them. vi Vita 2009....................................................B.S. Animal Sciences, Zhejiang University, China 2011....................................................M.S. Animal Sciences, Purdue University 2011 to present ...................................Graduate Research Associate, Department of ............................................................Animal Sciences, The Ohio State University Publications Pan, D., & Yu, Z. (2013). Intestinal microbiome of poultry and its interaction with host and diet. Gut Microbes, 5(1), 108-119. Fields of Study Major Field: Animal Sciences Focus: Poultry Intestinal Microbial Ecology vii Tabel of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv Chapter 1: Introduction ........................................................................................................1 Chapter 2: Literature Review ...............................................................................................5 2.1 Intestinal microbiome of poultry ...............................................................................5 2.2 Interactions between gut microbiome and poultry host ............................................8 2.2.1 Nutritional interactions ......................................................................................8 2.2.2 Microbiome affects intestinal morphology and physiology ............................13 2.2.3 Microbiome and immunity ..............................................................................15 2.3 Interactions between gut microbiome and diet .......................................................20 viii 2.3.1 Dietary Components Affect Gut Microbiome .................................................20 2.3.2