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Investigation of Microbiota in Health and Disease of Poultry University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2019 Investigation of Microbiota in Health and Disease of Poultry Bishnu Adhikari University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/etd Part of the Animal Diseases Commons, Bacteriology Commons, Microbial Physiology Commons, Molecular Biology Commons, and the Poultry or Avian Science Commons Recommended Citation Adhikari, Bishnu, "Investigation of Microbiota in Health and Disease of Poultry" (2019). Theses and Dissertations. 3371. https://scholarworks.uark.edu/etd/3371 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected]. Investigation of Microbiota in Health and Disease of Poultry A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Poultry Science by Bishnu Adhikari Tribhuvan University Bachelor of Veterinary Science & Animal Husbandry, 2010 Aarhus University Master of Science in Sustainable Animal Nutrition and Feeding, 2015 University of Debrecen Master of Science in Sustainable Animal Nutrition Agricultural Engineer, 2015 August 2019 University of Arkansas The dissertation is approved for recommendation to the Graduate Council. Young Min Kwon, Ph.D. Dissertation Director Billy M. Hargis, Ph.D. Jiangchao Zhao, Ph.D. Committee Member Committee Member Guillermo Tellez-Isaias, Ph.D. Committee Member ABSTRACT The microbiotas play vital roles in health and diseases of both humans and animals. 16S rRNA genes sequence analysis is one of the most popular and commonly used methods in the analysis of microbiotas associated with hosts. In this dissertation, the microbiotas of chickens (broilers, breeders, and layers) and turkeys were evaluated by 16S rRNA gene sequencing. Characterization of the culturable subpopulations of Lactobacillus in the chicken gut can serve as a valuable resource for probiotic development. In Chapter 2, Lactobacillus subpopulations recovered on MRS from chicken gut were defined comprehensively for the first time using 16S rRNA gene profiling, where they varied with different regions (cecum vs. ileum) and locations (lumen vs. mucosa) with in the same region. In Chapter 3, we investigated the effect of cell densities as determined by varying levels of sample dilution on the culture-enriched microbiota profiles using MRS agar medium as a model system. The dilution levels of original samples was found to alter the resulting culture-enriched microbiota profiles via unknown density-dependent mechanisms. In chapter 4, Bacillus isolates (B. subtilis and B. amyloliquefaciens) were used to evaluate their therapeutic and prophylactic effects against Salmonella Enteritidis, and found their potentialities to reduce S. Enteritidis colonization and improve the intestinal health in broiler chickens possibly through altering the composition and functions of gut microbiota. In chapter 5, we investigated the cecal microbiota and egg production in two strains of Hy-Line (Brown and W- 36) housed in conventional cages (CC) and enriched colony cages (EC), and noticed differences in egg production and cecal microbiota between strains and housing types. In chapter 6, we performed a comprehensive survey of the litter microbiotas using booty swab samples in the 5 commercial turkey farms of the Northwest Arkansas. The litter microbiotas were found to differ between farms, and flocks which were further affected by the ages of turkeys. In Chapter 7, we developed and evaluated the nested TaqMan probe based qPCR assay for the quantitative detection of Clostridium septicum that targets the alpha toxin gene (csa). ACKNOWLEDGEMENTS I am really honored to work under the supervision of my major advisor Dr. Young Min Kwon throughout my Ph.D. Truly speaking, it was one of the best decisions in my life to start Ph.D. in his lab, and I don’t have any words to describe what he had contributed for me to sharpen my academic career. I am indeed indebted with him and would like to give immense thanks for his exceptional guidance and supervisions. His humbleness, kindness, optimistic thinking, supportiveness, and moreover, understanding of things beyond academic life will always be remembered and tried to implement in my life too. In addition, I am feeling so lucky to get my excellent committee members. It’s my pleasure to have Dr. Tellez-Isaias (Memo) in my committee who have always been a source of inspiration and motivation to work hard for better contributions to the science. I am very thankful for his kind support, collaborations, encouragements and overall mentorship. Similarly, I would like to acknowledge Dr. Hargis whom I found as “Wikipedia” of knowledge, and his way of thinking and challenging the notions with facts always fostered me to study and explore more. I am also grateful to Dr. Zhao for providing me the expert opinion and suggestions whenever I needed in the field of Microbiome and Bioinformatics. Furthermore, I would like to offer my sincere gratitude to Dr. Tieshan Jiang for his endless work to make the lab well managed and provide technical assistances whenever necessitated. Likewise, my heartfelt gratitude was for my friendly, cooperative, and helpful labmates; Dr. Deepti Samarth, Dr. Yichao Yang, Dr. Rabindra Mandal, Anita Mandal, Dr. Adil Al-Ogaili, Dr. Sardar Abdullah, Dr. Nicole Calhoun, Dr. Yoonjung Kho, Abigeal Omolewu, and Mohammed Aljuwayd, in addition, for my awesome Nepalese friends and families of Northwest Arkansas. I would also like to express my profound thanks to Ms. Donna Sue Delozier for her role as a guardian for many students including me, and all other nice and helpful faculty, staff members, and my colleagues of the Center of Excellence for Poultry Science. Last but not the least, I am very proud of my parents, my wife Sumana, son Subin, daughter Saanvi, my brother Deepak and his family, my seven angel sisters, in-laws families, and all my relatives and friends in Nepal who are always backbone of me, and would like to share this happiest moment with the greatest appreciation. DEDICATION I would like to dedicate this dissertation to my beloved parents; Mrs. Laxmi Devi Adhikari and late Mr. Basanta Adhikari, whose continuous selfless love, support, and encouragement helped me to achieve this degree. TABLE OF CONTENTS CHAPTER ONE ……………………………………………………………………………..........1 1.1 Review of Literature ………………………………………………………………………...1 1.1.1 Common Terminologies Used in the Microbial Community Analysis …………………1 1.1.2 Status and Limitations of the Current Research on Animal Gut Microbiota ……………2 1.1.3 Characterization of Microbial Community by 16S rRNA Genes Analysis ……………...5 1.1.3.1 Library Preparation and Sequencing ………………………………………………5 1.1.3.2 Computational Analysis of 16S rRNA Gene Sequences …………………………..8 1.1.3.2.1 Quality Filtering and Preprocessing of Reads ………………………………..8 1.1.3.2.2 Taxonomic Composition Analysis …………………………………………..8 1.1.3.2.3 Microbial Diversity Analysis ………………………………………………10 1.1.3.2.3.1 Alpha Diversity Analysis …………………………………………….10 1.1.3.2.3.2 Beta Diversity Analysis ………………………………………………12 1.1.3.2.3.3 Principal Coordinate Analysis (PCoA) ………………………………15 1.1.3.2.4 Functional Prediction of 16S rRNA Gene Sequences ………………………16 1.1.4 Research Approaches to Improve 16S rRNA Based Sequencing Resolution …………17 1.1.5 An Introduction to Clostridial Dermatitis (Cellulitis) in Turkey ……………………….19 1.1.6 An Introduction to Food Borne Pathogens with Emphasis on Salmonella ……………..20 1.2 References …………………………………………………………………………………22 1.3 Tables and Figures …………………………………………………………………………33 CHAPTER TWO ………………………………………………………………………………...35 2.1 Abstract ……………………………………………………………………………………36 2.2 Introduction ……………………………………………………………………………......38 2.3 Materials and Methods ……………………………………………………………………..40 2.3.1 Sample Collection and Processing …………………………………………………….40 2.3.2 DNA Extraction and PCR ……………………………………………………………...41 2.3.3 Data Analysis …………………………………………………………………………..42 2.4 Results ……………………………………………………………………………………..43 2.4.1 Taxonomy Assignment ………………………………………………………………...43 2.4.1.1 Phylum Level …………………………………………………………………….43 2.4.1.2 Genus Level ……………………………………………………………………...43 2.4.1.3 Species Level …………………………………………………………………….44 2.4.1.4 OTU Heatmap at Species Level …………………………………………………44 2.4.2 Alpha Diversity ………………………………………………………………………..45 2.4.3 Beta Diversity ………………………………………………………………………….45 2.5 Discussion …………………………………………………………………………………45 2.6 Author Contributions ……………………………………………………………………...50 2.7 Conflict of Interest Statement ……………………………………………………………..51 2.8 Acknowledgements ………………………………………………………………………..51 2.9 Funding …………………………………………………………………………………….51 2.10 References ………………………………………………………………………………..51 2.11 Figures ……………………………………………………………………………….…...56 CHAPTER THREE ……………………………………………………………………………...62 3.1 Abstract ……………………………………………………………………………………63 3.2 Introduction ………………………………………………………………………………..64 3.3 Materials and Methods …………………………………………………………………….66 3.3.1 Cecal Sample Collection and Processing ………………………………………………66 3.3.2 DNA Extraction and PCR ……………………………………………………………...67 3.3.3 Data Analysis …………………………………………………………………………..67 3.4 Results ……………………………………………………………………………………..68 3.4.1 Summary of Sequencing Analysis and Composition of Microbiotas ………………….68 3.4.2 Comparison of Alpha Diversity ………………………………………………………..69 3.4.3 Impact of the Dilution
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