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Exploring the Breadth and Depth of Diversity Within the Canine Gut Microbiome

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Exploring the Breadth and Depth of Diversity Within the Canine Gut Microbiome EXPLORING THE BREADTH AND DEPTH OF DIVERSITY WITHIN THE CANINE GUT MICROBIOME by DANIEL HAND A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences The University of Birmingham September 2010 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT The mammalian gut microbiota is an essential factor in intestinal function and thus overall health. In the post genomic era, culture independent studies into the gut microbiota, particularly that of humans have allowed great leaps forward in knowledge of this ecosystem. Furthermore, recent advances in sequencing technologies have allowed acceleration and broadening of work in this research field. Despite this, the canine gut microbiome has remained relatively uncharacterised. This work investigates the faecal microbiota of a diverse multi-breed and multi-location group of 79 dogs, by amplifying and sequencing the 16S rRNA gene from these dogs using both Sanger sequencing of clone libraries and high throughput pyrosequencing. A robust census of the canine faecal microbiota was undertaken. The most abundant genera were the Bacteroides, Prevotella, Cetobacterium, Fusobacterium, Sutterella and Megamonas. A limited core microbiome was defined in 90% of the study population; this represented less than 0.5% of richness but more than 37.4% of abundance. Influences of host sex, diet and age were investigated but were found not significant. Some evidence was found for breed associated richness differences, most marked in Labrador retrievers and miniature Schnauzers. Furthermore, the microbiota of the Labradors appeared to cluster separately from the other breeds. DEDICATION This PhD thesis is dedicated to Harry and Ruth Abbott, proud and supportive grandparents, who always wished the best for their grandchildren. ACKNOWLEDGMENTS I would like to acknowledge my supervisors Charles W Penn and Corrin Wallis for giving me the chance to study for this PhD. Their advice, support and perseverance were invaluable. I would also like to acknowledge Mathew Gibbs, Alan Martin and the WALTHAM® Centre for Pet Nutrition (WCPN) lab team for all the help they provided. A number of colleagues at Birmingham University were especially helpful; Gemma Blackwell, Deborah Eaves, Mala Patel, Chrystala Constantinidou, Jon Hobman, Antony Jones, Lorraine Wallace, Jonathan James, Mohammad Ahasan, Olga Timoshenko and Al-Ali Abdulaziz. I would like to acknowledge the inspirational lectures delivered by my undergraduate lecturers Charles W Penn, Julia Lodge, Clive Sweet and in particular Jon Hobman that ignited my interest in microbiology. I would like to acknowledge my father, Geoff Hand and my friend Andrew Hall for the support they provided during the last year of my PhD study. This project would not have been possible without funding from BBSRC andWCPN. NON- AUTHOR CONTRIBUTIONS Chapters 5 and 6 presented as academic papers were written by Daniel Hand, Charles W Penn and Corrin Wallis. The Introductions and discussions were mostly written by Charles W Penn, the material and methods by Daniel Hand, the results were written by Daniel Hand and Charles W Penn. Some of the statistical analyses described in Chapter 6 were performed by George Gettinby and Alison Colyer. In the practical work, stools from non WCPN animals were collected by Wendy Brown, University of New England, Australia and Corrin Wallis, WCPN. The DNA extractions from all dogs except the miniature Schnauzer were performed by lab workers at WCPN, as was clone library construction for the Papillon, cocker spaniel and Labrador retriever breeds. TABLE OF CONTENTS CHAPTER ONE: GENERAL INTRODUCTION ..........................................................................................1 1.1 CHARACTERISING THE GUT MICROBIOTA .............................................................................1 1.2 HIGH THROUGHPUT SEQUENCING TECHNOLOGIES ........................................................... 10 1.3 MICROBIOLOGY OF THE GUT .................................................................................................. 13 1.3.1 COMMENSALS, MUTUALISTS AND PARASITES .................................................... 13 1.3.2 THE CANINE GUT MICROBIOTA ............................................................................... 15 1.4 PHD PROJECT DESCRIPTION ..................................................................................................... 18 1.4.1 AIMS AND HYPOTHESES ............................................................................................ 20 1.4.2 OUTLINE OF WORK PRESENTED .............................................................................. 21 CHAPTER TWO: GENERAL MATERIALS AND METHODS ................................................................ 22 2.1 CANINE SAMPLE POPULATION ................................................................................................ 22 2.1.1 SAMPLE COLLECTION ................................................................................................ 24 2.1.2 FAECAL DNA EXTRACTION ...................................................................................... 24 2.2 PCR PRIMERS ............................................................................................................................... 25 2.3 AGAROSE GEL ELECTROPHORESIS......................................................................................... 25 2.4 BIOINFORMATICS. ...................................................................................................................... 26 2.4.1 RIBOSOMAL DATABASE PROJECT (RDPII) ............................................................. 26 2.4.2 RDP CLASSIFIER .......................................................................................................... 26 2.4.3 CLUSTALX .................................................................................................................... 27 2.4.4 RAREFACTION ANALYSIS ......................................................................................... 27 2.4.5 CHAO1 MINIMUM RICHNESS ESTIMATE ................................................................ 27 2.4.6 SHANNON-WIENER DIVERSITY INDEX ................................................................... 28 CHAPTER THREE: CHARACTERISATION OF MINIATURE SCHNAUZER FAECAL MICROBIOTA VIA 16S CLONE LIBRARY SEQUENCING. .................................................................... 29 3.1 INTRODUCTION........................................................................................................................... 29 3.2 MATERIALS AND METHODS ..................................................................................................... 31 3.2.1 FAECES COLLECTION ................................................................................................. 31 3.2.2 FAECAL DNA EXTRACTION ...................................................................................... 31 3.2.3 16S RRNA GENE PCR ................................................................................................... 31 3.2.4 CLONING ....................................................................................................................... 33 3.2.5 PLASMID DNA EXTRACTION .................................................................................... 36 3.2.6 ENDONUCLEASE RESTRICTION DIGESTS ............................................................... 37 3.2.7 SEQUENCING ................................................................................................................ 38 3.2.8 PHYLOGENETIC ANALYSIS/ BIOINFORMATICS .................................................... 38 3.2.8.1 QUALITY PROCESSING OF SANGER READS ........................................... 38 3.2.8.2 OTU DETERMINATION AND STATISTICS ................................................ 39 3.2.8.3 TAXONOMIC ASSIGNMENTS ..................................................................... 39 3.3 RESULTS AND DISCUSSION ...................................................................................................... 39 CHAPTER FOUR: BREED SPECIFIC CHARACTERISATION OF CANINE FAECAL MICROBIOTA BY SANGER SEQUENCING. ........................................................................................................................ 57 4.1 INTRODUCTION........................................................................................................................... 57 4.2 MATERIALS AND METHODS ..................................................................................................... 58 4.2.1 FAECES COLLECTION ................................................................................................. 58 4.2.2 FAECAL DNA EXTRACTION ...................................................................................... 58 4.2.3 16S RRNA GENE PCR ................................................................................................... 58 4.2.4 CLONING ......................................................................................................................
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