Characterization of the Fecal Microbiota of Commercial Mink (Neovison vison) by Nicole Compo A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Veterinary Science in Pathobiology Guelph, Ontario, Canada © Nicole Compo, August, 2017 ABSTRACT Characterization of the Fecal microbiota of Commercial Mink (Neovison vison) Nicole Compo Advisor: University of Guelph, 2017 Dr. Patricia V. Turner Mink, obligate carnivores, are the fifth largest livestock commodity group in Canada, with more than 3 million pelts produced annually and an industry valuation at almost $100 million (2015). To date, research in the field of microbiota; the collective, interacting genomes and the symbiotic microorganisms in the gastrointestinal tract, has focused primarily on herbivores and omnivores, with comparatively few studies carried out in carnivores. The goal of the current study was to characterize the fecal microbiota of farmed mink, Neovison vison. Fecal samples (n=366) were collected from adult females and weaned kits during two consecutive summers in 2014 and 2015 and adult females just before winter breeding in 2016. The V4 regions of the 16S rRNA genes were amplified, sequenced, and classified from extracted fecal bacterial DNA. The most predominant phyla were Firmicutes and Proteobacteria, with all other phyla together comprising <5% of the sequences identified. The relative abundance of Proteobacteria was found to be much higher than previous reports in mammals. Additionally, ranges in the relative abundances of both the predominant and many non-predominant taxa were found to be very large, suggesting that mink are tolerant to a broader community of organisms than has been seen in other mammals. Few differences were identified at each taxonomic level between adult females and weaned kits, though several genera within the families Lactobacillales and Clostridiales were identified to be differentially abundant in weaned kits (p<0.05). The years 2014 and 2016, the two periods that would have had the fewest number of animals in common, had the greatest number of taxa that were different. Farms were generally similar in the predominant phyla and genera from 2014 to 2015. The microbiota can likely be impacted by a range of dietary, environmental, and treatment factors, none of which are understood in mink. This body of work will contribute significantly to our understanding of how these factors impact the fecal microbiota of farmed mink, and the carnivore GI microbiota more generally, with the hope of ultimately utilizing this information to optimize the health, production, and welfare of mink. DEDICATION I dedicate this work to my parents, my anchors…my Mommy and DD, whose endless love, support, and laugh-until-I-cry moments have gotten me through the stormiest of waters. iii ACKNOWLEDGEMENTS The completion of my program would undoubtedly not have been possible without a great number of people. From help and advice to laughter and encouragement, their roles were diverse and important in ways that I didn’t always realize. First, my advisory committee. To my advisor, Pat Turner, thank-you for your willingness to share your limitless knowledge with me, encouragement of my creative and independent thoughts, and always pushing me to be better and to do more. You’ve been an incredible resource and I am lucky to have been able to learn under you. To Scott Weese, your patience, kindness, and insight made navigating the world of microbiome and, ESPEPCIALLY, mothur, manageable. I may never have gotten through the analysis without you. Many thanks to David Pearl for all your help and guidance with the statistics for the mink post-mortem project. Finally, to Andrew Winterborn, who is both a mentor and a friend. Thank-you for always believing in me more than I believed in myself. You’ll never know how much it meant to me. Thank-you to the staffs of the Weese and Turner labs, especially Jutta and Joyce, you were always there when I wanted to poke my eye out with a pipette when my PCR didn’t work after days of pippetting. You’ve answered my many millions of questions with patience and enthusiasm, so thank-you for training and guiding me through this project. Next, my quad-mates, and greater scope room peeps, without whom this thesis likely would have been finished many months ago. Thank-you for the laughter, support, and, most importantly, friendship. I wish you all the best in wherever your endeavours take you. I hope we will find reason to come together again. To my one and only deskie, Chris, I appreciate someone to talk football with in a country where I’m surrounded by hockey fans. You’ve been a great friend and I look forward to our many monkey path consults. To my LLP, who knows who she is but would hate the recognition: thank-you for being you. The fur butts and I will miss you immensely, as I don’t see you ever taking a vacation, much less to an island of sunshine, beaches, and every meal outside. I’ll smuggle pickles for you any time. And, finally, my amazing family. I’ve got a team of parents whose emotional and, most importantly, financial support made pursuing my dream of being a laboratory animal veterinarian possible. When I came up with this crazy idea of moving to Canada, my mother said, “What!? Are you sure? You know we’re not going to come visit, right?” I definitely wasn’t sure then, but I am now. I have grown so much as both a person and veterinarian because of their support. To iv the little bro, who will forever be the rational to my emotional, the common sense to my absent- mindedness, and the artist to my scientist, thank-you for always answering my random calls in the middle of the night when I just want to say hi and need a laugh. To Katlyn, who, in so many ways, is a mini-me. I so look forward to watching you conquer the world of medicine; you’re going to be amazing whatever you choose to do and wherever you choose to do it. And to my perfect nephew, Jude, I’m so happy I’ll soon be closer and get to see more of you growing up. They’re my best friends and favorite people to be with and without them, this journey would not have been possible. I love you guys so much. One final thanks to my financiers: the Canadian Mink Breeders Association and the Ontario Ministry of Agriculture, Food, and Rural Affairs. None of this would have been possible without them. v DECLARATION OF WORK PERFORMED All work presented in this thesis was completed by myself, with the following exceptions: • Collection of fecal samples was completed by Brian Tapscott • Initial receipt and packaging of samples was conducted by Dr. Jutta Hammermueller and Sabrina Stevens • Aliquoting was done by myself and Alison Thomas • 16S rRNA gene sequencing using the Illumina MiSeq was conducted by Jeffrey Gross of the University of Guelph’s Advanced Analysis Centre, Guelph, ON • Post-mortem examinations of mink kits included in Appendix A were conducted by Drs. Patricia V. Turner, Marina Brash, and Ms Amanda Storer • Microbiological evaluation of enteric tissue samples in Appendix A was completed by Dr. Durda Slavic vi TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iv DECLARATION OF WORK PERFORMED .......................................................................... vi TABLE OF CONTENTS ........................................................................................................... vii LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ...................................................................................................................... x LIST OF APPENDICES ............................................................................................................. xi ABBREVIATIONS ..................................................................................................................... xii CHAPTER ONE: INTRODUCTION TO MINK AND THE GASTROINTESTINAL MICROBIOTA ................................................................................ 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Bookmark not defined. 1.1 INTRODUCTION .............................................................................................................. 1 1.1.1 Production Cycle in Captivity ........................................................................................ 1 1.1.2 Anatomy and Physiology of the Mink Gastrointestinal Tract ....................................... 2 1.1.3 Diet ................................................................................................................................. 2 1.2 INFECTIOUS ENTERIC AGENTS OF FARMED MINK ........................................... 3 1.3 THE ENTERIC MICROBIOTA ....................................................................................... 5 1.3.1 Methods of Characterizing the Enteric Microbiota ....................................................... 6 1.3.2 Role of the Bacterial Gastrointestinal Microbiota in Health and Disease ..................... 7 1.4 THESIS OBJECTIVES AND HYPOTHESES .............................................................. 15 1.4.1 Hypotheses ................................................................................................................... 15 1.4.2 Study objectives