Epidemiology of Clostridium perfringens and Clostridium difficile among Ontario broiler chicken flocks By Hind Kasab-Bachi A thesis presented to the University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Population Medicine Guelph, Ontario, Canada © Hind Kasab-Bachi, May, 2017 ABSTRACT Epidemiology of Clostridium Perfringens and Clostridium Difficile Among Ontario Broiler Chicken Flocks Hind Kasab-Bachi Advisor University of Guelph, 2017 Dr. Michele Guerin This thesis is an investigation of the epidemiology and antimicrobial susceptibility of C. perfringens and C. difficile, and the use of antimicrobials, among commercial broiler chicken flocks in Ontario. Data were obtained from the Enhanced Surveillance Project, a large-scale, cross-sectional study (July 2010 to January 2012). Caecal and colon/caecal swabs (5-pooled samples from 15 birds per flock) from 231 randomly selected flocks were collected and delivered to the laboratory for microbiological testing. Flock data were collected from producers using a questionnaire. Clostridium perfringens was frequently recovered from flocks. The netB gene was found in a moderate number of isolates and flocks. All of the C. perfringens isolates were classified as type A, except for one type E. Clostridium perfringens isolates had reduced susceptibility to ceftiofur, erythromycin, tylosin tartrate, clindamycin, oxytetracycline, tetracycline, and bacitracin. The most commonly used antimicrobials in the feed were polypeptides and ionophores. The most commonly used antimicrobials in the water were penicillins and sulfonamides. A small proportion of flocks received cephalosporins at the hatchery. Our study identified a number of factors associated with the presence of C. perfringens (and netB among C. perfringens positive flocks), and C. perfringens resistance to ceftiofur, tylosin, erythromycin, and clindamycin, oxytetracycline, tetracycline, and bacitracin. Factors included some feed mills, use of feed containing antimicrobials, feed problems, season, average duration of monitoring the flock per visit, and presence of a garbage bin at the barn entrance. Clostridium difficile was infrequently isolated from flocks. Genes encoding for toxin A and/or B, and ribotypes 001 and 014, were detected in isolates. Isolates had reduced susceptibility to ceftiofur, tylosin tartrate, erythromycin, penicillin, oxytetracycline, tetracycline, clindamycin, ampicillin, imipenem, and cefoxitin. Our study identified the baseline prevalence, genetic characteristics, and antimicrobial susceptibility of C. perfringens and C. difficile, and described the use of antimicrobials, among Ontario broiler chicken flocks. Our study also identified biosecurity, and management practices factors, including antimicrobials, significantly associated with the presence of C. perfringens (and netB), and with C. perfringens resistance to several antimicrobials. Our study findings can be used to inform future disease intervention studies. ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Michele Guerin for giving me the opportunity to join her team of students. I learned a great deal from Dr. Guerin both on an academic and personal level. I learned the importance of teamwork, organization, and the value of high quality work. Her expertise, guidance, and continued demand for excellence taught me to always challenge myself. I would also like to thank Dr. Scott McEwen for his support and guidance. Dr. McEwen’s expertise in the area of antimicrobial use and resistance has been invaluable. I would also like to thank Dr. David Pearl for sharing his expertise in epidemiology, support, and guidance. Dr. Pearl’s reassuring words really helped me to get back on track during stressful days. I would also like to thank Dr. Durda Slavic for sharing her expertise in microbiology, and also for her guidance and support. Dr. Slavic’s door has always been open to answer my questions, and I truly appreciate all the guidance she offered. I would also like to thank Dr. Andreas Boecker for his guidance and support. I truly value all the knowledge I gained from attending his agricultural economics class. I would also like to thank Dr. Scott Weese and Dr. Patrick Boerlin for sharing their expertise in microbiology and antimicrobial resistance. This study would not have been possible without the dedication of key individuals. I would like to thank the broiler farmers and slaughter plants for their collaboration. Many thanks go to Dr. Marina Brash for sharing her technical expertise in sample collection. Many thanks also go to the Enhanced Surveillance Project graduate students, Michael Eregae and Eric Nham, for sample and data collection. I would also like to thank research assistants Elise Myers, Chanelle Taylor, Heather McFarlane, Stephanie Wong, Veronique Gulde, and laboratory technician, Amanda Drexler, for their contributions. IV I would also like to thank all the individuals in the Population Medicine building for the wonderful memories I have made over the years. To mention a few names, I would like to thank Glen Cassar, Karen Richardson, and Bryan Bloomfield for teaching me how to play Euchre, and fellow graduate students Katherine Bottoms, Andreia Arruda, Tara Roberts, Julianna Ferreira, and research assistant Tatiana Petukhova for their friendship. I would also like to thank all the funding agencies that helped make this study possible. Financial support for the project has been provided by the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) - University of Guelph Partnership, OMAFRA - University of Guelph Agreement through the Animal Health Strategic Investment fund (AHSI) managed by the Animal Health Laboratory of the University of Guelph, the Poultry Industry Council, and the Chicken Farmers of Ontario. Student stipend support was provided by the Ontario Veterinary College MSc Scholarship and OVC incentive funding. Of course, this work would not have been possible without the support and love of my family. I would like to thank my father, Dr. Zakaria, for all his support and guidance during this program. My father is, and will always be, my rock and role model. Many thanks go to my mother, Roua, for her support and guidance, and most importantly, for all the tasty meals she prepared for me. Thanks to my brother, Mohamed, and my sister, Hadil, for their support and friendship. Being in university at the same time has thrown many challenges our way, but these challenges have only made our bond stronger. I would also like to thank my loving grandmother, Sabiha, for her endless support. Thanks to my best friend Alya Ali Othman, whose words of encouragement have always steered me in the right direction, and whose companionship has been essential to my success. V This thesis is dedicated to my family, Zakaria, Roua, Mohamed, and Hadil, and my best friend, Alya, for their love and support. VI STATEMENT OF WORK This thesis is part of the Enhanced Surveillance Project, a large-scale cross-sectional study that aimed to investigate the epidemiology of 13 pathogens in Ontario commercial broiler chicken flocks. The sampling period for this project was from July 2010 to January 2012. This thesis is focused on investigating the epidemiology and antimicrobial susceptibility of two bacterial pathogens, C. perfringens and C. difficile. When I joined the team in September 2010, sample and data collection for this project were underway. Dr. Michele Guerin, the principal investigator, designed this project in collaboration with experts in the fields of poultry diseases and microbiology. Dr. Michele Guerin also designed the questionnaire used to collect information for this project, and Dr. Michael Eraegae, who was a PhD student in 2010 and a research team member, contributed additional questions to the questionnaire. When I first joined the research team, I took some time to learn the different components of the broiler industry, and to familiarize myself with the project study design. After training in the various methods of sample and data collection, I started to join the team in administering the questionnaire to farmers during face-to face interviews. I also participated in sample collection at the slaughter plants, and processing samples for submission to the Animal Health Laboratory. My main task in this project was to determine the baseline prevalence, genetic characteristics, and antimicrobial susceptibility of C. perfringens and C. difficile among Ontario broiler chicken flocks. The antimicrobial use chapter in this thesis was inspired by the drive to explore different methods of analyzing antimicrobial use data. I was also responsible for conducting all statistical and data analysis related to C. perfringens and C. difficile under the supervision of my advisor, Dr. Guerin and my other committee members (Drs. McEwen, Pearl, Slavic, and Boecker).. VII TABLE OF CONTENTS CHAPTER ONE ........................................................................................................................... 1 Introduction, literature review, study rationale, and objectives .............................................. 1 INTRODUCTION .......................................................................................................................... 1 LITERATURE REVIEW ............................................................................................................... 3 Clostridium perfringens .................................................................................................................