Ecology and Epidemiology of Campylobacter jejuni in Broiler Chickens A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Hae Jin Hwang IN PARTIALL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Dr. Randall Singer, Dr. George Maldonado June 2019 © Hae Jin Hwang, 2019 Acknowledgements I would like to sincerely thank my advisor, Dr. Randall Singer, for his intellectual guidance and support, great patience, and mentorship, which made this dissertation possible. I would also like to thank Dr. George Maldonado for his continuous encouragement and support. I would further like to thank my thesis committee, Dr. Richard Isaacson and Dr. Timothy Church, for their guidance throughout my doctoral training. I thank all my friends and colleagues I met over the course of my studies. I am especially indebted to my friends, Dr. Kristy Lee, Dr. Irene Bueno Padilla, Dr. Elise Lamont, Madhumathi Thiruvengadam, Dr. Kaushi Kanankege and Dr. Sylvia Wanzala, for their support and friendship. Heartfelt gratitude goes to my family, for always believing in me, encouraging me and helping me get through the difficult and stressful times during my studies. Lastly, I thank Sven and Bami for being the best writing companions I could ever ask for. i Abstract Campylobacteriosis, predominantly caused by Campylobacter jejuni, is a common, yet serious foodborne illness. With consumption and handling of poultry products as the most important risk factor of campylobacteriosis, reducing Campylobacter contamination in poultry products is considered the best public health intervention to reduce the burden and costs associated with campylobacteriosis. To this end, there is a need to improve our understanding of epidemiology and ecology of Campylobacter jejuni in poultry. The overall goal of this dissertation is to answer knowledge gaps regarding Campylobacter ecology and epidemiology in broiler chickens with an emphasis on the pre-harvest components of the broiler production system. The objectives included: (1) to assess current interventions and control measures practiced by the U.S. broiler industry to reduce Campylobacter contamination on broiler chicken products; (2) to investigate indirect selective pressures responsible for the persistence of fluoroquinolone-resistant Campylobacter jejuni in broiler chickens; and (3) to describe the temporal changes in broiler litter bacterial microbiota with respect to bird age, flock cycle, antibiotic use and presence of Campylobacter on farms. For the first objective, we surveyed key stakeholders of the U.S. broiler industry, including poultry veterinarians, farm managers and processing plant managers. The survey respondents reported the use of various pre- and post-harvest interventions that are recommended by the USDA-FSIS to decrease Campylobacter contamination in broiler chickens and on carcasses. Yet, the survey results also revealed the lack of understanding of Campylobacter epidemiology among the respondents, indicating that further education and training programs are necessary to improve understanding of Campylobacter among the key stakeholders which can lead to a reduction of Campylobacter contamination and improvement in food safety. To ii investigate indirect selective pressures allowing persistence of Campylobacter jejuni in broiler chickens, we tested two hypotheses: limited bioavailability in poultry and activity of microcin B17. However, we found that the hypotheses tested were unlikely to be responsible for the persistence of fluoroquinolone-resistant Campylobacter jejuni despite the lack of fluoroquinolone use in the industry. Lastly, we investigated the temporal changes in the broiler litter microbiota by sampling seven commercial broiler farms over two flock cycles. We found that flock cycle and age of bird were two significant farm variables driving the bacterial diversity and composition of the litter microbiota. Additionally, when the litter bacterial microbiota was compared between Campylobacter- positive and Campylobacter-negative samples, a lower abundance of Lactobacillus was observed among the positive samples. Overall, the work presented in this dissertation demonstrates collective efforts to improve our understanding of Campylobacter jejuni in broiler chickens using multidisciplinary approaches. iii Table of Contents Acknowledgements .............................................................................................................. i Abstract ............................................................................................................................... ii Table of Contents ............................................................................................................... iv List of Tables ...................................................................................................................... v List of Figures ................................................................................................................... vii Chapter One: Literature Review ......................................................................................... 1 Chapter Two: Survey of the U.S. broiler industry regarding pre- and post-harvest interventions targeted to mitigate Campylobacter contamination on broiler chicken products ............................................................................................................................. 16 Summary ....................................................................................................................... 16 Introduction ................................................................................................................... 17 Methods ......................................................................................................................... 20 Results ........................................................................................................................... 22 Discussion ..................................................................................................................... 29 Chapter Three: Investigation of indirect selective pressures conferring fitness advantages in fluoroquinolone-resistant Campylobacter jejuni in vitro ............................................. 50 Summary ....................................................................................................................... 50 Introduction ................................................................................................................... 51 Materials and Methods .................................................................................................. 53 Results ........................................................................................................................... 63 Discussion ..................................................................................................................... 65 Chapter Four: Characterization of temporal changes in the bacterial microbiota of conventional broiler litter .................................................................................................. 80 Summary ....................................................................................................................... 80 Introduction ................................................................................................................... 81 Methods ......................................................................................................................... 83 Results ........................................................................................................................... 88 Discussion ..................................................................................................................... 95 Chapter Five: Conclusion ............................................................................................... 141 Bibliography ................................................................................................................... 145 Appendices ...................................................................................................................... 166 Appendix A. Survey Questionnaire. ........................................................................... 166 iv List of Tables Table 2.1. Descriptions of the processing plants and broiler farms that participated in the survey. ............................................................................................................................... 36 Table 2.2. List of pre-harvest interventions and management practices implemented on- farm to reduce foodborne pathogen colonization in broiler chickens. (n* = total number of responses from broiler farm mangers, n† = total number of responses from poultry veterinarians)..................................................................................................................... 37 Table 2.3. Opinions on the effectiveness of pre- and post-harvest interventions and management practices implemented on farm and in the processing plant to reduce Campylobacter contamination in broiler chickens and carcasses. .................................... 38 Table 2.4. List of post-harvest interventions implemented in the processing plant to minimize microbial contamination on broiler chicken products. ..................................... 40 Table 2.5. New recommendations or changes that have been implemented within the past year to meet the USDA-FSIS performance standards for