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Development of Intranasal Bacterial Therapeutics to Mitigate the Bovine Respiratory Pathogen Mannheimia Haemolytica

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Development of Intranasal Bacterial Therapeutics to Mitigate the Bovine Respiratory Pathogen Mannheimia Haemolytica University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2019-11 Development of Intranasal Bacterial Therapeutics to Mitigate the Bovine Respiratory Pathogen Mannheimia haemolytica Amat, Samat Amat, S. (2019). Development of Intranasal Bacterial Therapeutics to Mitigate the Bovine Respiratory Pathogen Mannheimia haemolytica (Unpublished doctoral thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/111258 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Development of Intranasal Bacterial Therapeutics to Mitigate the Bovine Respiratory Pathogen Mannheimia haemolytica by Samat Amat A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES CALGARY, ALBERTA NOVEMBER, 2019 © Samat Amat 2019 Abstract The emergence of multidrug-resistant pathogens associated with bovine respiratory disease (BRD) presents a significant challenge to the beef industry, as antibiotic administration is commonly used to prevent and control BRD in commercial feedlot cattle in North America. Thus, developing antibiotic alternatives such as bacterial therapeutics (BTs) to mitigate BRD is needed. Recent studies suggest that the nasopharyngeal (NP) microbiota, particularly lactic acid-producing bacteria (LAB), are important to bovine respiratory health and may be a source of BTs for the inhibition of BRD pathogens. The research presented in this thesis aimed to develop intranasal BTs to mitigate the BRD pathogen Mannheimia haemolytica and promote NP microbiota stability in feedlot cattle. Results from Study 1 showed that commercial probiotic bacteria were able to inhibit M. haemolytica growth and its adherence to epithelial cells. Study 2 revealed that the NP microbial community structure and relative abundance of LAB families underwent significant changes when cattle transported from the farm to an auction market, then to feedlot. Many of the LAB families were inversely correlated with the BRD-associated Pasteurellaceae family, and isolates from Lactobacillaceae, Streptococcaceae and Enterococcaceae families inhibited growth of M. haemolytica in vitro. This study provided evidence of potential antagonistic competition taking place between LAB and BRD-associated pathogens within the respiratory tract. Following these studies, using a targeted approach based on criteria evaluating M. haemolytica inhibition, adherence to turbinate cells, and immunomodulation, 6 Lactobacillus strains from an initial group of 178 bacterial isolates originating from nasopharynx of cattle were identified as the best BT candidates (Study 3). Intranasal inoculation of these BTs reduced colonization by M. haemolytica and induced modulation of respiratory microbiota in dairy calves experimentally challenged with M. haemolytica (Study 4). Finally, the longitudinal effects of intranasally administered BTs on 2 the NP microbiota and the prevalence of BRD pathogens including Mannheimia were evaluated in post-weaned beef calves (Study 5). A single dose of intranasal BTs induced longitudinal modulation of the NP microbiota while showing no adverse effects on animal health and growth performance. With further characterization of inoculant dose and time of inoculation, the BTs may have potential for application as an antimicrobial alternative for mitigation of M. haemolytica in beef cattle. 3 Acknowledgements The combined efforts of many great people and organizations have made the successful completion of the research work presented in this thesis. First and foremost, I would like to gratefully acknowledge my supervisor Dr. Trevor Alexander for his guidance, support and encouragement throughout my PhD program. I have been lucky to have a supervisor who always trusted me and gave me the freedom to expand my research experience and collaboration network, as well as to pursue some of my own research ideas which were not within the scope of my thesis project. Dr. Alexander has made significant influence on my research career particularly on my academic writing and organizing research-related thoughts. I truly appreciate all the effort and long hours that Dr. Alexander has put into finalizing my thesis. I will always remember him by his following words and advice: “Samat, I have faith in you, you can do it” “Sky is the limit” “Please see the big picture, but also pay attention to the details on how to get there” and “Never use the same word twice in a sentence”. Next, I would like to thank my supervisor Dr. Edouard Timsit for his support, guidance, and constructive criticism during the course of my program. Dr. Timsit has helped me to foster my logical thinking skills. I am thankful for his help during my candidacy exam preparation, and for sending me for additional training. I gratefully acknowledge my supervisory committee including Drs. Tim McAllister, Andre Buret and Joroen De Buck. I truly appreciate all the time and constructive feedback they provided for my project. I am also very thankful to my supervisory committee for giving me the second chance to redo my Candidacy exam. Although it was a painful experience in failing the first attempt, this failure was a wakeup call for me and led me to realize how much I don’t know about my “craft”. During the next 6 months of Candidacy exam preparation, I have increased knowledge on my subject which was very useful for my thesis project. This candidacy exam experience has also taught me an important life lesson and made me to realize the meaning of Steve Job’s quote “You can't connect the dots looking forward; you can only connect them looking backward”. 4 I also extend a special thank you to Dr. Frank van deer Meer who agreed to join my supervisor committee as co-supervisor at the late stage of my program, and provided guidance in thesis writing. I would also like to thank Dr. Danica Baines, who provided special assistance during my cell culture work. My sincere thanks also go to Dr. Devin Holman for his excellent contribution to the bioinformatics analysis, manuscript preparation, and helping me to learn bioinformatics. I also sincerely thank Dr. Timothy Schwinghamer for his special help in statistical analysis particularly with path modeling analysis. I wish to thank Dr. Matthew Workentine for his assistance in some bioinformatics analysis. I am also grateful to Jay Yanke, Grant Duke, and Darell Vedres for their technical support in biochemical tests, microscopic imaging, and GC analysis, respectively. I express my appreciation to the following lab team members including Dr. Long Jin, Pamela Caffyn and Leandra Schneider for their technical support during my animal trials and laboratory analysis. I also thank the feedlot crew at the Lethbridge Research and Development Centre, and animal care staff at the Veterinary Science Research Station, and Pathologists at Diagnostic Services Unit, College of Veterinary Medicine, University of Calgary for their technical support during the animal trials. I also thank my friends Dennis and Marie-Jeanne Will, Marvin Genno, and Bev Lanz for the support they provided me and my family during my PhD program. My special thanks also goes to my MSc. supervisor Dr. Steve Hendrick, who kindly provided me several hours of lectures on Epidemiology and feedlot practice which helped me to get ready for my candidacy exam. I am sincerely grateful to my wife Mikrigul for her tremendous support throughout my study. Without her support, I would not have been able to concentrate on my research work as much as I did and spend as much time in the lab and office as I spent. She made more time for my work by taking care of our kids in most of the evenings and weekends. I also thank my two beautifful daughters, Subina and Nargiza for bringing me more joy and strength, and for being so forgiving to their dad 5 when he missed many nights to put them to sleep and missed attending some of their weekend activities. I am extremely indebted to my parents, brothers, and sisters Wurgul and Nurgul Amat for their endless support during the pursuit of my education. I also acknowledge the financial support provided by the Alberta and Agiculture Forestry, Beef Cattle Research Council, and Agriculture and Agri-Food Canada (AAFC). I am grateful for the Canadian Natural Science and Engineering Research Council (NSERC) for providing me with the NSERC doctoral scholarship. Last, but not the least, I would like to thank the support I received from the friendly and supportive research community at the AAFC Lethbridge Research and Development Centre. My thanks also goes to Department of Production Animal Health, and the Office of Graduate Study at the Faculty of Veterinary Science, University of Calgary. 6 Dedication I am dedicating this thesis to the few special people in my life. First, to my dad Amat Niyaz and my mom Hansahan Yasin who have taught me the value of working hard, and to always follow my dreams. The advice my dad gave me at the early stage of my life: “Hustle silently and your work will speak for itself” and “You are the only one who can
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