University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2021-03-24 Characterizing the microbiota of bovine digital dermatitis Caddey, Benjamin Caddey, B. J. (2021). Characterizing the microbiota of bovine digital dermatitis (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/113188 master 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 Characterizing the microbiota of bovine digital dermatitis by Benjamin Jordan Caddey A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES CALGARY, ALBERTA MARCH, 2021 © Benjamin Jordan Caddey 2021 Abstract Bovine digital dermatitis (DD) is a skin disease affecting cattle worldwide. As a significant contributor to infectious lameness, DD is increasingly an economic and animal welfare concern for dairy and beef producers. DD is a polymicrobial disease, with many different types of anaerobic bacteria strongly associated with lesions. No causative agents or etiopathogenesis mechanisms of DD are yet accepted due to the multiple different species present and microbiota variation among individual lesions. The involvement of Treponema in lesion development is generally accepted, whereas what combination of other anaerobic bacteria are involved is currently debated. Insufficient and incomplete identification and characterization of these additional species are a limiting factor in DD pathogenesis research. This thesis aimed to fill the gaps in knowledge of these additional anaerobes throughout DD lesions, while providing the first comprehensive description of DD microbiota in feedlot beef cattle. Through high throughput sequencing and culturing of DD tissue biopsies, we identified Treponema, Mycoplasma, Fusobacterium spp., Porphyromas levii, and Bacteroides pyogenes as potential DD pathogens, and used a multiplex qPCR for absolute quantification and reproducible characterization of species population dynamics. We identified T. medium, P. levii, and T. phagedenis as a group strongly associated with early lesion stages, thus potentially being involved in lesion formation. Through a meta-analysis of all publicly available DD metagenomic studies, we identify Treponema, Mycoplasma, Fusobacterium, and Porphyromonas as the primary DD-associated microbiota. We recommend focusing future DD research efforts on culturing and characterizing species of these groups in an effort to establish etiopathogenesis mechanisms of DD. ii Preface This thesis contains two manuscripts prepared for submission. The following manuscripts included in this thesis are: Chapter 2 Caddey, B, Orsel, K, Naushad S, Derakhshani H, De Buck J. 2020. Identification and quantification of bovine digital dermatitis-associated microbiota across lesion stages in feedlot beef cattle. Chapter 3 Caddey B, De Buck J. 2020. Meta-analysis of bovine digital dermatitis microbiota reveals distinct microbial community structures associated with lesions. iii Statement of Contribution Ben Caddey (BC), Jeroen De Buck (JDB), and Karin Orsel (KO) designed the study for chapter 2. Biopsy sampling protocols for the collection of skin biopsies were designed by BC, JDB, and KO. Sampling was conducted by BC, KO, Anice Thomas, Julian Cortes, Lacey Wynnyk, and Tina Petersen. Biopsy handling and processing, including anaerobic culture of Treponema and other anaerobes, DNA extraction, and sample preservation were performed by BC. Purified biopsy DNA was sequenced against the V3V4 hypervariable region of the 16S rRNA gene by the McMaster Genomics Facility. BC performed all processing and analysis of metagenomic sequencing data. Anaerobic bacteria from DD biopsies were isolated and identified by BC, and DNA extractions were performed by BC before Hooman Derakhshani sequenced and assembled draft genomes of isolates. Species-specific genes for qPCR targeting were identified by Sohail Naushad and BC. The novel multiplex qPCR was designed and validated by BC, and all qPCR reactions and analysis were performed by BC. The study for Chapter 3 was designed by BC and JDB. All studies eligible for inclusion were identified by BC, and data collection and analysis was performed by BC. Data interpretation for both chapters was conducted by BC and JDB. All manuscripts and chapters in this thesis were written by BC under the guidance and feedback of JDB. iv Acknowledgements First and foremost, I would like to thank my supervisor, Dr. Jeroen De Buck, for his role in this transformative journey over the past couple years. The moulding of your supervision has led to immense personal growth, and developed confidence in my aptitude for research. Thank you for trusting in my capabilities and allowing me the freedom to explore my research interests without hesitation. I will fondly remember our meetings that were always filled with creative and ambitious dialogue, and occasionally overflowing with out-of-this-world ideas, usually requiring one of us to bring the other back to reality. Thank you to my supervisory council. Your knowledge, experience, and insights were invaluable to this work and my personal development. Thank you to Dr. Doug Morck for helping kickstart my progress on this journey by providing anaerobic cultures so that I could get well-acquainted with fastidious organisms. Dr. Karin Orsel, many-many thanks for your constant support and shared interest in my dairy-crazy passions. You were pivotal in improving my critical thinking, scientific reasoning, and conceptualization of research on a grander scale, always with a producer-first mentality. Also, thank you to all the veterinarians, producers, and feedlot workers that supported our research and for your continued enthusiasm. To my lab mates, thank you for the years of light-heartedness and support to create a welcoming lab environment. To Rakel Arrazuria, you inspired a rigorous criticism for every step of the scientific process. Your mentorship went above and beyond, and I can’t thank you enough for your friendship and support. To my fellow graduate students and the UCVM, who facilitated a community of support and teamwork. Thank you, Lisa Gamsjaeger, for the mutual support v during the rollercoaster ride that is graduate school. I am incredibly grateful for your friendship and all the adventures we had that made Calgary feel like home. To all my equally crazy mountaineering friends, thank you for all the amazing and insane trips outside of the city. The mountains will never stop calling. To my parents and grandparents, you have all supported my goals and ambitions unconditionally, even when it means losing a pair of hands for baling small-square hay in 40 ºC weather for a couple summers! You all motivate me to be my best every day. Thank you, all. vi Dedication To Ron and Pat Jackson, who have always been there for me, every step of the way. vii Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Statement of Contribution .......................................................................................................... iv Acknowledgements ....................................................................................................................... v Dedication .................................................................................................................................... vii Table of Contents ....................................................................................................................... viii List of Tables ................................................................................................................................. x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Chapter 1: Introduction ............................................................................................................. 14 Bovine digital dermatitis ........................................................................................................... 14 DD in beef cattle ....................................................................................................................... 15 Related lesions .......................................................................................................................... 15 Lesion scoring ........................................................................................................................... 16 Risk factors ................................................................................................................................ 17 Mitigation and treatment strategies