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Using Comparative Genomics to Identify Virulence Traits and Vaccine Candidates in Mannheimia Haemolytica

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Using Comparative Genomics to Identify Virulence Traits and Vaccine Candidates in Mannheimia Haemolytica Using comparative genomics to identify virulence traits and vaccine candidates in Mannheimia haemolytica A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfilment of the Requirements For the Degree of Doctor of Philosophy In the Department of Large Animal Clinical Sciences Western College of Veterinary Medicines University of Saskatchewan Saskatoon By Cassidy Klima Copyright Cassidy Klima, June 2015. All rights reserved Permission to Use In presenting this thesis/dissertation in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis/dissertation or parts thereof for financial gain shall not be allowed with-out my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: Head of the Department of Large Animal Clinical Sciences Western College of Veterinary Medicine University of Saskatchewan Saskatoon, Saskatchewan, S7N 5B4 Canada OR Dean College of Graduate Studies and Research University of Saskatchewan 107 Administration Place Saskatoon, Saskatchewan, S7N 5A2 Canada i Abstract Bovine respiratory disease (BRD) is the principal cause of morbidity and mortality among feedlot cattle. Mannheimia haemolytica is consistently implicated in this condition, but treatment options are diminishing with the rise of antimicrobial resistance and intensifying consumer pressure to reduce reliance on conventional therapies. Thus, sustainable alternatives like vaccination are required. In this study, the phenotypic and genotypic diversity of BRD pathogens were examined with the objective to identify vaccine targets using reverse vaccinology, an innovative approach to identify antigens via genomic sequence. Preliminary surveillance confirmed M. haemolytica serotype 2 isolates were predominant in healthy animals (75.5%) while serotypes 1 (70.7%) and 6 (19.5%) were common in diseased animals. Pathogens of BRD, including M. haemolytica, Pasteurella multocida and Histophilus somni were also isolated from North American BRD mortalities, and compared using pulsed-field gel electrophoresis and antimicrobial susceptibility. Concurrently, polymerase chain reaction detection of bacterial and viral agents confirmed that M. haemolytica with bovine viral diarrhea virus were the most prevalent. Whereas isolates from live cattle were found to have a relatively low level of resistance, several pathogens from the mortalities were found to contain integrative conjugative elements (ICE) conferring resistance to seven antimicrobial classes. These ICEs were transferred via conjugation to other bacterial species, emphasizing the need for alternative antimicrobial therapies. Collectively, data from these investigations informed the selection of 11 diverse M. haemolytica strains for whole genome sequencing and comparative analyses. Several bacteriophage associated genes and CRISPR-Cas regulated gene expression systems were identified and are likely contributing to virulence in M. haemolytica. Coding sequences across all genomes were screened using pan-genome analysis, identifying 291 candidates with cell-surface associated signatures. Using a cell-free translation system and enzyme-linked immunosorbent assay the candidates were screened against serum from cattle challenged with serovar 1, 2 or 6 of M. haemolytica, and ranked according to immunogenicity. The top five vaccine candidates included Ssa1, ComE, a solute binding protein, an outer membrane protein, and the periplasmic component of an ABC transporter. With further characterization, these unique antigenic candidates could be developed into a vaccine to effectively reduce the dependence on antimicrobial therapies. ii Acknowledgments I would like to gratefully acknowledge my graduate studies supervisors, Drs. Tim McAllister and Steve Hendrick for all of their guidance, support and patience. I would also like to acknowledge my advisory committee including Drs. Andrew Potter, Trevor Alexander and Gregory Penner. I appreciate all of the time and wisdom each member provided during the course of this project. Thank you to all of the technical support staff at Agriculture and Agri-Food Canada including Dawn Gray, Ruth Barbieri, Fred Van Herk, Wendi Smart and Lyn Patterson. To Ronda Carlson, Cindy Johnson and Bernie Genswein from the IT department at the Lethbridge Research Center, I cannot express enough appreciation for all of the support given. To Dr. Rahat Zaheer and Shaun Cook, who have both been so integral to the work done here, I express the most heartfelt gratitude. To all of the students and support staff that helped pick me up along the way, thank you so much for all of your time, effort and encouragement. Sample collection for this study was facilitated by Feedllot Health Management Services; their contributions are very much appreciated. I gratefully acknowledge the financial support provided by Alberta Livestock and Meat Association and The Alberta Livestock Genome Program. iii Dedication To all of my family who have supported me in countless ways for many, many years. And, for Pam, Justin, Alicia and Paul C. You guys are the best anyone could ask for. iv Table of contents Permission to Use ............................................................................................................................. i Abstract ............................................................................................................................................ ii Acknowledgments ............................................................................................................................iii Dedication ........................................................................................................................................ iv List of Tables .................................................................................................................................... ix List of Figures ....................................................................................................................................x List of Abbreviations ........................................................................................................................ xi 1 Literature review ........................................................................................................................... 1 1.1 Introduction to bovine respiratory disease and the North American cattle industry ........................ 1 1.1.1 Occurrence and economics .......................................................................................................... 1 1.1.2 Bovine respiratory disease complex ............................................................................................ 1 1.1.2.1 Viral agents of BRD .............................................................................................................. 1 1.1.2.1.1 Bovine herpes virus 1 (BHV-1) ......................................................................... 1 1.1.2.1.2 Bovine respiratory syncytial virus (BRSV) ........................................................ 2 1.1.2.1.3 Bovine parainfluenza 3 virus (BPI3V)............................................................... 2 1.1.2.1.4 Bovine viral diarrhoea virus (BVDV) ................................................................ 3 1.1.2.2 Bacterial agents of BRD ........................................................................................................ 3 1.1.2.2.1 Mycoplasma bovis ........................................................................................... 3 1.1.2.2.2 Histophilus somni ............................................................................................. 4 1.1.2.2.3 Pasteurella multocida ...................................................................................... 4 1.1.2.2.4 Mannheimia haemolytica ................................................................................ 4 1.2 Management of BRD ........................................................................................................................... 6 1.2.1 Preconditioning ............................................................................................................................ 6 1.2.2 Vaccination ................................................................................................................................... 6 1.2.3 Antimicrobial Use ......................................................................................................................... 7 1.3 Using the Genome for Vaccine design ................................................................................................ 8 1.3.1 Reverse vaccinology ....................................................................................................................
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