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Understanding the Interaction Between Mycoplasma Bovis and Bovine Respiratory Macrophages

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Understanding the Interaction Between Mycoplasma Bovis and Bovine Respiratory Macrophages UNDERSTANDING THE INTERACTION BETWEEN MYCOPLASMA BOVIS AND BOVINE RESPIRATORY MACROPHAGES A Thesis Submitted to the College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Veterinary Microbiology University of Saskatchewan Saskatoon By TERESIA W. MAINA © Copyright. Teresia. W. Maina, March 2019. All rights reserved. PERMISSION TO USE 1 In presenting this thesis/dissertation in partial fulfillment of the requirements for a Post-graduate 2 degree from the University of Saskatchewan, I agree that the Libraries of this University may 3 make it freely available for inspection. I further agree that permission for copying of this 4 thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by 5 the professor or professors who supervised my thesis/dissertation work or, in their absence, by 6 the Head of the Department or the Dean of the College in which my thesis work was done. It is 7 understood that any copying or publication or use of this thesis/dissertation or parts thereof for 8 financial gain shall not be allowed without my written permission. It is also understood that due 9 recognition shall be given to me and to the University of Saskatchewan in any scholarly use 10 which may be made of any material in my thesis/dissertation. 11 Requests for permission to copy or to make other uses of materials in this thesis/dissertation in 12 whole or part should be addressed to: 13 Head of the Department of Veterinary Microbiology 14 University of Saskatchewan 15 Saskatoon, Saskatchewan S7N 5B4 16 Canada 17 OR 18 Dean 19 College of Graduate and Postdoctoral Studies 20 University of Saskatchewan 21 116 Thorvaldson Building, 110 Science Place 22 Saskatoon, Saskatchewan S7N 5C9 Canada 23 24 i ABSTRACT 25 Mycoplasma bovis is the most pathogenic bovine mycoplasma in Europe and North America. It 26 forms an important component in the bovine respiratory disease (BRD) complex, a multifactorial 27 disease of feedlot cattle that causes major economic loss. Naturally occurring M. bovis persist in a 28 herd over an extended period and can be consistently identified not only in lesions but also 29 commonly in healthy lungs and those with pneumonia indicating that M. bovis is capable of 30 persisting in lungs of recovered animals. This suggests that M. bovis can modulate alveolar 31 macrophage effector functions, although the mechanisms employed by M. bovis are not well 32 understood. In this thesis, I determined how M. bovis modulates bovine alveolar macrophage 33 (BAM) effector functions. I used primary alveolar macrophages infected with M. bovis Mb1 to 34 demonstrate that the production of nitric oxide and cytokines was impaired. I observed a 35 reduction in the production of nitric oxide but not a decrease in gene transcription of iNOS; and 36 no stimulation of the pro-inflammatory cytokine TNF-α coupled with an increase of IL-10 37 expression in infected BAMs. These results suggest modulation of the immune response by M. 38 bovis. To better understand other strategies of immune suppression by M. bovis to evade the host 39 immune responses, I focused on the ability of M. bovis to modulate macrophage apoptosis during 40 infection. Previously, studies on M. bovis infection have reported it to exert both pro- and anti- 41 apoptotic effects on a diversity of cell types, including neutrophils, lymphocytes, monocytes and 42 macrophages. In this thesis, I sought to understand the mechanism of inhibition of apoptosis 43 using a BoMac bovine macrophage cell line to identify the apoptosis pathways modulated by M. 44 bovis infection. The results indicate that M. bovis strain Mb1 is able to delay STS-induced 45 apoptosis in BoMac cells, activate the NF-kβ pathway and induce up-regulation of the pro- 46 survival genes Bcl-2 and Bcl-XL. Additionally, upon infection M. bovis inhibits the activity of 47 caspases 3, 6, and 9, reduces ROS production and inhibits DNA fragmentation. These results 48 support the observation that M. bovis inhibit apoptosis for survival and potentially facilitate 49 bacterial survival, replication and transmission. 50 The results presented in this thesis taken together detail how M. bovis modulates the macrophages 51 effector functions and apoptosis to survive within the bovine host. The studies also improve the 52 understanding of factors that contribute to virulence; dissemination and immune evasion of a 53 pathogen is critical knowledge to contemplating new vaccines and therapeutics. 54 55 ii ACKNOWLEDGEMENTS 56 The work presented in this thesis would not have been possible without support from many 57 different individuals to whom I am greatly indebted. 58 Firstly, I am grateful to my supervisor, Dr. Jose Perez-Casal for tirelessly working with me to 59 conceive the ideas that shaped this work, for constant direction and helpful insights along the 60 way, and for reading the thesis and giving useful suggestions in record time. 61 I also would like to thank the members of my Research Committee: Dr. Baljit Singh, Dr. Heather 62 Wilson, Dr. Janet Hill, Dr. Jeffrey Chen, Dr. Scott Napper, and Dr. Volker Gerdts for their 63 valuable insightful comments and constructive critique that undoubtedly improved the quality of 64 this work. To the team back in Kenya, Dr. Jan Naessens and Dr. Hezron Wesonga thank you for 65 the opportunity to participate in this interdisciplinary-international group which immensely 66 contributed to my professional development and team dynamics. 67 I also would like to thank the members of Jose’s lab and aggregates, especially Tracy Prysliak to 68 whom I am heavily indebted to for expert help in the lab with protocols, experiments and animal 69 trial assistance; and Dr. Steve Jimbo, Dr. Musa Mulongo, and Dr. Muhammed Suleman for their 70 constant rescues. To my colleagues at VIDO for their continued support; every seminar, lab 71 meeting, “office” talks and chit chatting contributed for my professional and personal growth, 72 and made everything more enjoyable and fun! To Yurij Popowych for the support and all the 73 hours he put in performing and analysing my flow cytometry work and the VIDO-InterVac 74 animal care group for their patience during the animal trials and isolation of BAMs. Special 75 thanks to Dr. Matthias Schweizer (Universität Bern), for the gift of the BoMac cell line. 76 Last but not least, to my family back at home “Dad, Mum, Mike, and Njau,” I am sorry I missed 77 most of the important get-togethers. Thank you for being my support system and prayer warriors 78 that helped me to keep going. My gratitude also extends to Lisa, Patsy, and Dave (R.I.P), Rey, 79 and Dr. Moh Hamid’s family, for being my family away from home throughout my grad school 80 and beyond. Your kindness will never be forgotten! 81 82 83 iii TABLE OF CONTENTS 84 PERMISSION TO USE ................................................................................................ i 85 ABSTRACT .................................................................................................................. ii 86 ACKNOWLEDGEMENTS ......................................................................................... iii 87 LIST OF FIGURES .................................................................................................. viii 88 LIST OF ABBREVIATIONS ..................................................................................... ix 89 CHAPTER 1. Introduction and literature review ....................................................... 1 90 1.1. Mycoplasmas ..................................................................................................................................... 1 91 Classification ......................................................................................................................................................... 1 92 Structure ................................................................................................................................................................. 1 93 Habitat .................................................................................................................................................................... 2 94 Multiplication and cell invasion ............................................................................................................................ 2 95 1.2 Mycoplasma bovis ............................................................................................................................... 5 96 Epidemiology ......................................................................................................................................................... 6 97 Pathogenesis and virulence .................................................................................................................................... 6 98 Modulation of immune responses to M. bovis ...................................................................................................... 9 99 Diagnostics .......................................................................................................................................................... 11 100 Treatment ............................................................................................................................................................. 13 101 Control ................................................................................................................................................................
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