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IMPROVING THE DIAGNOSTIC METHODS AND PROCESSES FOR THE IDENTIFICATION AND CHARACTERIZATION OF BRACHYSPIRA 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 DHARMASIRI GAMAGE RUWINI SULOCHANA KULATHUNGA © Copyright Dharmasiri Gamage Ruwini Sulochana Kulathunga, April 2021. All rights reserved. Unless otherwise noted, copyright of the material in this thesis belongs to the author. PERMISSION TO USE In presenting this thesis in partial fulfillment 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 in any manner, in whole or in part, for scholarly purposes may be granted by the professor who supervised my thesis work, or in their absence, permission may be granted 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 or parts thereof for financial gain shall not be allowed without 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. 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 Veterinary Microbiology Western College of Veterinary Medicine University of Saskatchewan 52 Campus Drive Saskatoon, Saskatchewan S7N 5B4 Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan S7N 5C9 Canada i ABSTRACT There are no standardized antimicrobial susceptibility test methods or interpretative criteria for Brachyspira spp. and laboratories rely heavily on in-house methods that yield highly variable test results. Despite advances in diagnostic technologies, treatment choices for swine dysentery are most often empiric, which can lead to increasing levels of antimicrobial resistance. The objectives of this study were (1) to develop a standardized agar dilution method and describe the antimicrobial susceptibility of a collection of Brachyspira isolates, (2) to develop a feasible methodology for antimicrobial susceptibility testing to include in the diagnostic workflow; including an analysis of accuracy and cost for different diagnostic approaches, (3) to develop assay selection decision trees based on the diagnostic objectives and (4) to investigate the genetic determinants of resistance to protein synthesis inhibitory drugs. To obtain consistent minimum inhibitory concentrations (MICs) between replicates in susceptibility testing for all Brachyspira spp. the media (TSA based), temperature (42°C), and the starting inoculum size (1-2 × 108 CFU/ ml) were determined. A standard antibiotic susceptibility test (agar dilution) with good reproducibility (80-100%) was developed. According to the test results B. hyodysenteriae and B. hampsonii isolates were profoundly susceptible to pleuromutilin drugs while some of B. pilosicoli isolates indicated high MICs (MIC > 8 µg/ml) for pleuromutilin drugs. Moreover, highly variable susceptibility test results for tylosin, tylvalosin, and lincomycin were observed amongst all species. The material and technical cost for all the diagnostic tests performed in the WCVM diagnostic laboratory were calculated, including a modified (miniaturized) agar dilution test. Further, decision trees were developed to select the most cost-effective test bundle based on the diagnostic objective. Using whole genome sequencing, isolates with higher MICs to macrolides were found to carry either a resistant gene called lnuC or have SNPs (A2058G or A2059G both) while isolates with higher ii MICs for pleuromutilin drugs were found to have the tvaB gene. None of these resistance genes or SNPs were identified in isolates with lower MICs for those drugs. A multiplex PCR was developed to screen the two resistance genes (lnuC and tvaB) and predict the phenotypic resistance of clinical isolates in future. In conclusion, overall findings and novel test methods that were developed will help to improve the diagnostic workflow for the identification of Brachyspira- associated diseases including swine dysentery and support veterinarians for rapid diagnosis and evidence-based selection of antimicrobials to improve the treatment outcome. iii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my supervisor Dr. Joe Rubin, for his continued guidance, dedication, and support throughout my PhD program. His dynamism, vision, sincerity, and motivation have deeply inspired me, and it was a great privilege and honour to work and study under his guidance. I would also like to thank all my graduate committee members Dr. J. Harding, Dr. S. Gow, Dr. M. Links and my graduate committee chair Dr. J. Hill for their support and assistance during my research study and writing of this thesis. Also, recognition is extended to Dr. S. Parker for her support in statistical data analysis. Very special thanks go to Champika and Michelle for all their support in my research. I would also like to acknowledge my lab mates (Roshan, Kazal, Dongyun, Bev, and Rachel), office mates (Rajnish, Emilie, Tami and Kela), the staff at GMP, Linda, Lana and all my other friends for their help and kind friendship during my PhD program. Most importantly, I dedicate this thesis to my parents D.G. Kulathunga (father), I. Dissanayake (Mother), husband (Amila) and my loving two sisters for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without them. Finally, I wish to thank my two wonderful daughters, Prabashi and Methushi who provide unending love and patience during my studies. Finally, gratitude is extended to the funding agencies Swine Innovation Porc., Elanco Animal Health, NSERC, Devolved Scholarship, Department of Veterinary Microbiology, SK Innovation for financial support during this study. iv TABLE OF CONTENTS PERMISSION TO USE ............................................................................................................................... i ABSTRACT ................................................................................................................................................. ii ACKNOWLEDGEMENTS ...................................................................................................................... iv TABLE OF CONTENTS ........................................................................................................................... v LIST OF TABLES ................................................................................................................................... viii LIST OF FIGURES .................................................................................................................................... x LIST OF ABBREVIATIONS .................................................................................................................. xii 1. INTRODUCTION AND LITERATURE REVIEW ........................................................................ 1 1.1 Brachyspira-associated diseases of pigs ....................................................................................... 1 1.1.1 Brachyspira taxonomy .......................................................................................................... 1 1.1.2 Re-emergence of Brachyspira-associated diseases in Canada .............................................. 4 1.1.3 Pathogenesis and clinical disease in pigs .............................................................................. 5 1.1.4 Diagnostic methods of Brachyspira-associated diseases ...................................................... 7 1.1.5 Prevention and control of swine dysentery ......................................................................... 18 1.1.6 Treatment (Kulathunga and Rubin 2017) ........................................................................... 20 1.2 Antimicrobial drug action and resistance .................................................................................... 21 1.2.1 General introduction to antimicrobial therapy .................................................................... 21 1.2.2 Antimicrobial drugs use in swine medicine ........................................................................ 27 1.3 Antimicrobial susceptibility testing ............................................................................................ 28 1.3.1 Classification of antimicrobial susceptibility tests .............................................................. 28 1.3.2 Specific challenges to susceptibility testing of Brachyspira (Kulathunga and Rubin 2017) .. ............................................................................................................................................. 33 1.3.3 Summary of susceptibility test methods that have been used for Brachyspira (Kulathunga and Rubin 2017) .................................................................................................................................
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