Template C v3.0 (beta): Created by J. Nail 06/2015 Advancing our understanding of the Edwardsiella By TITLE PAGE Stephen Ralph Reichley A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Veterinary Medical Science in the College of Veterinary Medicine Mississippi State, Mississippi August 2017 Copyright by COPYRIGHT PAGE Stephen Ralph Reichley 2017 Advancing our understanding of the Edwardsiella By APPROVAL PAGE Stephen Ralph Reichley Approved: ____________________________________ Matthew J. Griffin (Co-Major Professor) ____________________________________ Mark L. Lawrence (Co-Major Professor) ____________________________________ Terrence E. Greenway (Committee Member) ____________________________________ Lester H. Khoo (Committee Member) ____________________________________ David Wise (Committee Member) ____________________________________ R. Hartford Bailey (Graduate Coordinator) ____________________________________ Kent H. Hoblet Dean College of Veterinary Medicine Name: Stephen Ralph Reichley ABSTRACT Date of Degree: August 11, 2017 Institution: Mississippi State University Major Field: Veterinary Medical Science Major Professors: Matthew J. Griffin and Mark L. Lawrence Title of Study: Advancing our understanding of the Edwardsiella Pages in Study 211 Candidate for Degree of Doctor of Philosophy Diseases caused by Edwardsiella spp. are responsible for significant losses in wild and cultured fishes around the world. Historically, Edwardsiella tarda has been considered the most phenotypically and genotypically heterogeneous member of the genus. Investigations into intraspecific variability of E. tarda demonstrated isolates previously classified as E. tarda actually represent three genetically distinct yet phenotypically ambiguous groups, leading to the adoption of E. piscicida and E. anguillarum as discrete taxa. Current genomic investigations have demonstrated significant differences between these organisms. To this end, real-time quantitative PCR assays were developed to quickly and accurately detect the pathogens in pond water, fish tissue and broth culture. Additionally, whole genome sequencing was performed for representative isolates of each Edwardsiella spp. Furthermore, forty-seven Edwardsiella isolates, representing all five taxa, from different hosts and a wide temporal and geographic range were analyzed using commercial microbial identification kits, repetitive sequence-mediated polymerase chain reaction, fatty acid methyl ester analysis, antimicrobial resistance profiles, in addition to 16S, gyrB, sodB and plasmid sequencing. This analysis demonstrated key differences in gene sequences and plasmid profiles among these important bacterial pathogens and further supported contemporary taxonomic classifications. Additionally, a real-time multiplex PCR was developed to accurately discriminate between all Edwardsiella spp. affecting fish; namely E. ictaluri, E. piscicida, E. tarda and E. anguillarum. Moreover, recent publications suggest E. piscicida is more commonly associated with disease outbreaks in Mississippi catfish aquaculture than E. tarda or E. anguillarum. To this end, several different challenge models were evaluated. Comparative virulence was assessed, along with histopathological lesions and posterior kidney clearance rates in channel catfish (Ictalurus punctatus). Diagnostic case submissions suggest E. piscicida is more commonly associated with disease outbreaks in blue catfish (Ictalurus furcatus) x channel catfish hybrids compared to channel catfish. This led to investigations into the relative pathogenicity of E. piscicida in hybrid and channel catfish, which demonstrated a significantly lower median lethal dose (LD50) for E. piscicida in hybrid catfish; an important finding given the increased production of hybrid catfish in U.S. farm-raised catfish in the southeastern United States. DEDICATION This dissertation is dedicated to my family. Their support, love, inspiration and understanding throughout my life and especially during the years of my PhD program made all of this possible. ii ACKNOWLEDGEMENTS I would like to thank Dr. Matt Griffin for his willingness to mentor me during my externship as a veterinary student and involving me then in his research projects. That experience piqued my interest in research and was the focal point of considering a PhD program. I thank you for welcoming me back to your lab two years later as a PhD student and for everything you have done for me over the past four years. I sincerely appreciate your patience with me throughout my program, the great discussions we have had, your constant sage advice and all the information you have bestowed upon me. In addition to my dissertation work, I am very appreciative for you including me in many of the other projects within your lab; these experiences greatly expanded my knowledgebase and professional network. I would also like to thank Dr. Mark Lawrence, my co-major professor, for welcoming me into his lab and providing me office and lab space in Starkville. Your guidance and mentorship were pivotal during my PhD program and I will always be grateful to you for the opportunities you have given me. Thanks also to Dr. Lester Khoo. I appreciate you taking the chance on a veterinary student with no fish experience and your immense hospitality during my externship and throughout my PhD program. Thanks also to my other committee members Dr. Terry Greenway and Dr. David Wise. Your mentorship, instruction and friendship during my time in the Delta were much appreciated. iii I would also like to extend my sincere thanks and gratitude to the faculty and staff in the Department of Pathobiology and Population Medicine, Department of Basic Science and the Thad Cochran National Warmwater Aquaculture Center for their time and support. A special thanks to Cyndi Ware, James Steadman and Marsha Lewis for all their help in the lab. Thanks also to Dr. Graham Rosser who taught me so much along my journey and was always there for support and guidance. There were many other graduate students and post-docs who helped along the way, you know you are and I am very grateful. iv TABLE OF CONTENTS DEDICATION ........................................................................................................ ii ACKNOWLEDGEMENTS ................................................................................... iii LIST OF TABLES ................................................................................................. ix LIST OF FIGURES .............................................................................................. xii CHAPTER I. INTRODUCTION .....................................................................................1 1.1 Introduction ...................................................................................1 1.1.1 U.S. catfish aquaculture ...........................................................1 1.1.2 Members of the Edwardsiella .................................................3 1.1.3 Reclassification of Edwardsiella tarda ...................................9 1.2 Research Objectives: ...................................................................14 1.2.1 Objective 1: ...........................................................................14 1.2.2 Objective 2: ...........................................................................14 1.2.3 Objective 3: ...........................................................................14 1.2.4 Objective 4: ...........................................................................14 1.2.5 Objective 5: ...........................................................................14 1.3 Research Approaches ..................................................................15 1.3.1 Objective 1 Approach ............................................................15 1.3.2 Objective 2 Approach ............................................................15 1.3.3 Objective 3 Approach ............................................................16 1.3.4 Objective 4 Approach ............................................................17 1.3.5 Objective 5 Approach ............................................................18 1.4 Potential Impact ...........................................................................18 1.5 References ...................................................................................19 II. REAL-TIME POLYMERASE CHAIN REACTION ASSAYS FOR THE DETECTION AND QUANTIFICATION OF EDWARDSIELLA TARDA, EDWARDSIELLA PISCICIDA, AND EDWARDSIELLA PISCICIDA-LIKE SPECIES IN CATFISH TISSUES AND POND WATER ...................................................................................................27 2.1 Introduction .................................................................................27 2.2 Materials and methods .................................................................29 v 2.2.1 Bacterial cultures and isolation of genomic DNA .................29 2.2.2 Design of primer and probe sets ............................................32 2.2.3 Generation of PCR standards ................................................33 2.2.4 Quantitative PCR ...................................................................33 2.2.5 PCR specificity, sensitivity, repeatability, and reproducibility .......................................................................33 2.2.6 Detection of target DNA from broth culture, fish tissue, and pond