Identification of Novel Flavobacteria from Michigan and Assessment of Their Impacts on Fish Health
Total Page：16
File Type：pdf, Size：1020Kb
IDENTIFICATION OF NOVEL FLAVOBACTERIA FROM MICHIGAN AND ASSESSMENT OF THEIR IMPACTS ON FISH HEALTH By Thomas P. Loch A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Pathology 2012 1 ABSTRACT IDENTIFICATION OF NOVEL FLAVOBACTERIA FROM MICHIGAN AND ASSESSMENT OF THEIR IMPACTS ON FISH HEALTH By Thomas P. Loch Flavobacteriosis poses a serious threat to wild and propagated fish stocks alike, accounting for more fish mortality in the State of Michigan, USA, and its associated hatcheries than all other pathogens combined. Although this consortium of fish diseases has primarily been attributed to Flavobacterium psychrophilum, F. columnare, and F. branchiophilum, herein I describe a diverse assemblage of Flavobacterium spp. and Chryseobacterium spp. recovered from diseased, as well as apparently healthy wild, feral, and famed fishes of Michigan. Among 254 fish-associated flavobacterial isolates recovered from 21 fish species during 2003-2010, 211 of these isolates were Flavobacterium spp., and 43 were Chryseobacterium spp. according to ribosomal RNA partial gene sequencing and phylogenetic analysis. Both F. psychrophilum and F. columnare were indeed associated with multiple fish epizootics, but the majority of isolates were either most similar to recently described Flavobacterium and Chryseobacterium spp. that have not been reported within North America, or they did not cluster with any described species. Many of these previously uncharacterized flavobacteria were recovered from systemically infected fish that showed overt signs of disease and were highly proteolytic to multiple substrates in protease assays. Polyphasic characterization, which included extensive physiological, morphological, and biochemical analyses, fatty acid profiling, and phylogenetic analyses using Bayesian and neighbor-joining methodologies, confirmed that there were at least eight clusters of isolates that belonged to the genera Chryseobacterium and Flavobacterium, which represented eight novel species. Experimental challenge studies to fulfill Koch’s postulates for 16 representative Flavobacterium and Chryseobacterium spp. isolates in five 2 economically and ecologically important fishes of the Great Lakes demonstrated that the majority of these isolates caused pathological lesions in infected fish, and the bacteria spread to vital organs (i.e., brain, spleen, liver, and kidneys), which resulted in cumulative mortalities ≥ 80%. Gross pathological changes associated with experimental infection varied by isolate and host species, but were consistent with a bacterial septicemia. Median lethal dose experiments conducted with a Chryseobacterium sp. isolate that is proposed as a novel species, C. aahli sp. nov., suggested that this bacterium was only mildly pathogenic to fish under laboratory conditions. Similar experiments conducted with a Flavobacterium sp. also proposed as a novel species, F. spartani sp. nov., indicated that this bacterium was comparatively more pathogenic. Histopathological changes associated with experimental F. spartani sp. nov. infection in its original host, the Chinook salmon (Oncorhynchus tshawytscha), included severe proliferative branchitis, lymphocytic and histiocytic myositis, multifocal necrosis within the kidney and liver, lymphocytic hepatitis, renal tubular degeneration and necrosis, and multifocal edema within the granular cell layer of the cerebellar cortex and brainstem. The findings of this study underscore the complexity of etiologies associated with flavobacteriosis and suggest that negative impacts that multiple previously undescribed and/or novel flavobacteria and chryseobacteria can have on Michigan fish stocks. 3 ACKNOWLEDGEMENTS First, I express utmost gratitude to my Major Advisor, Dr. Mohamed Faisal. I will never forget our first meeting at the Foundation and with it, the sense that he would teach me great things. During the last 7 years, I have witnessed firsthand Dr. Faisal’s guidance, friendship, research prowess, and work ethic. Through his interminable energy, endless patience, willingness to listen, and tireless nights spent investing in me, he has produced a lifelong believer in hard work, diligence, and hunger for knowledge. Indeed, when I walked through his door, I knew very little about fish health and after 7 years of learning so much from him, I realize I know even less. I am ready for what my career will bring due to his diligence, care, and hard work. Next, I thank my Guidance Committee members, Dr. Rocco Cipriano, Dr. Scott Fitzgerald, Dr. Ken Cain, and Dr. Travis Brenden, each of whom made their expertise available to me and provided a much-needed balance and perspective. Without their contributions, I would never have been able to successfully complete this work. I am also thrilled that we have forged what I anticipate will be life-long friendships. Many thanks are also due to my friends at the Michigan Department of Natural Resources for their collaborations and assistance with fish and sample collections; especially to Jim Aho, Randy Espinoza, Matt Hughes, Dan Sampson, Aaron Switzer, Jon Jackoviak, Scott Heintzelman, Ed Eische, Jan VanAmberg, Martha Wolgamood, and Gary Whelan. I am also extremely grateful to the Great Lakes Fishery Trust (20101-1147) and USDA-APHIS (10-9100-1293) for their funding of this research. I would also like to thank past and present members of the Michigan State University - Aquatic Animal Health Laboratory for their friendship, countless memories, and support. It’s amazing iv how much time we spent together, making mountains of work turn into mountains of fun through laughter and good-times, but life eventually makes us go our separate ways. Nevertheless, Wei Xu, Carolyn Schulz, Robert Kim, Andrew Winters, Elena Millard, P. Gary Egrie, Michelle Gunn, and so many others, we will forever be like family until we are old, grey, and beyond! Last, but of course not least, I thank my family. To my sisters, Lisa and Christine, and my brother, Eric Chu; thanks for always being so supportive, even when it meant not seeing me for long periods of time. To my mother and father, Norma and Thomas, words cannot express how much I love you; you are the best parents a son could ever have! To my twins, Ethan and Hailee, and our newest addition, Penelope, you are the lights of my life and give me strength when I feel weak. I love you so much and am so proud of the people you are becoming; now let’s make up on some lost camping and fishing trips! And most of all, thanks to the love of my life, my forever sunshine when skies are grey, my wife Jamie, who I have leaned on more than anyone throughout this process. v TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………………..……ix LIST OF FIGURES……………………………………………………………………………..xii GENERAL INTRODUCTION AND OVERVIEW……………………………………………...1 CHAPTER 1 REVIEW OF LITERATURE…………………………………………......................................4 I. History of Flavobacteriosis in Fish…………………………………………………..……....4 II. The Family Flavobacteriaceae…………………………..……………………………….....7 III. The Genus Flavobacterium…………………………………………………………………9 History of the genus…………………………………………………………................9 Characteristics of the genus Flavobacterium……………………………………….10 Culture and identification…………………………………………………….............10 Ecology………………………………………………………………………………….11 Flavobacterium spp. as pathogens………………………………………………….13 Flavobacteriosis in fish………………………………………………………………..13 “Other” Flavobacterium spp. associated with diseased fishes……………………14 IV. The Genus Chryseobacterium…………………………………………………………….17 History of the genus…………………………………………………………..............17 Characteristics of the genus Chryseobacterium...…………………………………17 Culture and identification…………………………………………………….............18 Ecology…………………………………………………………………………………18 Chryseobacterium spp. as pathogens…...………………………………………….20 Chryseobacterium infections in fish…..……………………………………………..21 Antimicrobial resistance in Chryseobacterium spp…………...…………………...23 V. The Challenges of Working with Members of the Family Flavobacteriaceae………...23 CHAPTER 2 DIVERSITY OF FISH-ASSOCIATED FLAVOBACTERIA OF MICHIGAN……...............26 I. Introduction…………………………………………………………………………………..26 II. Materials & Methods………………………………………………………………………...27 Fish and bacterial isolation…………………………………………………..............27 Characterization of recovered isolates………………………………………………28 16S rRNA gene amplification………………………………………………..............29 Phylogenetic analyses………………………………………………………..............29 III. Results……………………………………………………………………………...............30 IV. Discussion………………………………………………………………………………….43 V. Acknowledgements………………………………………………………………………....49 VI. Chapter 2 Appendix (A)……………………………………………………………………62 CHAPTER 3 EMERGENCE OF CHRYSEOBACTERIUM SPP. INFECTIONS IN MICHIGAN FISHES………………………………………………………………………………………..105 I. Introduction………………………………………………………………………………….105 II. Materials & Methods……………………………………………………………………….106 vi 16S rRNA gene amplification & phylogenetic analysis…………………………..106 Polyphasic characterization of 8 representative Chryseobacterium spp. isolates………………………………………………………………………..............106 Morphological, physiological, & biochemical characterization………….107 Antibiotic susceptibility testing……………………………………………..108 Fatty acid profiling…………………………………………………………...108 Phylogenetic analyses based on near complete 16S rDNA…………….108 Experimental challenge studies…………………………………………………….109 Fish……………………………………………………………………………109 Determination of growth kinetics…………………………………………..109