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Culture of the Bacterial Gill Disease Organism, Flavobacterium Branchiophilum and Strain Differences Relevant to Epizoology

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Culture of the Bacterial Gill Disease Organism, Flavobacterium Branchiophilum and Strain Differences Relevant to Epizoology Culture of the Bacterial Gill Disease Organism, Flavobacterium branchiophilum and Strain Differences Relevant to Epizoology by Iwona Skulska A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © Iwona Skulska, October 2014 ABSTRACT Culture of the Bacterial Gill Disease Organism, Flavobacterium branchiophilum and strain differences relevant to epizoology. Iwona Skulska Advisor: Dr. Roselynn M.W. Stevenson University of Guelph, 2014 The isolation, growth, and long-term storage of Flavobacterium branchiophilum is difficult. The aims of this work were to define optimal culture and storage media that would allow for growth of F. branchiophilum and determine if there were phenotypic and genotypic differences amongst isolates. Flavobacterium branchiophilum grew best on Anacker and Ordal’s medium that was supplemented with millimolar amounts of KCl, CaCl2, and MgCl2; with growth and colony visibility further improved with the addition of charcoal; 53 isolates were isolated from Ontario on this improved medium. Cultures of F. branchiophilum were viable over a period of 2 years when suspended in 5% (w/v) skim milk powder in Cytophaga Salts Broth or 10% DMSO in Cytophaga Salts Broth and stored at -75°C. By transmission electron microscopy, all isolates examined possessed surface structures resembling pili, and had evidence of membrane vesicles (MV) and tubules. The gyrB gene had a 7% difference in the 820 nucleotides sequenced, which was observed in 10 isolates from a particular case of BGD in brook trout and 4 other isolates. Differences between sequences of atpA and tuf from ONT case 6199 and 4 other isolates were also detected. Eventually, qPCR may be used to identify potential reservoirs of F. branchiophilum from the hatchery environment. Acknowledgments First, I would like to thank my advisor, Dr. Roselynn Stevenson, for all the advice, encouragement, patience, understanding, and support both prior to and during my Masters. You have helped me to grow and inspired me be the best that I can be both professionally and personally. I would also like to thank my committee members, Dr. Lucy Mutharia and Dr. Jan MacInnes, for all the advice throughout the course of my Masters. The members of the Stevenson/Mutharia/Kaushik Labs, both past and present, most notably Melinda Raymond, Steve Lord, Olivier Tremblay, and Tony Facciuolo for all their advice, support, friendship, and for making my time in the lab a fun and amazing experience. Additional thanks go to my office mates Christian Carlucci, Adam Rocker, Michelle Daigneault, and Dr. Amber Park for their advice and friendship. And last but definitely not least, thanks to my family and friends for their love and support, and for putting up with all the microbiology and fish talk. A special thanks goes to Deb Flett, Mariola Manning and Adam Kirstine, your constant encouragement helped keep me positive and gave me the confidence to get this far. I couldn’t have done it without your support! All work reported in this thesis was completed by Iwona Skulska, except for the formulation of Charcoal Salts Cytophaga Agar which was carried out by Melinda Raymond and Transmission Electoron Microscopy which was carried out by Dianne Moyle. Funding for this research was provided by the Fish Culture Section, Ontario Ministry of Natural Resources. iii Table of Contents Acknowledgments.............................................................................................................iii List of Tables.....................................................................................................................vi List of Figures..................................................................................................................vii Abbreviations..................................................................................................................viii 1. Introduction....................................................................................................................1 2. Literature Review..........................................................................................................3 2.1 Bacterial Gill Disease....................................................................................... 3 2.2 Flavobacterium branchiophilum.......................................................................4 2.3 The Genus Flavobacterium..............................................................................10 2.4 Culture of F. branchiophilum..........................................................................11 2.5 Disease Features...............................................................................................15 2.6 Strain Variance and Virulence.........................................................................17 2.7 Genetics............................................................................................................17 2.8 Environmental Reservoirs................................................................................19 2.9 Prospectus........................................................................................................20 3. Materials and Methods................................................................................................21 3.1 Media...............................................................................................................21 3.2 Bacterial Cultures and Growth.........................................................................21 3.3 Bacterial Culture Storage.................................................................................24 3.4 Isolation of F. branchiophilum from Fish........................................................25 3.4.1 Diagnosis...........................................................................................25 3.4.2 Flavobacterium branchiophilum Isolation.......................................25 3.4.3 Microscopy and Cell Measurements.................................................25 3.5 16S rRNA PCR and Sequencing......................................................................26 3.5.1 DNA Isolation and 16S rRNA PCR.................................................26 3.5.2 16S rRNA Sequencing......................................................................26 3.6 Quantitative PCR.............................................................................................28 3.6.1 Primer Design...................................................................................28 3.6.2 Panel of Isolates for qPCR................................................................31 3.6.3 Primer Specificity Testing................................................................31 3.6.4 Quantitative PCR Cycling Parameters..............................................32 3.7 Primer Design and Sequencing of gyrB, atpA, tuf, and sprB amplicons.........32 3.8 Tissue Culture..................................................................................................34 iv Table of Contents 4. Results...........................................................................................................................35 4.1 Isolation and Culture........................................................................................35 4.2 Cell Morphology..............................................................................................35 4.3 Culture Media Comparisons............................................................................36 4.4 Long-term Culture Storage..............................................................................37 4.5 Genetic diversity of Isolates.............................................................................38 4.6 qPCR detection of F. branchiophilum.............................................................42 4.7 Cell surface structure and Attachment.............................................................42 5. Discussion.....................................................................................................................65 References.........................................................................................................................78 Appendix A.......................................................................................................................84 Appendix B.......................................................................................................................87 Appendix C.......................................................................................................................92 Appendix D.......................................................................................................................96 Appendix E.......................................................................................................................97 v List of Tables Table 1. F. branchiophilum strains isolated from BGD infected fish gills reported in the literature.......................................................................6 Table 2. Formulations reported to support growth of Flavobacterium branchiophilum.................................................................8 Table 3. Formulations of media used for the culture of Flavobacterium species.............................................................................22 Table 4. Isolates from the FHL culture collection used in this study................................................................................................23
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