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Characterization of the Fish Pathogen Flavobacterium Psychrophilum Towards Diagnostic and Vaccine Development

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Characterization of the Fish Pathogen Flavobacterium Psychrophilum Towards Diagnostic and Vaccine Development Characterization of the Fish Pathogen Flavobacterium psychrophilum towards Diagnostic and Vaccine Development Elizabeth Mary Crump B. Sc., University of St. Andrews, Scotland, 1995 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in the Department of Biochemistry and Microbiology O Elizabeth Mary Crump, 2003 University of Victoria All rights reserved. This dissertation may not be reproduced in whole or in part, by photocopying or other means, without the permission of the author. Supervisor: Dr. W.W. Kay Abstract Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrophile, Flavobacterium psychrophilum, is the etiological agent of rainbow trout fry syndrome (RTFS) and bacterial cold water disease (BCWD), septicaemic diseases which heavily impact salmonids. These diseases have been controlled with limited success by chemotherapy, as no vaccine is commercially available. A comprehensive study of F. psychrophilum was carried out with respect to growth, speciation and antigen characterization, culminating in successful recombinant vaccines trials in rainbow trout fry. Two verified but geographically diverse isolates were characterized phenotypically and biochemically. A growth medium was developed which improved the growth of F. psychrophilum, enabling large scale fermentation. A PCR-based typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates. In collaborative work, LPS O- antigen was purified and used to generate specific polyclonal rabbit antisera against F. psychrophilum. This antiserum was used to develop diagnostic ELISA and latex bead agglutination tests for F. psychrophilum. F. psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low MW carbohydrate antigen, as well as several protein antigens. Surface exposed antigens were revealed by a combination of irnmuoflourescence microscopy, immunogold transmission and thin section EM They were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTAIpolymyxin B agarose beads as well as by extrinsic labeling of amines with sulpho-NHS-biotin and glycosyl groups with biotin hydrazide. The predominant -16 KDa antigen was identified as low MW LPS, whereas high MW LPS containing 0-antigen was not as prevalent on whole cells but was abundant in culture supernatants. Genomic DNA was isolated from F. psychrophilum and used to construct an expression library in lambda ZAP 11. The library was screened with rabbit anti-F. psychrophilum serum. The respective DNA inserts in the immunoreactive clones were sequenced providing 15 kb of novel DNA sequence encoding 13 hypothetical proteins. Two open reading frames encoding a 91 amino acid HU-beta-like protein (FP91), and a 166 amino acid ribosomal L10-like protein (FP 166), were cloned and expressed as fusion proteins in E. coli. Rainbow trout convalescent antisera strongly recognized both MW classes of LPS as well as a predominant -20 kDa protein. The 20 kDa antigen was separated by 2D gel electrophoresis, isolated and subject to proteolysis. Peptide fragments were analysed by quadrupole time-of-flight mass spectrometry. Fragmented peptide spectra were generated and peptide sequences obtained. Degenerate PCR was used to amplify a 537 bases corresponding to179 amino acids; the PCR product was cloned and expressed as a fusion protein in E. coli. The recombinant proteins were tested in rainbow trout fjr for their ability to confer protective immunity against F. psychrophilum. All proteins were shown to have some protective effect. In an attempt to boost immunity, a T cell epitope from measles virus was incorporated into the recombinant vaccines. The presence of the T cell epitope affected the protection of each protein differently, nevertheless, successful recombinant vaccine candidates were developed. Table of Contents Abstract .................................................................................................................ii Table of Contents ...................................................................................................iv List of Tables ......................................................................................................... xi List of Figures ....................................................................................................... xii .. List of Figures ....................................................................................................... XII List of Abbreviations .......................................................................................... xiv .. Acknowledgements............................................................................................. XVII Dedication .......................................................................................................... xix General Introduction .............................................................................................. 1 Taxonomy ...............................................................................................................................2 Aquaculture and Disease ........................................................................................................ 7 Diseases caused by F . psychrophilum .................................................................................. 10 Vaccinology of Fish .............................................................................................................. 14 The Immune System of Teleost Fish .................................................................................... 18 Materials and Methods .........................................................................................28 Bacterial strains. growth and characterization................................................................ 28 Growth conditions .......................................................................................................... 29 Carbohydrate metabolism ............................................................................................... 30 Large scale growth.......................................................................................................... 30 Biochemical and physiological characterization of isolates ........................................... 31 Protein analysis ...................................................................................................................32 SDS PAGE ...................................................................................................................... 32 Gel staining .....................................................................................................................32 Proteinase K treatment ....................................................................................................32 Acetone precipitation ......................................................................................................32 [14c] Palmitate labeling of F. psychrophilum lipoproteins .............................................33 Biotinylation of cell-surface proteins ............................................................................. 33 Triton X-114 phase partitioning of F. psychrophilum.................................................... 34 Two dimensional gel electrophoresis and mass spectrometry ........................................35 Staining of 2D gels with Sypro Ruby .............................................................................36 Staining of 2D gels with Coomassie Brilliant Blue G-250 .............................................37 Reduction, alkylation and tryptic digests of 2D gel spots ..............................................37 MALDI-TOF mass spectrometry.................................................................................... 38 Nanospray tandem mass spectrometry (MSIMS) and peptide sequencing..................... 39 Virtual mass mapping ..................................................................................................... 39 Electroblotting of 2D gels onto PVDF membrane for N-terminal sequencing ......................................................................................................................40 Protein expression in E. coli (of pETC clones) ............................................................... 40 Isolation and quantification of inclusion bodies ............................................................. 40 Quantification of expressed protein concentration ........................................................ 41 DNA analysis ....................................................................................................................... 41 Plasmids used in this study ............................................................................................ -41 Purification of chromosomal DNA from F. psychrophilum ........................................... 42 Routine isolation of F . psychrophilum chromosomal DNA for PCR ............................. 43 Isolation of plasmid DNA ..............................................................................................-43 Restriction enzyme digests .............................................................................................44 Agarose gel electrophoresis ...........................................................................................
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