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Lipopolysaccharide Serotyping of Vibrio Vulnificus. Stephen Jeffrey Martin Louisiana State University and Agricultural & Mechanical College

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Lipopolysaccharide Serotyping of Vibrio Vulnificus. Stephen Jeffrey Martin Louisiana State University and Agricultural & Mechanical College Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1989 Lipopolysaccharide Serotyping of Vibrio Vulnificus. Stephen Jeffrey Martin Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Martin, Stephen Jeffrey, "Lipopolysaccharide Serotyping of Vibrio Vulnificus." (1989). LSU Historical Dissertations and Theses. 4731. https://digitalcommons.lsu.edu/gradschool_disstheses/4731 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS The most advanced technology has been used to photo­ graph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. 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University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9002158 Lipopolysaccharide serotypingVibrio of vulnificus Martin, Stephen Jeffrey, Ph.D. The Louisiana State University and Agricultural and Mechanical Col., 1989 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 LIPOPOLYSACCHARIDE SEROTYPING OF VIBRIO VULNIFICUS A dissertation Submitted to the Graduate Faculty of the Louisiana State Univesity and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by Stephen Jeffrey Martin A.A., Grand View College, 1977 B.A., Drake University, 1979 M.A., Drake University, 1986 May, 1989 DEDICATION To my parents, whose hard work and constant support have given me the opportunity to follow my dreams. ii ACKNOWLEDGEMENT A sincere thank you is extended to my major professor, Dr. R.J. Siebeling, the enthusiasm and patience shown during the course of this research and the preparation of this dissertation were much appreciated. His approach to teaching and his sense of humor will be long remembered. I would like to thank Drs. J.M. Jaynes, V.R. Srinivasan, L.T. Hart, E.C. Achberger, A. Hammond, and K.L. Schnorr for serving on my committee. I would also like to thank Dr. K.L. Schnorr for his instruction and expertise in cell culture and for the use of his tissue culture laboratory at the beginning of this work. This research was supported in part by the Louisiana Sea Grant College Program, a part of the National Oceanic and Atmospheric Administration, U.S. Department of Commerce (grant NA 85 AA-D-56141). The Louisiana Program is administered by the Center for Wetland Resources, Louisiana State University, Baton Rouge, Louisiana. Table of Contents Page Dedication................................................... ii Acknowledgement............................................ iii Table of Contents........................................... iv List of Figures............................................. vi List of Tables............................................. vii Abstract..................................................... ix Introduction.................................................. l Chapter I Isolates of Vibrio vulnificus Used in the Development of LPS Serotyping Scheme....... 16 Abstract.....................................17 Introduction................................ 18 Materials and Methods...................... 20 Results and Discussion..................... 27 Chapter II Production of Vibrio vulnificus Serovar- Specific Polyclonal Antisera...............44 Abstract.....................................45 Introduction................................ 46 Materials and Methods...................... 48 Results and Discussion..................... 53 Chapter III Serotyping of Vibrio vulnificus Using Lipopolysaccharide Specific Monoclonal Antibodies.................................. 56 Abstract.....................................57 Introduction................................ 58 Materials and Methods...................... 60 Results......................................66 Discussion.............. 76 Literature Cited............................................ 78 Vita......................................................... 90 v LIST OF FIGURES Figure I. 1. Tranlucent and opaque phenotypes of V. vulnificus strain VvlOOSH on alkaline peptone agar, pH 8.0................... LIST OF TABLES Table Introduction 1. Vibrio species isolated from clinical specimens and their most common associated diseases............................................. 3 Chapter I. 2. Louisiana Vibrio vulnificus isolates, patient information, and strains designations............ 21 3. Biochemical characteristics used to screen Vibrio vulnificus isolates.........................28 4. Hemolytic patterns for representative Vibrio vulnificus human isolates observed on rabbit blood agar (rRBC) and mouse blood agar (mRBC) plates...............................................30 5. Virulence of selected environmental and human V. vulnificus isolates for Balb/c mice...............32 6. Time course mortality study of seven human V. vulnificus isolates................................ 33 7. Frequency of switching from opaque to translucent and translucent to opaque phenotypes of Vibrio vulnificus.......................................... 37 8. The sensitivity of Vibrio vulnificus isolates to the bactericidal effects of apo-lactoferrin...... 42 Chapter II 1. O-tube agglutination and PHA titers produced in rabbits immunized with whole cell vaccines and PS- protein carrier conjugates prepared from V. vulnificus 1005.................................. 54 Chapter III 1. Agglutination patterns for 5 anti-V. vulnificus sera raised in rabbits immunized with formalinized whole cell vaccines and tested against vaccine strains............................ 67 2. Slide agglutination patterns for five affinity purified anti-V. vulnificus-PS sera............... 68 3. Serological specificity of monoclonal antibodies versus polyclonal antisera for LPS (0-serovar specific) antigens..................... 70 vii Table Chapter III 4. Hybridomas producing V. vulnificus lipopolysaccharide specific monoclonal antibody............... 72 5. Proposed serological varieties of V. vulnificus based on lipopolysaccharide specific monoclonal antibodies........................................ 73 6. ELISA titers for opaque and translucent V. vulnificus whole cells and LPS extracted from opaque and translucent V. vulnificus cells vs. polyclonal and monoclonal antibodies........................................ 75 viii Abstract Current schemes used to serotype Vibrio vulnificus are based on direct bacterial agglutination using polyclonal rabbit antisera. During the course of serotyping Louisiana V. vulnificus isolates we failed to observe a reproducible agglutination profile for these isolates. The major obstacle in producing serovar specific sera has been the failure to produce high titered anti-LPS sera in rabbits. Anti-LPS serum from rabbits immunized with formalin-killed, heat-killed, or acetone dried whole cell vaccines and LPS- carrier conjugates uniformly produced titers < 320. Separation of anti-LPS activity from whole sera by affinity chromatography also yielded low titered antisera. A typing system predicated on the detection of LPS-associated O antigens using monoclonal antibodies (Mab) is proposed for V. vulnificus. Hybridomas have been established which produce antibodies that react with LPS purified from V. vulnificus. V. damsela. and V. hollisae. Vibrio vulnificus shows considerable heterogeneity within the genus. Twenty- four Mabs produced to date, react with 10 of 16 (61%) of V. vulnificus strains tested by LPS ELISA. Monoclonal antibodies react more strongly with the translucent than the opaque phenotype o'f V. vulnificus using whole cell ELISA. Monoclonal antibodies reactive by LPS ELISA with V. damsela and V. hollisae LPS are non-reactive with LPS prepared from V. vulnificus. Introduction In recent years there has been a dramatic increase
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