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Bacteriological and Toxigenic Studies on Vibrio Species Isolated from The Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1983 Bacteriological and Toxigenic Studies on Vibrio Species Isolated From the Blue Crab (Callinectes Sapidus) and the Purification of Toxic Factors Produced by Vibrio Fluvialis (Louisiana). Susan Lynne Payne Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Payne, Susan Lynne, "Bacteriological and Toxigenic Studies on Vibrio Species Isolated From the Blue Crab (Callinectes Sapidus) and the Purification of Toxic Factors Produced by Vibrio Fluvialis (Louisiana)." (1983). LSU Historical Dissertations and Theses. 3859. https://digitalcommons.lsu.edu/gradschool_disstheses/3859 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 This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted. The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction. 1.The sign or “target” for pages apparently lacking from the document photographed is “Missing Page(s)”. If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity. 2. 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These prints are available upon request from the Dissertations Customer Services Department. 5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed. Uni International 300 N. Zeeb Road Ann Arbor, MI4B106 8318022 Payne, Susan Lynne BACTERIOLOGICAL AND TOXIGENIC STUDIES ON VIBRIO SPECIES ISOLATED FROM THE BLUE CRAB (CALLINECTES SAPIDUS) AND THE PURIFICATION OF TOXIC FACTORS PRODUCED BY VIBRIO FLUVIALIS The Louisiana Slate University and Agricultural and Mechanical ColPH.D. 1983 University Microfilms International 300 N. Zeeb Road, Ann Arbor, M I 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages ^ 2. Colored illustrations, paper or print____ 3. Photographs with dark background / 4. Illustrations are poor copy______ 5. Pages with black marks, not original copy______ 6. Print shows through as there is text on both sides of page______ 7. Indistinct, broken or small print on several pages______ 8. Print exceeds margin requirements______ 9. Tightly bound copy with print lost in spine_____ 10. Computer printout pages with indistinct print______ 1 1 . Page(s)________lacking when material received, and not available from school or author. 12. Page(s)____________seem to be missing in numbering only as text follows. 13. Two pages numbered___________. Text follows. 14. Curling and wrinkled pages______ 15. Other_____________________________________________________ University Microfilms International BACTERIOLOGICAL AND TOXIGENIC STUDIES ON VIBRIO SPECIES ISOLATED FROM THE BLUE CRAB (CALLINECTES SAPIDUS) AND THE PURIFICATION OF TOXIC FACTORS PRODUCED BY VIBRIO FLUVIALIS A dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by Susan L. Payne B. S., Southeastern Massachusetts University, 1978 May, 1983 DEDICATION This dissertation is dedicated to my husband, Ross, who has suffered through the many hours of work which is represented here and who supplied loving support throughout this study. ii AKNOWLEDGEMENT I would like to express thanks and sincere appreciation to Dr. R. J. Siebeling for the encouragement, support, and patience that he has given throughout this research and during the preparation of this dissertation. This work would not have been completed without his help and constant reassurance. I would also like to express sincere thanks to Drs. H. D. Braymer, V. R. Srinivasan, A. D. Larson, and S. H. Chang who freely gave their help and advice whenever asked. Special thanks to Dr. E. W. Blakeney whose door was always open and whose daily help was instrumental for the completion of the final phase of this research. I would also like to thank Dr. R. Montelaro for his help and advice, Melanie West for help with gel electrophoresis experiments, and Janet Evans for her expert help in the preparation of this dissertation. iii TABLE OF CONTENTS Page DEDICATION..................................................... 11 ACKNOWLEDGEMENT ............................................... ill TABLE OF C O N T E N T S ............................................. iv LIST OF F I G U R E S ............................................... v LIST OF TABLES................................................. vii ABSTRACT..........................................................viil PART I Bacteriological assay of blue crab hemolymph and feces for Vibrio c h o l e r a e ............................ 1 Introduction .................................... 2 Materials and Methods ................................ 9 R e s u l t s ............................................. 25 Discussion............................................ 43 PART II Assay of environmental Vibrio cholerae isolates for the production of cholera toxin using a ganglioside- ELISA t e c h n i q u e ...................................... 48 Introduction ......................................... 49 Materials and Methods ................................ 55 Results and Discussion ................................ 59 PART III Purification of a permeability factor from culture supernatant fluids from Vibrio fluvialis DJVP 6957 . 65 Introduction .......................................... 66 Materials and Methods ................................ 69 Results ....................... 80 Discussion............................................ 119 LITERATURE CITED ................................................ 123 VITA ........................................................... 130 iv LIST OF FIGURES Figure Page 1. Methods for the collection of hemolymph and fecal samples.................................................. 14 2. Method developed for the identification of Vibrio cholerae isolates ....................................... 20 3. Incidence of Vibrio cholerae versus halophilic Vibrio species in crab hemolymph ........................ 27 4. Incidence of Vibrio cholerae versus halophilic Vibrio species in crab f e c e s ......... 31 5. Incidence of Vibrio species in crab hemolymph and f e c e s....................................... 33 6. Sensitivity of the ganglioside-ELISA for cholera to x i n ....................... ........................... 62 7. Scheme for the purification of IF/PF activity from Vibrio fluvialis supernatant fluids ...................... 73 8. Gel filtration of crude concentrated culture filtrates from Vibrio fluvialis DJVP 6957 on Sephadex G-100 85 9. DEAE-Cellulose ion exchange chromatography of Vibrio fluvialis DJVP 6957 shaking culture supernatant f l u i d s ........................... 88 10. Gel filtration chromatography of DEAE IF activity peak II on Sephadex G - 1 0 0 ................................ 90 11. SDS-PAGE analysis of IF activity separated by DEAE- Cellulose and Sephadex G-100 chromatography .............. 92 12. Rechromatography of pooled DEAE IF activity peaks from Vibrio fluvialis DJVP 6957 fermentor culture supernatant fluids on DEAE-Cellulose .................... 95 13. SDS-PAGE analysis of IF activity from fermentor culture supernatant fluids recovered by DEAE- cellulose ion exchange chromatography .................... 97 14. Gel filtration chromatography of IF activity recovered from DEAE-Cellulose on Sephadex G-75 99 v Figure Page 15. Gel filtration chromatography of IF activity recovered from Sephadex G-75 on Sepharose 6 B .......... 101 16. SDS-PAGE analysis of IF activity peaks I and II recovered from Sepharose 6B showing three bands
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