Western Michigan University ScholarWorks at WMU Dissertations Graduate College 4-2000 Identification and Classification of Coryneform Bacteria Isolated from Bovine Mammary Glands Jeffrey L. Watts Western Michigan University Follow this and additional works at: https://scholarworks.wmich.edu/dissertations Part of the Biology Commons, and the Dairy Science Commons Recommended Citation Watts, Jeffrey L., "Identification and Classification of Coryneform Bacteria Isolated from Bovine Mammary Glands" (2000). Dissertations. 1490. https://scholarworks.wmich.edu/dissertations/1490 This Dissertation-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Dissertations by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. IDENTIFICATION AND CLASSIFICATION OF CORYNEFORM BACTERIA ISOLATED FROM BOVINE MAMMARY GLANDS by Jeffrey L. Watts A Dissertation Submitted to the Faculty of The Graduate College in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Department of Biological Sciences Western Michigan University Kalamazoo, Michigan April 2000 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IDENTIFICATION AND CLASSIFICATION OF CORYNEFORM BACTERIA ISOLATED FROM BOVINE MAMMARY GLANDS Jeffrey L. Watts, Ph. D. Western Michigan University, 2000 Coryneform bacteria are frequently isolated from bovine mastitis and these infections are associated with economic losses. Corynehacterium bovis, a lipid- requiring species, has been the most frequently isolated coryneform from the milk of infected bovine mammary glands. However, the taxonomic status of this organism is uncertain. In the current study, a polyphasic approach was used to identify coryneform bacteria isolated from bovine mastitis and determine the phylogenetic relationships among the identified species. A total of 212 coryneform bacteria isolated from bovine mastitis was obtained from mastitis reference laboratories in the United States and Canada. Presumptive identification based upon Gram-stain, oxidase, catalase, and Tween 80 stimulated growth classified 183 isolates as Corynehacterium species. Eighty-seven strains were selected for species level identification by 16S rRNA gene sequencing, the Biolog system and the API Coryne system. Fifty strains were identified as Corynebacterium bovis by 16S rRNA gene similarity studies: the Biolog and API Coryne systems identified 54.0 and 88.0% of these strains, respectively. Antimicrobial susceptibility testing of 46 C. bovis and 14 C. amylocolatum strains determined these organisms were susceptible to ampicillin, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. oxacillin, cephalothin, ceftiofur, a combination of penicillin and novobiocin, erythromycin, clindamycin, pirlimycin, tetracycline, florfenicol, enrofloxacin, sarafloxacin, danofloxacin, and premafloxacin but not tilmicosin. Finally, phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and phylogenetic analyses performed. All strains identified as C. bovis exam ined clustered with the reference strains indicating that C. bovis is a well defined species within the genus Corynehacterium. Rep-PCR of these strains indicated that only minor genetic variation exists within strains of C.bovis. Corynehacterium bovis ATCC 13722 was determined to be most closely related to Brevibacterium helvolum. Based on phylogenetic analyses, this organism was placed in the genus Brevibacterium as Brevibacterium neaveae sp. nov. Results of this study confirm that the coryneforms isolated from bovine mammary glands are a heterogeneous group of organisms. Furthermore, direct sequencing of the 16S rRNA gene appears to be the most accurate method for identification ofCorynehacterium species isolated from bovine mastitis. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INFORMATION TO USERS This manuscript has been reproduced 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. 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Bell & Howell Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 9963070 Copyright 2000 by Watts, Jeffrey Lynn All rights reserved. UMI UMI Microform9963070 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Copyright by Jeffrey L. Watts 2000 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS The ability of a person to successfully complete a doctoral program at a later stage in their career depends greatly on support from a number of persons. First. I would like to thank and am greatly appreciative of the time and effort given to me by my advisor. Dr. Silvia Rossbach. Through her willingness to mentor a student working in an area different from her own. she has demonstrated the professional agility to address a broad range of research areas. I would also like to thank the members of my committee, Dr. Lenard Ginsberg, Dr. Robert Eisenberg, and Dr. Chuck Ford. In particular, Dr. Ford has served as a source of encouragement and support during my program. I would also like to thank Pharmacia & Upjohn Animal Health for supporting my program. I would also like to thank my many colleagues at P&U for their support over the years. In particular, I want to thank Dr. David Lowery and Ms. Janet Teel for their help in training me in the molecular techniques needed for this work. I also want to thank Ms. Cathy Ditto for performing the Rep-PCR experiments in the study. I also want to thank Dr. Robert J. Yancey, Jr. for encouraging me to complete my doctorate and Dr. Richard C. Wardley for providing the time and resources to complete this program. Last and most important, I would like to thank my family. In particular, I would like to thank my son, Eric, and daughter, Danielle, for sacrificing their time to ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgments - Continued allow me to complete this work. This is truly time that cannot be returned. To my wife, Vickie, I offer my greatest thanks. Her sacrifice, dedication, and understanding through this program have been my strength. At a time in my life that I had resigned myself to never completing a doctorate, she kept the dream alive. Jeffrey L. Watts iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ACKNOWLEDGMENTS............................................................................................................ii LIST OF TABLES ....................................................................................................................... vi LIST OF FIGURES.....................................................................................................................vii CHAPTER I. INTRODUCTION ............................................................................................................ I Bovine Mastitis and the Corynebacteria .......................................................... 1 Overview of Bovine M astitis................................................................... 1 Mastitis Pathogens.......................................................................................2 The Role of Corynebacteria in Bovine Mastitis..................................5 Classification of the Corynebacteria.................................................................8 Historical Perspective.................................................................................8 Taxonom y of C.bovis .........................................................................................14 Study