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University of Florida Thesis Or Dissertation Formatting NONTUBERCULOUS MYCOBACTERIA IN FLORIDA By STEPHANIE CASSANDRA YARNELL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Stephanie C. Yarnell 2 To my parents, Howard and Donna Yarnell 3 ACKNOWLEDGMENTS I would like to thank the members of the Emerging Pathogens Institute for their help- Youliang Qiu, Jason Blackburn, Kevin Fennelly, Michael Lauzardo, Judith Johnson, Glenn Morris, and Andrew Kane, as well as, the present and past members of the Marine Animal Disease Laboratory for their help - Linda Archer, Jim Wellehan, and Hendrik Nollens. This work could not have been done without them. Stephen Hsu and Skip Harris deserve additional thanks for the immense moral support. Funding for this project was provided, in part, by an Opportunity Fund Award (Project Number 00091337) from the University of Florida, Office of Research, the University of Florida Emerging Pathogens Institute, the College of Public Health and Health Professions, and the MD/PhD Program. Further technical support was provided by Steve McNulty, Barbara Elliott, Linda Bridge, Ravikiran, and Richard Wallace of the University of Texas, Tyler, Texas. I thank my committee for their mentorship- my chair, Judith Johnson, and Glenn Morris, Andrew Kane, Michael Lauzardo, as well as the IDP representative, Richard Condit. My parents, Howard and Donna, deserve special thanks for being incredibly supportive as I continue down my seemingly endless educational path. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 General Background ............................................................................................... 15 Cell Structure .......................................................................................................... 16 Medical ................................................................................................................... 19 Respiratory Infections ....................................................................................... 21 Hypersensitivity Pneumonitis ............................................................................ 25 Cystic Fibrosis (CF) .......................................................................................... 26 Disseminated Disease ...................................................................................... 28 Immunology ...................................................................................................... 31 Soft tissue/ Cutaneous Manifestations ............................................................. 33 Buruli Ulcer ....................................................................................................... 34 Traumatic Wound/ Surgery ............................................................................... 36 Chronic Bowel Disease .................................................................................... 38 Cervical Lymphadenitis .................................................................................... 42 Wildlife and Veterinary Diseases ...................................................................... 43 Intracellular Growth .......................................................................................... 45 Environmental Distribution ...................................................................................... 46 Water ...................................................................................................................... 48 Water Quality .......................................................................................................... 50 DOC ................................................................................................................. 51 Salt Tolerance .................................................................................................. 51 Dissolved Oxygen ............................................................................................ 52 Temperature ..................................................................................................... 53 pH ..................................................................................................................... 54 Drinking Water ........................................................................................................ 55 Geographical Mapping ............................................................................................ 65 Final Thoughts ........................................................................................................ 67 5 2 NONTUBERCULOUS MYCOBACTERIA DETECTED USING NOVEL QPCR PROBES ................................................................................................................. 69 Background ............................................................................................................. 69 Materials and Methods ............................................................................................ 71 Primer Development ......................................................................................... 71 Serial Dilutions ................................................................................................. 73 Extraction by Physical Disruption ..................................................................... 74 Extraction by Chemical Disruption .................................................................... 75 Comparison of qPCR and Traditional PCR ...................................................... 75 Results .................................................................................................................... 75 Primer Sensitivity and Specificity ...................................................................... 75 Chemical versus Physical Disruption ............................................................... 76 Culture Confirmation ........................................................................................ 76 Comparison of qPCR and Traditional PCR ...................................................... 77 Discussion .............................................................................................................. 77 3 NONTUBERCULOUS MYCOBACTERIA IN FLORIDA SURFACE AND MUNICIPAL WATERS ............................................................................................ 86 Background ............................................................................................................. 86 Materials and Methods ............................................................................................ 87 Sampling for Mycobacterial DNA ...................................................................... 88 DNA Extraction ................................................................................................. 88 Quantitative PCR (q-PCR) ................................................................................ 88 Conformation of Near-Threshold qPCR Data ................................................... 89 Culture Confirmation ........................................................................................ 90 Water Chemistry ............................................................................................... 91 Statistics ........................................................................................................... 91 Results .................................................................................................................... 91 Quantification ................................................................................................... 91 PCR and Sequencing ....................................................................................... 92 Culture Confirmation ........................................................................................ 93 Water Quality .................................................................................................... 93 Discussion .............................................................................................................. 94 4 DEMOGRAPHICS OF NONTUBERCULOUS MYCOBACTERIA CASES IN FLORIDA, 2006-2008 ........................................................................................... 104 Background ........................................................................................................... 104 Materials and Methods .......................................................................................... 106 Data Source ................................................................................................... 106 Data Analysis ................................................................................................. 107 Multivariate Analysis ......................................................................................
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