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PHD Dissertaiton by Zhen Tao.Pdf (3.618Mb) Vibrios associated with marine samples from the Northern Gulf of Mexico: implications for human health by Zhen Tao A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 3, 2013 Keywords: recreational fishing, Vibrio vulnificus, public health Copyright 2013 by Zhen Tao Approved by Covadonga R. Arias, Chair, Full Professor of Fisheries and Allied Aquacultures Thomas A. McCaskey, Professor of Animal Science Calvin M. Johnson, Professor of Pathology Stephen A. Bullard, Assistant Professor of Fisheries and Allied Aquacultures Abstract In this dissertation, I investigated the distribution and prevalence of two human- pathogenic Vibrio species (V. vulnificus and V. parahaemolyticus) in non-shellfish samples including fish, bait shrimp, water, sand and crude oil material released by the Deepwater Horizon oil spill along the Northern Gulf of Mexico (GoM) coast. In my study, the Vibrio counts were enumerated in samples by using the most probable number procedure or by direct plate counting. In general, V. vulnificus isolates recovered from different samples were genotyped based on the polymorphism present in 16S rRNA or the vcg (virulence correlated gene) locus. Amplified fragment length polymorphism (AFLP) was used to resolve the genetic diversity within V. vulnificus population isolated from fish. PCR analysis was used to screen for virulence factor genes (trh and tdh) in V. parahaemolyticus isolates yielded from bait shrimp. A series of laboratory microcosm experiments and an allele-specific quantitative PCR (ASqPCR) technique were designed and utilized to reveal the relationship between two V. vulnificus 16S rRNA types and environmental factors (temperature and salinity). In summary, research data showed that the human pathogen V. vulnificus is commonly found in non-shellfish samples (fish, bait shrimp and tar ball) of the Northern GoM coast. Moreover, I discovered a higher percentage of strains of great virulence potential in fish and shrimp than those previously reported in oysters. I proved that 16S type B strains outcompete type A strains at warmer temperatures explaining why more ii cases of vibriosis due to this pathogen occur at the end of summer. Finally, the effects of the Deepwater Horizon oil spill significantly increased the presence of V. vulnificus in beach samples. Overall, my research shows that recreational activities conducted in the Northern GoM coast have an intrinsic risk of exposure to V. vulnificus. ii Acknowledgments First, I would like to express the deepest appreciation to my committee chair Professor Dr. Cova R. Arias. Without her guidance and persistent help, this dissertation would not have been possible. I would also like to thank my committee members, Dr. Thomas A. McCaskey, Dr. Calvin M. Johnson, and Dr. Stephen A. Bullard for their time and expertise. I thank all my lab mates I worked with throughout my time here: Ryan Wood, Matt Lewis, Óscar Olivares-Fuster, Stacey LaFrentz, Andrea Larsen, Wenlong Cai, Haitham Hussien Mohammed and Jeff Holder. My research would not have been possible without their helps. I would also like thank the administration staff and colleagues in the Department of Fisheries and Allied Aquacultures for the help over the years. I would like to express my gratitude to the Chinese Scholarship Council for their financial support through my four-year PhD study. Finally, I would like thank to my parents and elder brother. They were always supporting me and encouraging me with their best wishes. iii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv List of Tables .................................................................................................................... vii List of Figures .................................................................................................................... ix List of Abbreviations ......................................................................................................... xi Chapter 1. Literature review ..............................................................................................1 Chapter 2. Objectives .......................................................................................................36 Chapter 3. Prevalence and population structure of Vibrio vulnificus on fishes from the northern Gulf of Mexico .................................................................................38 Abstract ................................................................................................................38 Introduction ..........................................................................................................39 Material and methods ...........................................................................................41 Results ..................................................................................................................46 Discussion ............................................................................................................50 References .............................................................................................................56 Chapter 4. Occurrence of Vibrio vulnificus and V. parahaemolyticus in bait shrimp from Alabama and Mississippi ..............................................................................73 Abstract ................................................................................................................73 Introduction ..........................................................................................................73 iv Material and methods ...........................................................................................74 Results and discussion ..........................................................................................77 References .............................................................................................................81 Chapter 5. High numbers of Vibrio vulnificus in tar balls collected from oiled areas of the north-central Gulf of Mexico following the 2010 BP Deepwater Horizon Oil Spill ....................................................................................................90 Abstract ................................................................................................................90 Introduction ..........................................................................................................91 Material and methods ...........................................................................................92 Results and discussion ..........................................................................................94 References .............................................................................................................96 Chapter 6. Effect of temperature and salinity on the dynamics of Vibrio vulnificus 16S rRNA genotypes revealed by allele-specific quantitative PCR ....................101 Abstract ..............................................................................................................101 Introduction ........................................................................................................102 Material and methods .........................................................................................104 Results ..................................................................................................................112 Discussion ...........................................................................................................114 References ...........................................................................................................119 Chapter 7. Summaries and conclusion .............................................................................136 Appendix 1. Diversity of culturable bacteria from blood vascular system of lesser electric rays (Narcine bancroftii) captured in the northern Gulf of Mexico off Alabama .......................................................................................................139 v List of Tables Chapter 3 Table 3-1 ................................................................................................................61 Table 3-2 ...............................................................................................................62 Table 3-3 ...............................................................................................................63 Table 3-4 ...............................................................................................................65 Chapter 4 Table 4-1 ...............................................................................................................85 Table 4-2 ................................................................................................................87 Table 4-3 ................................................................................................................88 Table 4-4 ................................................................................................................89 Chapter 5 Table 5-1 ...............................................................................................................98
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