Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2010 In vivo and in vitro pathogenesis of Francisella asiatica in tilapia nilotica (Oreochromis niloticus) Esteban Soto Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation Soto, Esteban, "In vivo and in vitro pathogenesis of Francisella asiatica in tilapia nilotica (Oreochromis niloticus)" (2010). LSU Doctoral Dissertations. 2796. https://digitalcommons.lsu.edu/gradschool_dissertations/2796 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 Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. IN VIVO AND IN VITRO PATHOGENESIS OF FRANCISELLA ASIATICA IN TILAPIA NILOTICA (OREOCHROMIS NILOTICUS) 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 Interdepartmental Program in Veterinary Medical Sciences through the Department of Pathobiological Sciences by Esteban Soto Med.Vet., Universidad Nacional-Costa Rica, 2005 M.Sc., Mississippi State University, 2007 August, 2010 ACKNOWLEDGEMENTS The main reason why I’m being able to present this dissertation is because of all the help and advices received by many people along these years. Firstly and foremost I thank my wife Tati for always believing in me and giving me all the support I needed. To my dad and mom, thanks for being a perfect example of integrity and perseverance. It is very easy to have personal heroes having both of you as parents. My brothers Quique and Teto, thanks for always having my back, and always giving me a great excuse to go back to Costa Rica. I’m also greatly indebted to my advisor Dr. John P. Hawke, who sacrificed time, energy, and effort to help me realize my educational goal. Your support, friendship and guidance both as advisor and as person will always be remembered and I will always be in debt for all the help provided. I would like to express all my appreciation to committee members Dr. Ron Thune, Dr. Kevin Macaluso, Dr. Philip Elzer, and Dr. Donald Baltz who provided assistance and advice during the research of these projects. I also greatly thank all the help, friendship, and technical assistance of Judy Wiles, Denise Fernandez, Dr. Matt Rogge, Dr. Wes Baumgartner, Judith Beekman, Christie Landry, Johnny Shelly, Natthida Petchampai, Dr. Piyanate Sunyakumthorn, Dr. Walairat Pornwiroon, Dr. Chutima Thepparit, and Michael Kearney from the Pathobiological Sciences Department, LSU School of Veterinary Medicine. I also gratefully thank Dr. Juan A. Morales from the Escuela de Medicina Veterinaria in the Universidad Nacional of Costa Rica for providing a new perspective on veterinary science, and help me get started in the fish health field. I thank, Dr. Jagit Ludu and Dr. Francis Nano at the Department of Biochemistry and Microbiology of the University of Victoria, B.C.; Dr. Xhavit Zogaj and Dr. Karl E. Klose from the South Texas Center for Emerging Infectious Diseases and Department of Biology, University of Texas San Antonio, San Antonio, TX for sharing some of the plasmids used in this study. ii I also thank Dr. Anne-Berit Olsen from the National Veterinary Institute, Bergen, Norway and Dr. John Hansen from the Interdisciplinary Program in Pathobiology, University of Washington, Seattle, WA, for sharing some of the strains used in the study. I also thank Dr. B. Arulanandam and Dr. J.-J. Yu, from the Department of Biology, University of Texas at San Antonio for sharing some of the DNA used in this study. I also appreciate all the help provided by Dr. Matt Griffin and Dr. Patricia Gaunt from the Thad Cochran National Warmwater Aquaculture Center for their skillful technical assistance; and Dr. Richard Shelby (USDA/ARS, Auburn, AL) for sharing the mouse anti-tilapia IgM antibodies used in this study. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ……………………………………………………….……………ii LIST OF TABLES ……………………………………………………………………….…...vi LIST OF FIGURES …………………………………………….……………………………..vii ABSTRACT ……………………………………………………………..………………………x CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ………………..……….…...1 Tilapia and Tilapia Aquaculture ....…………..…………………………………..…..1 Tilapia Bacterial Diseases ...…………………………………………………………...2 Rickettsia Like-Organisms and Piscirickettsia Like-Organisms ..…………………....2 Genus Francisella …………………………………………………………………..…6 Fish Francisellosis ………………………………………………………………..……8 Taxonomic Classification of Francisella sp. Isolated from Fish ..………………..…12 Francisella tularensis Pathogenesis …………………………………………………....14 Francsiella tularensis Pathogenicity Island ………………………………………..…..17 Objectives …………………………………………………………………………....19 Literature Cited …………………………………………………………………....22 CHAPTER 2. FRANCISELLA SP., AN EMERGING PATHOGEN OF TILAPIA (OREOCHROMIS NILOTICUS) IN COSTA RICA ………………………………………..…..30 Introduction ………………………………………………………………………..…..30 Materials and Methods …………………………………………………………....32 Results …………………………………………………………………………....36 Discussion ………………………………………………………………………..…..43 Literature Cited ………………………………………………………………..…..46 CHAPTER 3. ATTENUATION OF THE FISH PATHOGEN FRANCISELLA SP. BY MUTATION OF THE IGLC GENE …………………………………………………..………..50 Introduction ……………………………………………………………………..……..50 Materials and Methods ……………………………………………..……………..53 Results ………………………………………………………………………………..…..59 Discussion …………………………………………………………………………....65 Literature Cited ………………………………………………………………..…..68 CHAPTER 4. INTERACTION OF FRANCISELLA ASIATICA WITH TILAPIA (OREOCHROMIS NILOTICUS) INNATE IMMUNITY ………………………..…………..72 Introduction ………………………………………………………………………..…..72 Materials and Methods ………………………………………………………..…..75 Results ………………………………………………………………………..…..81 Discussion ……………………………………………………………………..…..…89 Literature Cited ………………………………………………………..………..…96 iv CHAPTER 5. ATTENUATED FRANCISELLA ASIATICA IGLC MUTANT INDUCES PROTECTIVE IMMUNITY TO FRANCISELLOSIS IN TILAPIA …………………..102 Introduction …………………………………………………………………………..102 Materials and Methods …………………………………………………………..104 Results …………………………………………………………………………………..109 Discussion …………………………………………………………………………..115 Literature Cited …………………………………………………………………..117 CHAPTER 6. DEVELOPMENT OF A REAL-TIME PCR ASSAY FOR IDENTIFICATION AND QUANTIFICATION OF THE FISH PATHOGEN FRANCISELLA NOATUNENSIS SUBSP. ORIENTALIS ………………...………………………………………………..121 Introduction …………………………………………………………………………..121 Materials and Methods …………………………………………………………..124 Results …………………………………………………………………………..131 Discussion …………………………………………………………………………..134 Literature Cited …………………………………………………………………..137 CHAPTER 7. COMPARISON OF IN VITRO AND IN VIVO EFFICACY OF FLORFENICOL FOR TREATMENT OF FRANCISELLA ASIATICA INFECTION IN TILAPIA …………..141 Introduction …………………………………………………………………………..141 Materials and Methods …………………………………………………………..143 Results …………………………………………………………………………..148 Discussion …………………………………………………………………………..155 Literature Cited …………………………………………………………………..158 CHAPTER 8. CONCLUSIONS …………………………………………………………..162 Literature Cited …………………………………………………………………..169 APPENDIX 1. JOURNAL OF FISH DISEASE PERMISSION …………………………..172 APPENDIX 2. JOURNAL OF AQUATIC ANIMAL HEALTH PERMISSION …………..173 APPENDIX 3. INFECTION AND IMMUNITY PERMISSION …………………………..174 APPENDIX 4. DISEASES OF AQUATIC ORGANISMS PERMISSION …………………..175 VITA …………………………………………………………………………………..176 v LIST OF TABLES Table 2.1 Estimates of evolutionary divergence between 16S rRNA sequences of members of the genus Francisella and representative warmwater fish pathogens and isolate LADL 07-285A ……………………………………………………………………………42 Table3. 1 Description of strains, plasmids and primers used in this study ……………………54 Table 3.2 Summary of Francisella sp. LADL 07-285A LD50 Virulence Assays and % mortalities calculated by the method of Reed-Muench ……………………………………63 Table 6.1 Bacterial DNA used in this study …………………………………………………..126 Table 6.2 TaqMan primers and probe used in this study …………………………………..128 Table 6.3 Quantification of Francisella noatunensis subsp. orientalis from inoculated PBS and spleen …………………………………………………………………………………..132 Table 7.1 Minimum inhibitory concentration (MIC) of florfenicol against Francisella asiatica and Escherichia coli 25922 isolates obtained from culture fish ……………….....149 Table 7. 2 Histopathological lesions in selected tissues from medicated and non-medicated tilapia 30 days post-challenge with F. asiatica …………………………………………..151 vi LIST OF FIGURES Figure 2.1 Splenomegaly and renomegaly with widespread multifocal white nodules in infected nile tilapia (Oreochromis niloticus) ………………………………………..…………..37 Figure 2.2 Histopathological findings of Francisellosis in nile tilapia (Oreochromis niloticus) (a) Hyperplasic and hypertrophic primary and secondary gill lamella (b) Pericarditis with granulomatous cellular infiltrate …………………………………………………………....38 Figure 2.3 Francisella sp. bacteria seen intra and extracellularly with Giemsa stain (100X)…...38 Figure 2.4 PCR amplification of Francisella sp. isolates recovered from tilapia (Oreochromis niloticus) …………………………..………………………………………..40 Figure 2.5 Evolutionary relationships of 24 taxa based on partial 16S rRNA sequences of members of the genus