Studies on the Epidemiology, Vaccination, Susceptibility, and Treatment of Columnaris Disease in Fishes by Haitham H. Mohammed 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 1, 2015 Keywords: Columnaris disease, Vaccine, Flavobacterium columnare, Genomovar, Microbiome, Nigella sativa Copyright 2015 by Haitham H. Mohammed Approved by Covadonga R. Arias, Chair, Professor of Fisheries, Aquaculture, and Aquatic Sciences Stephen A. Bullard, Associate Professor of Fisheries, Aquaculture, and Aquatic Sciences Eric Peatman, Associate Professor of Fisheries, Aquaculture, and Aquatic Sciences Mark L. Lawrence, Professor of Basic Sciences, College of Veterinary Medicine, Mississippi State University Abstract In this dissertation, I explored the intraspecies genetic heterogeneity within Flavobacterium columnare isolates collected during particularly severe columnaris outbreaks observed at the E.W. Shell Fisheries Center, Aquatic Experiment Research Station at North Auburn, AL, USA. These outbreaks affected both farmed and sport fish species that were raised in ponds located under the same hydrologic unit from spring 2010 throughout summer 2012. All the recovered isolates were ascribed to genomovar II of F. columnare following restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene. Fingerprinting using amplified fragment length polymorphism (AFLP) revealed genetic diversity among the genomovar II isolates and persistence of certain clones throughout the watershed. Antimicrobial susceptibility testing of the isolates demonstrated resistance to some of the commonly used antimicrobial agents in aquaculture. I also compared the vaccine efficacy of newly generated stable and safe rifampicin resistant genomovar II mutants to that of the attenuated mutant strain in the commercial vaccine (AQUAVAC-COLTM, Merk), which is derived from a genomovar I (the less virulent genomovar) F. columnare strain. Based on relative percent survival rates in channel catfish, zebrafish, and Nile tilapia, both genomovar I and II mutants were equally protective against columnaris disease when a genomovar I strain of F. columnare was used to challenge the vaccinated fish. However, administration of the new genomovar II attenuated mutant as vaccine conferred greater ii protection against columnaris disease caused by a genomovar II strain than that provided by the genomovar I mutant. Because healthy microbiome on the skin and gills of fish was reported to benefit the hosts and prevent infections by opportunistic pathogens by hindering their invasion and/or stimulating the fish’s immune system, I investigated the effect of disrupting the surface microbiome of channel catfish by short term exposure to one of the commonly used surface-acting disinfectants in aquaculture: potassium permanganate (PP) (KMnO4). I tested the susceptibility of fish treated with PP to columnaris disease. Dysbiosis of the surface microbiome was analyzed by ribosomal intergenic spacer analysis (RISA) and pyrosequencing. Chemical treatment with PP altered the composition of the external microbiome and increased catfish mortality following experimental challenge with F. columnare. Finally, I tested the in vitro and in vivo efficacy of Nigella sativa, a medicinal plant commonly used in folk medicine by many nations to treat a wide range of diseases, against columnaris disease. I found that N. sativa oil possess a potent antibacterial activity against all genomovars of F. columnare by disc diffusion method. Moreover, supplementation of fish diets with N. sativa seeds or oil were protective to zebrafish and channel catfish against columnaris disease in controlled laboratory challenges. In summary, data obtained during my dissertation emphasized the importance of genomovar II isolates as causative agents of high mortality-columnaris disease in aquaculture and recreationally valued fish species. I tested two new promising methodologies to prevent and control columnaris disease and proved that caution should be used when treating external diseases in fish to avoid damaging the external microbiome. iii Acknowledgments First and foremost I thank God for giving me the strength, courage and patience to pursue my dreams and for all the blessings. I would like to express my deepest and everlasting gratitude to my major advisor Dr. Cova Arias for giving me the opportunity to join her lab. Without her guidance, mentoring, unconditional support, and tireless editing; I may have never been able to finish my dissertation work. Her role from the very beginning and throughout my study is beyond words of recognition. I am also grateful to my graduate committee members, Dr. Stephen (Ash) Bullard, Dr. Eric Peatman, and Dr. Mark L. Lawrence for their time, expertise, and insightful suggestions. Thanks to everyone in the School of Fisheries, Aquaculture and Aquatic Sciences, from the department head to professors, students, and the administrative staff for allowing me access to countless resources and for their help over the years. My doctoral studies would not be possible without the presence and help of several extraordinary people. Loads of thanks to our lab cat, Stacey LaFrentz, for her assistance in my projects and most importantly, for always being there when I needed her. I would like also to offer my appreciation to all the members of the Aquatic Microbiology Lab, past and present Arias Arrows: Ryan Wood, Andrea Larsen, Óscar Olivares-Fuster, Zhen Tao, Wenlong Cai, Candis Ray, Noemi Bujan, Francisca Burgos Valverde, Evelyn Willmon, Savannah Warren and, Jeff Holder. Thanks for making the dungeon in Upchurch Hall one of my favorite spots, for leaving me alone at night to do my crazy experiments and for the friendship you gave to me iv throughout this atypical journey. I owe you a lot and feel extremely fortunate to have you as my lab mates. You made my Auburn graduate experience memorable. Most importantly, I am and forever will be grateful to my family whose love and support made this possible. Utmost and sincere thanks to my beautiful wife Rasha and to my beloved gaggle of monsters, Salma, Hesham and my little hero Omar. I could not have made it here without your constant love and encouragement. Knowing that you love, trust and believe in me has given me the boost to endure when I wasn’t sure I could and sustained me to complete this work. I am uniquely and foremost grateful to my parents, Mr. Hussien and Mrs. Mona; my brothers, Mahmoud and Hammad; my sisters Mayada and Basma. They all made great personal sacrifices and inspired me to push myself beyond boundaries that would have been difficult to cross without their unlimited motivation and understanding. I also wish to extend my appreciation to Dr. Joe Newton and Priscilla Barger from the College of Veterinary Medicine, Auburn University, for the supply of so many catfish and to those who offered me any kind of aid, guidance, or advice, but I forget to mention their names. Last, but certainly not least, I want to thank the Egyptian Cultural and Educational Bureau (ECEB) for funding my graduate research scholarship. v Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables .................................................................................................................................. x List of Figures ................................................................................................................................ xi List of Abbreviations .................................................................................................................... xii CHAPTER 1. INTRODUCTION ................................................................................................... 1 Introduction ................................................................................................................................. 1 Objectives ................................................................................................................................... 3 Dissertation Organization ........................................................................................................... 4 References ................................................................................................................................... 5 CHAPTER 2. LITERATURE REVIEW ........................................................................................ 9 The Disease ................................................................................................................................. 9 The Pathogen ............................................................................................................................ 11 Epidemiology ............................................................................................................................ 19 Virulence Factors and Pathogenesis ......................................................................................... 26 vi Host Defense ............................................................................................................................