Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 12-8-2017 Understanding molecular mechanisms of host-Edwardsiella ictaluri interaction Nawar Hadi Al-Janabi Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Al-Janabi, Nawar Hadi, "Understanding molecular mechanisms of host-Edwardsiella ictaluri interaction" (2017). Theses and Dissertations. 5047. https://scholarsjunction.msstate.edu/td/5047 This Dissertation - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Template B v3.0 (beta): Created by J. Nail 06/2015 Understanding molecular mechanisms of host-Edwardsiella ictaluri interaction By TITLE PAGE Nawar Al-Janabi A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Veterinary Medical Sciences in the Department of Basic Sciences in the College of Veterinary Medicine Mississippi State, Mississippi December 2017 Copyright by COPYRIGHT PAGE Nawar Al-Janabi 2017 Understanding molecular mechanisms of host-Edwardsiella ictaluri interaction By APPROVAL PAGE Nawar Al-Janabi Approved: ____________________________________ Attila Karsi (Major Professor) ____________________________________ Mark L. Lawrence (Committee Member) ____________________________________ Lesya M. Pinchuk (Committee Member) ____________________________________ Daniel G. Peterson (Committee Member) ____________________________________ Larry A. Hanson (Graduate Coordinator) ____________________________________ Mark L. Lawrence Associate Dean College of Veterinary Medicine Name: Nawar Al-Janabi ABSTRACT Date of Degree: December 8, 2017 Institution: Mississippi State University Major Field: Veterinary Medical Sciences Major Professor: Dr. Attila Karsi Title of Study: Understanding molecular mechanisms of host-Edwardsiella ictaluri interaction Pages in Study: 118 Candidate for Degree of Doctor of Philosophy Catfish, the "king" of the U.S. aquaculture, is threatened by a severe, systemic bacterial disease known as enteric septicemia of catfish (ESC). This disease causes high mortality and massive economic losses in cultured channel catfish (Ictalurus punctatus) in the United States. E. ictaluri penetrates catfish intestinal epithelia quickly and establishes a systemic infection rapidly. However, our knowledge on catfish intestine and E. ictaluri interaction is very limited. In Particular, catfish intestinal immune responses and virulence genes needed by E. ictaluri to evade host defenses are not well understood. Hence, our long-term goal is to identify the molecular mechanisms of E. ictaluri-host interactions. The overall objectives of this study were to understand catfish immune responses to E. ictaluri infection and determine essential genes of E. ictaluri during the intestinal invasion. To accomplish the overall objectives of this research, intestinal ligated loops were constructed surgically in live catfish and loops were injected with wild-type E. ictaluri and two live attenuated E. ictaluri vaccine strains developed recently by our research group. We first determined catfish intestinal immune responses against E. ictaluri wild-type and live attenuated vaccine strains. Then, we analyzed the global gene ii expression patterns of wild-type E. ictaluri and vaccine strains during catfish intestinal invasion using high throughput RNA-Seq technology. Results showed a moderate level of neutrophil and B cell infiltration correlated with significantly lower expression of TNF-α, CD4-1, and CD8-α in the vaccine injected intestinal tissue compared to that of wild-type injected intestinal tissue. Further, RNA-Seq data analysis showed the prominent expression of genes related to bacterial secretion systems, ATP production processes, and multidrug resistance (MDR) efflux pumps in wild-type E. ictaluri. In contrast, the prominently expressed genes in vaccine strains were related to the phosphotransferase system and sugar metabolism processes. All these data suggest that our live attenuated vaccines are capable of triggering effective immune responses in catfish without causing damage to the host. iii DEDICATION I would like to dedicate this research to my parents Hadi Yaser Al-Janabi and Kareema Fanharawa, my sister Safa, and my brothers Mustafa, Ammar, and Yaser. iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my major professor Dr. Attila Karsi, who has provided excellent mentorship and support in all aspects of my research. I would also like to give my sincere thanks and appreciation to all my committee members, Dr. Mark Lawrence, Dr. Lesya Pinchuk, and Dr. Daniel Peterson, for their suggestions and encouragements. I would like to thank Dr. Hossam Abdelhamed and Dr. Jingjiun Lu for their support with lab work. I am thankful to Mrs. Michelle Banes and all my lab members, Safak, Ali, Iman, Hasan, and Salih for their advice and company. I would also like to thank Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University for providing me great environment, which is crucial for the successful completion of this dissertation. Finally, I would like to acknowledge the financial support from the Ministry of Higher Education of Iraq. My deepest gratitude goes to my parents Hadi Yaser Al-Janabi and Kareema Fanharawa for their great support and encouragement throughout my doctoral program. Without all of you, completion of this research would have been difficult. v TABLE OF CONTENTS DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENTS .................................................................................................v LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .............................................................................................................x CHAPTER I. INTRODUCTION AND REVIEW OF RELEVANT LITERATURE ................1 Edwardsiella ictaluri .............................................................................................1 Innate immune system in catfish ...........................................................................2 Pattern Recognition Receptors (PRRs) ...........................................................3 Antimicrobial Peptides (AMPs) ......................................................................5 Complement system ......................................................................................5 Lectin .............................................................................................................6 Cytokines .......................................................................................................7 Transferrin .....................................................................................................8 Mucus secretion .............................................................................................8 Adaptive immune system in catfish ......................................................................9 B lymphocytes and Immunoglobulins .............................................................9 T lymphocytes ...............................................................................................10 Significance of research and objectives ..............................................................11 II. THE INTESTINAL IMMUNE RESPONSES OF CHANNEL CATFISH AGAINST LIVE ATTENUATED EDWARDSIELLA ICTALURI VACCINES.............................................................................................14 Abstract ................................................................................................................14 Introduction .........................................................................................................15 Materials and Methods ........................................................................................18 Bacterial strains, media, reagents, and growth conditions ..........................18 Construction of catfish intestinal loops .......................................................18 Test of ligated intestinal loops efficiency ....................................................20 Determination of mucus secretion ...............................................................21 Determination of pathology in catfish small intestine .................................21 Immunohistochemistry (IHC) .....................................................................21 vi Total RNA extraction and cDNA synthesis ................................................22 Quantitative real time PCR and data analysis .............................................23 Statistical analysis .......................................................................................25 Results .................................................................................................................25 Construction and evaluation of ligated catfish intestinal loops ...................25 Catfish intestinal mucus secretion ...............................................................26 Histopathology of catfish intestine ..............................................................27