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Immunoprotective Mechanisms Associated with in Ovo Delivery

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Immunoprotective Mechanisms Associated with in Ovo Delivery IMMUNOPROTECTIVE MECHANISMS ASSOCIATED WITH IN OVO DELIVERY OF OLIGODEOXYNUCLEOTIDES CONTAINING CpG MOTIFS (CpG-ODN) AND FORMULATION OF CpG WITH NANOPARTICLES TO ENHANCE ITS EFFICACY IN NEONATAL BROILER CHICKENS A thesis submitted to the College of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Veterinary Pathology University of Saskatchewan Saskatoon By Thushari Anamari Gunawardana © Copyright Thushari A. Gunawardana, June 2018. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a postgraduate degree from the University of Saskatchewan, I agree that the libraries of this university may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professors who supervised my thesis work or in their absence, by the Head of the Department or the Dean of the college in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without any written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of the material in this thesis in whole or part should be addressed to: Head of the Department of Veterinary Pathology Western College of Veterinary Medicine University of Saskatchewan 52, Campus Drive Saskatoon, Saskatchewan S7N 5B4 Canada Or Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan S7N 5C9 Canada i ABSTRACT Oligodeoxynucleotides containing CpG motifs (CpG-ODNs) are known for their ability to stimulate vertebral immune system and provide protection against pathogens. Although CpG- ODN provides protection against bacterial infections in chickens mechanisms of immunoprotection remains elusive. The objective of this study was to identify mechanisms of immunoprotection of CpG-ODN following in ovo delivery in chickens . In the second chapter, we provide the mechanistic insights into CpG-ODN induced protection against Escherichia coli (E. coli). Multiplex cytokine gene analysis using QuantiGene Plex 2.0® technique revealed that CpG-ODN upregulates both Th1 and Th2 cytokines, as well as pro-inflammatory cytokines in both spleen and lung. In our study, Lipopolysaccharide-induced TNF factor-alpha factor (Litaf) stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN induced protection mechanisms. Flow cytometry analysis showed a marked increase of T lymphocyte and antigen presenting cells (APC) as well as enhanced expression of CD40 by APCs in spleen and lung after CpG-ODN treatment. This study demonstrated for the first time that CpG-ODN provides protection in neonatal chicks against E. coli infections by eliciting cytokine responses and enriching the immunological niches in spleen and lungs. In the third chapter, we show that CpG-ODN induces a dose-dependent influx of macrophages, CD4+ and CD8+ T-cell subsets in spleen and lungs of chicks that correlates with the immunoprotection against E. coli. We observed a dose-dependent enrichment of the immunological niches, wherein 25 µg and 50 µg of CpG-ODN induced significant changes in the immune profile of both spleen and lungs that correlated with their ability to resist E. coli infection. In the fourth chapter, we report that in ovo delivery of CpG-ODN formulated with CNT or lipid- surfactant potentiate the protective effect against E. coli infection. In conclusion, this study provides a greater understanding of cellular and molecular mechanisms for CpG-ODN induced antimicrobial immunity. And this significant advancement in the mechanistic insight will help in utilizing the full therapeutic potential of CpG-ODNs. Our work on CpG-ODN dose-dependent changes and enhanced protection against E. coli infection by nanoparticle formulations demonstrate the possible utilitization of CpG-ODN in the poultry industry as an alternative to antibiotics to prevent bacterial infections of neonatal chickens. ii ACKNOWLEDGEMENTS First and foremost, I express my sincere gratitude to my supervisor, Dr. Susantha Gomis, for his continuous support of my graduate studies, his patience, motivation, knowledge and understanding throughout my research program. I consider myself to be very fortunate to complete my PhD with such a supportive advisor and mentor. I would like to thank the other members of the committee, Dr.Marianna Foldvari, Dr. Suresh Tikoo, Dr. Philip Willson, Dr. Beverly Kidney and Dr. Elemir Simko for the assistance they provided at all levels of this research project. I would also like to thank all faculty members of the WCVM for their support and understanding throughout my research program. A very special thanks goes out to Drs. Khawaja Ashfaque Ahmed, Lisanework Ayalew and Shanika Kurukulasuriya for their help and guidance in all aspects of my thesis. I very much appreciate the continuous support of Shelly Popowich throughout my study. I am also thankful to Betty Chow-Lockerbie and Tara Zachar for all the help provided in successfully completing my research. I also thank Ian Shirley, all the staff members of Animal Care Unit of WCVM and Marina Vaneva Ivanova from University of Waterloo for their technical support. I am grateful to Sandy Mayes, Tyler Moss and Angela Turner for their help in academic and administrative areas. A special thank you goes to my fellow research team members, Kalhari Goonawardene, Ashish Gupta, Ruwani Karunarathna, Mengying Liu and my all my friends who have supported me in various ways. My heartfelt thank you goes to my parents, Premasiri Gunawardana and Jean Gunawardana, and my brother Nishantha for their love and support throughout my life. I am very much grateful to my loving husband, Kalpa for his constant support and encouragement, this thesis have never been possible without them. A special thanks to my baby son, Thehan for his lovely smile which was heartening during my thesis writing. I wish to thank for the financial support provided by Natural Sciences and Engineering Research Council of Canada, Western Economic Diversification Canada, Saskatchewan Chicken Industry Development Fund, Canadian Poultry Research Council, Alberta Livestock and Meat Agency Ltd. and Alberta Chicken Producers. iii DEDICATION I DEDICATE THIS THESIS TO MY LOVING PARENTS FOR THEIR CONSTANT LOVE AND SUPPORT iv TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................. i ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iii DEDICATION.............................................................................................................................. iv TABLE OF CONTENTS ............................................................................................................. v LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix LIST OF ABBREVIATIONS ...................................................................................................... x CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW .................................... 1 1.1 Introduction .................................................................................................................... 1 1.2 Overview of the avian immune system ......................................................................... 2 1.2.1 Avian immune organs ................................................................................. 2 1.2.2 Immune cells ............................................................................................... 4 1.3 Role of the immune system in protection against infectious diseases ........................ 6 1.3.1 Innate immune responses ............................................................................ 7 1.3.2 Adaptive immune responses........................................................................ 9 1.3.3 Mucosal immunity..................................................................................... 10 1.3.4 Neonatal immunity .................................................................................... 10 1.4 Common bacterial diseases in neonatal chickens ...................................................... 10 1.5 Immune stimulating agents as antibiotic alternatives for poultry ........................... 11 1.6 History of CpG-ODN ................................................................................................... 12 1.6.1 Mechanism of action of CpG-ODN .......................................................... 14 1.6.2 CpG-ODN safety ....................................................................................... 17 1.6.3 CpG-ODN applications ............................................................................. 18 1.6.4
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