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EFFECTS of CATECHOLAMINES on Yersinia Ruckeri and Yersiniosis in ATLANTIC SALMON (Salmo Salar)

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EFFECTS of CATECHOLAMINES on Yersinia Ruckeri and Yersiniosis in ATLANTIC SALMON (Salmo Salar) EFFECTS OF CATECHOLAMINES ON Yersinia ruckeri and yersiniosis in ATLANTIC SALMON (Salmo salar) by Nguyen Duc Quynh Anh Bachelor of Science in Aquaculture Master of Science in Aquaculture Institute for Marine and Antarctic Studies University of Tasmania Submitted in fulfilment of the requirements for the Doctor of Philosophy University of Tasmania December 2019 DECLARATIONS BY THE AUTHOR Statement of Originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Authority of Access This thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act of 1968. Statement of Ethical Conduct The research associated with this thesis abides by the international and Australia codes on animal experimentation. Animal ethics permits were obtained from the University of Tasmania Animal Ethics Committee for all aspects of the project where experiments involved live animals (Permit number A0017118) Signed: Date: 7th December 2019 Nguyen Duc Quynh Anh ii ACKNOWLEDGEMENTS The completion of this thesis would not have been possible without the guidance and precious support of several individuals. I would like to thank all who have directly or indirectly contributed to shape my career life during the last four years. First and foremost, I would like to express my deepest gratitude to my supervisors Assoc. Prof. Andrew Bridle, Prof. Barbara Nowak, and Dr. Philip Crosbie for their invaluable expertise, continuous guidance, support and tireless patience during my PhD. My special thank goes to Andrew, who was always available despite of his very busy schedule to give me useful feedback, suggestions in experimental designing, data analysis as well as new practical technique guidance. I will never forget how patient he was to try to explain something complicated for me. I would like to thank Barbara for her care and kind support especially at the beginning of my PhD life and her contribution in writing correction. I am also grateful to Phil for the big help in my experiment sampling (even at the midnight), careful reading and corrections for all my writing drafts as well as his touched care for my little son and my family. I would like to express my sincere thanks to Dr Mark Adams, Heindrick and Amin for their useful help in setting up my experiment at Aquaculture centre. Special thanks to Mei for your kindness and willingness to help whenever I asked even in research or normal life, thanks for being my good friend. I would also like to thank the Vietnamese Government for the finance support to give me opportunity to study and complete my PhD course in Australia. My thanks also go iii to all staffs of Faculty of Fisheries - Hue University of Agriculture and Forestry for their supporting during my study. I would also feel lucky and really appreciate to receive cares and concerns from “Thay Phuoc”, “chi Hue Linh”, “anh Tuan – chi Hang” and “co Hong”. Thank you for sharing your experience and helping me feel better whenever I had problems. To friends and colleagues: Ylenia, Lucas, Zack, Jimena, Simon, Aaron, Frankie, Em, Anna, Peter, Thu, Cuong, Trinh, Mai, Ngoc, Duc, Xuyen, Hoang, Nha, Mui and Thao, thanks for a nice friendship and social activities that made my life much more colourful during my stay in Launceston. I would like to thank my all family members from the bottom of my heart, for their understanding, endless and unconditional support and encouragement that helped me go through all difficulties during my study. I deeply gratitude to my dear parents who have looked after my son since he was few months old. Thanks for loving him as yours another son. Finally, to my little family, thank you my husband and my little boy for always being with me. You are always in my heart and we will be together soon. iv TABLE OF CONTENTS DECLARATIONS BY THE AUTHOR.............................................................................. ii ACKNOWLEDGEMENTS ................................................................................................. iii TABLE OF CONTENTS ....................................................................................................... v LIST OF FIGURES................................................................................................................ix LIST OF TABLES ............................................................................................................... xiv ABBREVIATIONS AND UNITS ..................................................................................... xvi EXPLANATORY NOTE REGARDING THESIS STRUCTURE ................................ xx ABSTRACT ..........................................................................................................................xxi Chapter 1. GENERAL INTRODUCTION ..........................................................................1 1. 1. Yersinia ruckeri INFECTION ...................................................................................1 1. 2. CHARACTERISTICS OF Yersinia ruckeri ............................................................5 1.2.1. Morphological and biochemical characteristics ................................................5 1.2.2. Virulence factors....................................................................................................8 1.2.2.1. Survival outside the host................................................................................8 1.2.2.2. Colonisation of the host .................................................................................9 1.2.2.3. Extracellular products (ECPs).................................................................... 10 1.2.2.4. Iron acquisition ............................................................................................ 14 1.2.2.5 Plasmids ......................................................................................................... 15 1.2.2.6. Other virulence factors................................................................................ 16 1.3. HOST STRESS HORMONES AND THEIR IMPACT ON GROWTH AND VIRULENCE FACTORS OF PATHOGENS .............................................................. 18 1.4. FISH GUT MICROBIOME..................................................................................... 25 1.5. THESIS AIMS........................................................................................................... 33 Chapter 2. EFFECTS OF CATECHOLAMINES ON GROWTH AND VIRULENCE OF Yersinia ruckeri .............................................................................................................. 34 2.1. INTRODUCTION..................................................................................................... 34 v 2. 2. MATERIALS AND METHODS ........................................................................... 36 2.2.1. Growth conditions for Yersinia ruckeri ........................................................... 36 2.2.2. Calibration curve for absorbance and cell density of Y. ruckeri .................. 37 2.2.3. Hormones ............................................................................................................ 37 2.2.4. Growth assays ..................................................................................................... 37 2.2.5. Lipase, phospholipase, caseinase and haemolysin assays ............................. 38 2.2.6. Siderophore assay ............................................................................................... 39 2.2.7. Biofilm formation assay .................................................................................... 40 2.2.8. Antimicrobial assay............................................................................................ 40 2.2.9. Statistical analysis .............................................................................................. 41 2.3. RESULTS .................................................................................................................. 42 2.3.1. Stress hormones increase the growth of Y. ruckeri in serum – supplemented medium ........................................................................................................................... 42 2.3.2. Lipase, phospholipase, caseinase and haemolysin assays ............................. 45 2.3.2.1. Lipase ............................................................................................................ 45 2.3.2.2. Caseinase ...................................................................................................... 47 2.3.2.3. Haemolysin................................................................................................... 48 2.3.3. Siderophore ......................................................................................................... 50 2.3.4. Biofilm formation ............................................................................................... 51 2.3.5. MIC assay ............................................................................................................ 52 2. 4. DISCUSSION..........................................................................................................
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