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JCU 55730-Malara-2017-Thesis.Pdf ResearchOnline@JCU This file is part of the following work: Malara, Danilo (2017) Photodynamic antimicrobial chemotherapy for pathogenic vibrio control in prawn hatcheries. PhD Thesis, James Cook University. Access to this file is available from: https://doi.org/10.25903/5bac064fe5557 Copyright © 2017 Danilo Malara The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owners of any third party copyright material included in this document. If you believe that this is not the case, please email [email protected] Photodynamic Antimicrobial Chemotherapy for Pathogenic Vibrio Control in Prawn Hatcheries Thesis submitted by Danilo Malara BSc University of Messina BSc (Hons) University of Messina MSc by Research University of Messina In October 2017 For the degree of Doctor of Philosophy In the College of Science and Engineering James Cook University Acknowledgements During my three and half years at James Cook University, I received support and encouragement from many people. This thesis is a team effort, therefore, I would like to express my gratitude to my advisory panel: Associate Professor Kirsten Heimann (JCU), Associate Professor Michael Oelgemöller (JCU) and Dr. Lone Høj (AIMS). It has been a great honour to be supervised by three globally recognized scientists in their field. My sincere thanks to Associate Professor Kirsten Heimann for your steadfast guidance and encouragement, as well as providing many opportunities and the space and time to try out my own ideas and to “create the story” using data generated from my experiment. Thank you Michael for your advice and your feedback; I learnt a great deal from you, especially in relation to how to prepare for conference and seminar presentations as well as on the chemistry of the compound used in this work. Lone, your contribution was vital for my progress. Your critical questioning helped me to better understand the issues and express my idea differently. Your microbiology and genetic expertise, as well as your training, taught me many new skills. I am very thankful to James Cook University for providing stipend funding (James Cook University Post Graduate scholarship), and AIMS@JCU for research funding through their annual Pilot Research Awards (2014, 2015 and 2016) and Travel Research Funding (2016). I am also very thankful to AIMS@JCU and James Cook University to provide a “Special Stipend Scholarship”, in this regards many thanks to Professor Helene Marsh, Dr. Libby Evans-Illidge, Dr. Cherie Motti, Melissa McLean, Lauren Gregory and all the people involved in this process. Many thanks to my NQAIF lab mates, all of you had an impact on my thesis. Some of you taught me how to use specific instruments. Thank you Dr. Florian Berner and Dr. Martino Malerba. Outside of the lab environment, your advice was constructive, pointing me in the right direction. Thanks Stan Hudson for you training and our long chat. Thanks also to Dr. Roger Huerlimann and Dr. Nick von Alvensleben for providing the calibration curves used in Chapter 4. Special thanks to Gabriella Citarrella, who worked as my volunteer for about 6 months. Your help was important for the thesis structure. Thanks also to all my Indian friends; we shared both good and i bad times in the lab, but all of us did our best to run the lab as a team. Thanks also to Dr. Brett Baillie, lab manager of PC2 laboratory at AIMS, your PCR training was important for the entire project. Despite the distance that separates us, I would like to say thank you to all the researchers at The Italian National Institute for Environmental Protection and Research (ISPRA) laboratory of Milazzo. Especially my old university colleague and friend, Dr. Cristina Pedà, we supported each other in these past few years, our long skype meetings and your precious advice in the tough periods, were very much appreciated. Thank you to principal scientists, Dr. Teresa Romeo and Dr. Pietro Battaglia, for your support and your suggestions. That I am here today is because of you. Everything you taught me and all that you advised me are still fresh in my mind and gave me the experience and the power to put my foot in the science door. Thank to Assoc. Prof. Leigh Owens (James Cook University) for providing the bacterial strain, and Crystal Bay Prawn Farm (Cardwell, North Queensland, Australia) for providing prawn post-larvae and commercial feed pellets used in this work. Professor Rhondda Jones (James Cook University) is thanked for statistical advice. Research conducted in this thesis comply with all ethic measures established by James Cook University and outlined by James Cook University Ethic Committee. Finally, but not least, I would like to say thank you to my family, starting with my Mum and my brothers. Although many kilometres away from me and living in my home country, they have always supported me and gave me the power to continue. A very big thanks to my wife Valeria, who selflessly gave me her unwavering support on this long voyage. Valeria took a step into the unknown when she decided to follow me to another city and start our life again. Most importantly, she gave me the most important thing in my life, my little daughter, Azzurra Sydney. Finally, I would like to dedicate this thesis to you, Dad. Thank you. Although you are no longer here for me to thank in person, I am here because of you. ii Statement of the Contribution of Others This thesis includes collaborative work with my supervisors, Associate Professor Kirsten Heimann (JCU), Associate Professor Michael Oelgemöller (JCU) and Dr. Lone Høj (AIMS). While the data collection was principally conducted by Danilo Malara, experimental design, data interpretation, data analysis, developing the approach and arguments as well as multiple manuscript drafts, were performed by myself, Danilo Malara, in collaboration with all of my advisory panel, as described in Table SCO.1. The research conducted in this thesis complies with all ethic measures established by James Cook University and outlined by James Cook University Ethic Committee. Table SCO.1: Contribution to thesis chapter and experimentation. DM= Danilo Malara, LH = Dr. Lone Høj, KH = A/Professor Kirsten Heimann, MO = A/Professor Michael Oelgemöller. Chapter Experimental Data Data analysis Approach and Manuscript n. design collection and arguments draft interpretation 2 DM, LH DM DM, LH, KH DM, LH, KH, DM, LH, KH, MO 3 DM, LH, KH DM DM, LH, KH DM, LH, KH DM, LH, KH, MO 4 DM, LH, KH DM DM, LH, KH DM, LH, KH DM, LH, KH, MO 5 DM, LH DM DM, LH, KH DM, LH, KH DM, LH, KH, MO iii Abstract In the last two decades, the aquaculture industry has grown significantly, providing fish, molluscs and crustaceans to the global market. Microbial pathogens are the principal cause of massive financial losses in many fish and crustacean farms. Disinfection using ozone, chlorine or UV irradiation are expensive and leave toxic by- products. Biosecurity threats through the introduction of wild brood-stock and live feed is a major concern for the aquaculture industry. Microalgae and/or Artemia nauplii (brine shrimp), have been identified as potential vectors for microbial pathogens that can result in high mortalities, particularly in hatcheries. Bacteria can proliferate rapidly in intensive aquaculture productions due to the high density of target animals, live feed organisms and build-up of biological waste that generate ideal conditions for many potential pathogens. The current inability to bio-secure farmed aquaculture animals through appropriate disinfection strategies of live feed without altering the quality of the final product is an area requiring urgent research, as it is the main barrier to cost-effective product development; the main hurdle to competitive expansions into national and international markets. Recently, photodynamic antimicrobial chemotherapy (PACT) has emerged as a promising water sterilisation technique. PACT uses the activation of photosensitisers by light to generate highly reactive oxygen species (ROS), which indiscriminately oxidises cell wall and cell membrane components (i.e. lipids, proteins, carbohydrates). After entry of the photosensitiser into cells, produced singlet oxygen also targets intracellular components such as organelles, membrane compartments, and nucleic 1 acids (i.e. DNA and RNA). Due to the indiscriminate action of O2, resistance cannot develop, unlike in the case of antibiotics. The choice of photosensitiser is crucial, and compounds that photobleach with time are preferred for aquaculture purposes to avoid build up in water or the farmed animal. Porphyrins are a group of natural or synthetic compounds that fit the photobleaching requirement, but to date no research has specifically investigated porphyrin-based sterilisation efficiency in prawn hatcheries. In addition, the porphyrin-based antimicrobial efficiency for the ones used in this research has not been tested against luminescent virulent strains of Vibrio iv harveyi-related species, the main causative agents for luminescent vibriosis, which causes large losses for finfish, bivalves and prawn industries. Each chapter of this thesis addresses specific objectives that, in conjunction with the other chapters, contribute to the principal aim of the research: investigate the potential of using PACT to control Vibrio bacteria in aquaculture. Chapter 2 details objectives relating to method development to select a suitable Vibrio harveyi-related strain to be used in the following chapters. While Chapter 1 reviews PACT history and its application with special interest to aquaculture, the method development chapter (chapter 2), pathogenicity towards prawns after injection and luminescence intensity of two selected Vibrio strains were evaluated. Challenge experiments were performed with Penaeus monodon and Koch’s postulates were fulfilled. Precise identification was obtained using molecular techniques including multiplex PCR, housekeeping gene analysis and construction of a phylogenetic tree. The strongly luminescent Vibrio sp ISO7, was demonstrated to be highly virulent towards P.
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