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Molecular Identification of Vibrio Harveyi-Related Bacteria and Vibrio Owensii Sp ResearchOnline@JCU This file is part of the following reference: Cano Gomez, Ana (2012) Molecular identification of Vibrio harveyi-related bacteria and Vibrio owensii sp. nov., pathogenic to larvae of the ornate spiny lobster Panulirus ornatus. PhD thesis, James Cook University. Access to this file is available from: http://eprints.jcu.edu.au/23845/ The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] and quote http://eprints.jcu.edu.au/23845/ Molecular identification of Vibrio harveyi-related bacteria and Vibrio owensii sp. nov., pathogenic to larvae of the ornate spiny lobster Panulirus ornatus Thesis submitted by Ana CANO-GÓMEZ B.Sc. (University of Cádiz, Spain) M.Appl.Sc. Biotechnology (James Cook University, Australia) In February 2012 for the degree of Doctor of Philosophy in the School of Veterinary and Biomedical Sciences James Cook University STATEMENT OF ACCESS I, the undersigned, author of this work, understand that James Cook University will make this thesis available for use within the University Library and, via the Australian Digital Theses network, for use elsewhere. I understand that, as an unpublished work, a thesis has significant protection under the Copyright Act and; I do not wish to place any further restriction on access to this work. Ana Cano-Gómez Febuary 2012 STATEMENT OF SOURCES DECLARATION I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of references is given. Ana Cano-Gómez February 2012 ii STATEMENT OF THE CONTRIBUTIONS OF OTHERS Contributors (affiliation): 1. Leigh Owens, main supervisor (Associate Professor Microbiology and Immunology, School of Veterinary and Biomedical Sciences, James Cook University, JCU) 2. Lone Høj, co-supervisor (Research Scientist, Australian Institute of Marine Science, AIMS) 3. Nikos Andreakis (Research Scientist, AIMS) 4. Mike Hall (Principal Research Scientist, AIMS) 5. Evan Goulden (PhD candidate, University of NSW-AIMS) 6. David Bourne (Research Scientist, AIMS) 7. AIMS@JCU scholarship (JCU) 8. The Australian Institute of Marine Science (AIMS) 9. Graduate Research Scheme Fund (JCU) 10. School of Veterinary and Biomedical Sciences (JCU) NATURE OF CONTRIBUTION Nature of assistance Contribution Contributors Intellectual support Proposal writing 1, 2, 4 Data analysis 1, 2, 3 Statistical support 1, 5 Editorial assistance 1, 2, 3, 4, 5, 6 Financial support University Fees 10 (AU$75 000) Field research 1, 7, 8, 9, 10 (~AU$25 000) Stipend 7 (AU$ 60 000) Conferences 7, 8, 10 (AU$2 000) Data collection Research assistance 8, 10 iii CONTRIBUTION TO PUBLICATIONS Chapter 2: Review of Literature. (Aquaculture 287, 1-10) Chapter 3: Description of Vibrio owensii sp. nov. (FEMS Microbiology Letters 302, 175-181). Chapter 4: Identification of Vibrio harveyi-related species by multilocus sequence analysis. (Systematic and Applied Microbiology 34, 561-565). Chapter Publication title Authors # 1 2 3 4 5 6 Molecular identification, typing and tracking of Vibrio x x x x 2 harveyi in aquaculture systems: current methods and future prospects 3 Vibrio owensii sp. nov., isolated from cultured x x x x crustaceans in Australia Multilocus sequence analysis provides basis for rapid x x x 4 and reliable identification of V. harveyi-related species and confirms previous misidentifications of important marine pathogens ELECTRONIC COPY I, the undersigned, the author of this work, declare that the electronic copy of this thesis provided to the James Cook University Library is an accurate copy of the print thesis submitted, within the limits of the technololgy available. Ana Cano-Gómez February 2012 iv ANIMAL ETHICS APPROVAL The research presented and reported in this thesis was conducted within the guidelines for research ethics outlined in the National Statment on Ethics Conduct in Research Involving Human (1999), the Joint NGMRC/AVCC Statement and Guidelines on Research Practice (1997), the James Cook University Policy on Experimentation Ethics, Standard Practices and Guidelines (2001) and the James Cook University Statement and Guidelines on Research Practice (2001). Approval number A1623. v ACKNOWLEDGEMENT The research presented in this thesis has been carried out at the School of Veterinary and Biomedical Sciences at James Cook University (JCU) and at the Australian Institute of Marine Science (AIMS) in Townsville. I would like to thank the AIMS@JCU joint venture and the Faculty of Medicine, Health and Molecular Science for giving me this opportunity and for funding the research during my PhD. I am grateful to the School of Veterinary Sciences and to AIMS for providing academic assistance and laboratory facilities. Foremost, I would like to express my sincere gratitude to my supervisor, Associate Professor Leigh Owens, for the continuous support of my PhD study and research, for his enthusiasm, patience and immense knowledge. He provided me with many helpful suggestions and important advice during the course of this work and I can say I have learnt something new from each of our conversations. I also wish to express my appreciation to my co-supervisors Dr. Lone Høj and Dr. Mike Hall from AIMS. Thank you for the warm welcome to the Institute, the guidance and support, the incredible assistance with scholarship applications and manuscripts, the stimulating discussions and the valuable advices. In addition to the members of my thesis committee, I want to thank several others who took keen academic interest in this study, providing valuable suggestions, ideas and discussions that improved the quality of the research, including Nikos Andreakis, Jim Burnell, Dianne Brinkman, Bryan Wilson, Linda Blackall, Matt Kenway, Graham Burgess, Evan Goulden, Laurie Reilly, Davina Gordon and Jenny Elliman. Special thanks are due to Chaoshu Zeng, Jim Burnell and Rocky de Nys, who provided initial career advice and guided me to the School and Leigh Owens. My thanks also goes to Helene Marsh, Barbara Pannach, Jasper Taylor, Madeleine van Oppen, Michelle Heupel, Lauren Gregory, Trish Gorbal, Lorraine Henderson, Savita Francis, Ken Taylor and Paul Parker for their services, assistance and kindness with multiple situations that came along during the candidature. I wish to express my appreciation to Rochelle Soo, Louise Veivers, Emily Wright, Beth Ballment, Karen Juntunen, Grant Milton, Justin Hochen, Juli Knap, Katie Holroyd, Orachun vi Hayakijkosol, Anthony Baker, Rusaini, Kerry Claydon, Kjersti Krabsetsve, David Abrego, JB Raina, Francois Seneca and Helen Long, for their assistance and patience in the laboratories and for sharing their knowledge. Special thanks are due to Kellie Johns for her assistance with the English of my scholarship applications, reports and first manuscripts. Thanks also to Mark Collins and Noppadol Prasertsincharoen for his assistance with the final editing of this thesis; also to Kathy La Fauce, Paul Muir, Greg Smith, Matthew Salmon, Grant Milton and Pacific Reef (Ayr) for isolating and/or providing bacterial strains, experimental animals or DNA samples. Thanks to Andrew Negri, Eneour Puill-Stephan and Jairo Rivera for offering me opportunities of collaborating in other research projects and taking me on their exciting trips. I also thank Brenda Govan, Jenny Elliman and Leigh Owens for the job opportunities at the School of Veterinary and Biomedical Sciences and Medicine. I would like to express my heartiest thanks to Laura Castell and Hugh Sweatman, Rose and Anya Myers, Nikos and Gabriella Andreakis, Rochelle Soo and Brett Shearer, Jemma Mulligan, Maria Altamirano and all their family members for their kindness and affection, and for never letting me feel that I was away from my family and country. I also thank the rest of my friends in Townsville for all the fun we had in the last three years. I am most grateful to my loved friends and family in Spain for always keeping in contact and for the good moments during holidays. Very special gratitude goes to Javier Gómez for his understanding and for joining me in Australia for a more fun and happier experience. He greatly supported me during the course of this work and helped me more than anyone to concentrate on completing this dissertation. Finally, I would like to express my greatest gratitude to my parents who have always supported me in every possible way throughout my life. They were my inspiration and energy during these years away from home and I would not have enjoyed this opportunity without their endless support. I am sure they will be the happiest to read this thesis, which I owe and dedicate to them. vii ABSTRACT Vibrio harveyi and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry worldwide. The ornate spiny lobster, Panulirus ornatus, is a potential valuable candidate as an aquaculture species but V. harveyi-related disease outbreaks during the extended larval life cycle are major constraints for the development of a breeding program for the aquaculture of this species at a commercial level. Bacterial identification methods such as phenotypic tests and 16S ribosomal RNA gene analysis fail to discriminate species within the V. harveyi group because these are phenotypically and genetically nearly identical. Multilocus sequence analysis (MLSA) was used to identify 36 V. harveyi-like isolates from the larval rearing system of P. ornatus and to re-evaluate the identity of other important Australian pathogens. Strains DY05 and 47666-1, isolated in northern Queensland from dying larvae of P. ornatus and Penaeus monodon prawns, clustered together and apart from currently recognised species. Biochemical tests, DNA-DNA hybridization, MLSA and fatty acid analyses confirmed that the two strains represent a new species of the V.
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