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Ectoparasites and Associated Pathogens Affecting Farmed Salmon During Marine Grow out in Chile and Australia

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Ectoparasites and Associated Pathogens Affecting Farmed Salmon During Marine Grow out in Chile and Australia 1 ECTOPARASITES AND ASSOCIATED PATHOGENS AFFECTING FARMED SALMON DURING MARINE GROW OUT IN CHILE AND AUSTRALIA By Laura González Poblete Marine Biologist Master in Aquaculture Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Institute for Marine and Antarctic Studies University of Tasmania March 2015 2 STATEMENT OF ORIGINALITY I declare that 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. 11/3/2015 (signature) (date) STATEMENT OF ACCESS This thesis is not to be made available for loan or copying for two years following the date this statement was signed. Following that time the thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. 11/3/2015 (signature) (date) 3 STATEMENT OF ETHICAL CONDUCT The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government’s Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University. 11/3/2015 (signature) (date) 4 ACKNOWLEDGEMENTS A supervisory team consisting of Prof. Barbara F. Nowak, Dr. Philip Crosbie, Dr. Andrew Bridle (IMAS, Launceston, University of Tasmania), Dr. Marcelo Cortez-San Martín and Dr. Ana María Sandino (Laboratorio de Virología, Centro de Biotecnología Acuícola, Universidad de Santiago, Santiago, Chile) provided advice on planning, experimental design, statistical analyses, interpretation of results and assisted with editing my thesis. Prof. Nowak, Dr. Cortez- San Martín and Dr. Sandino organised financial support and provided the logistics of research activities. Likewise I thank Dr. Andrea Rivas (Biochemist, Universidad de Santiago, Chile), Dr. Carmen Morales (Oceanography, Universidad de Concepción, Chile) and English assistance from University of Tasmania (Lucy, Steve and Stephen) for their comments on earlier versions of the manuscript. I would like to thank the collaboration and assistance of Richard Miller and company staff (Huon Region, Tassal Group Limited, Australia) as well as Richard Taylor (CSIRO, CMAR, Hobart, Australia) for allowing me to visit the Tasmanian salmon farms to collect data and samples for Chapter 2. To Dr. Melanie Leef, Dr. Victoria Valdenegro and Steward Dick thank you for implementing the experimental infection system with AGD that allowed the sampling for Chapter 3 of this thesis. Particularly to Dr. Andrew Bridle thank you very much for making me feels welcome and teaching me about DNA, RNA and PCRs. Also thank you both you Mark Pollinski and Dr. Bridle for your scientific feedback, constant support with troubleshooting protocols and solving problems. Also I must thank Dr. Philip Crosbie for teaching me about AGD isolation, culture and experimental infection. I am grateful to the Chilean Salmon Grower Association “SalmonChile”, Humberto Bertín and Marco Rozas for facilitating the collection of samples and providing environmental data from the farms for chapters 4 and 5. The research was supported by Becas Chile, the Australian Society for Parasitology, University of Tasmania, and the Centro de Biotecnología Acuícola, Universidad de Santiago de Chile I also acknowledge the guidance of Dr. Luis Cottet and Biochemist Nicolás Sandoval, and the technical assistance of María Teresa Castillo (Universidad de Santiago, Chile) in ISAV studies. I sincerely thank the technical staff that assisted in processing the samples, Karine Cadoret and Steve Hindrum. To all my fellow HRD and PhD candidates thank very much for your words of encouragement and ideas, Jacky Elizabeth, Sandra, Nicole, Dan, Victoria, Catarina, Megan, Deb, 5 Kaeden, Bikram, Thu, Rebecca, Milla, Diana, Melissa, Mean, Paul, Lucas, Ylenia, Gian Luca, Yuen Yue, Cris, Simon and Digory. Thank you very much to my mentors Juan Carvajal and Mario George-Nascimento, to my friends and family for supporting me whilst I was away Lucía, Emilia, Exequiel, Damon, Mago and Anita, Leonardo y Digna, Marianne, Irene, Quena Poblete, Chabe, Angelica, Marcela, Francine, Rose Marie, Ale, Margarita, Erika, Javier, Jano and Orietta Poblete, María Eugenia, Rafael, Pablo, Rebeca, Juan, Jorge, Miriam and Ricardo Otaiza. Finally thank you to my children Tomás and Mariana for always being with me. 6 Table of Contents STATEMENT OF ORIGINALITY ..............................................................................................................2 STATEMENT OF ACCESS .........................................................................................................................2 STATEMENT OF ETHICAL CONDUCT ..................................................................................................3 ACKNOWLEDGEMENTS ..........................................................................................................................4 List of Figures ...............................................................................................................................................9 List of Tables ............................................................................................................................................. 11 ABSTRACT ............................................................................................................................................... 12 CHAPTER 1 .............................................................................................................................................. 14 GENERAL INTRODUCTION .................................................................................................................. 14 1.1 Impact of diseases on salmon aquaculture in Chile compared to Australia ................................... 18 1.2. Caligus rogercresseyi and control strategies ................................................................................. 21 1.3. Piscirickettsiosis ............................................................................................................................. 23 1.4. Infectious salmon anaemia (ISA) ................................................................................................... 25 1.5. Aim of the thesis ............................................................................................................................ 26 CHAPTER 2 GILL ISOPOD Ceratothoa banksii AS A POSSIBLE RESERVOIR OF Neoparamoeba perurans AND AS A POTENTIAL PROBLEM FOR FARMED ATLANTIC SALMON Salmo salar IN AUSTRALIA ............................................................................................................................................. 27 2.1. INTRODUCTION ........................................................................................................................ 27 2.2. MATERIAL AND METHODS ..................................................................................................... 28 2.2.1 Fish sampling ......................................................................................................................... 28 2.2.2. Nucleic acid extraction ........................................................................................................... 31 2.2.3. Quantitative real-time PCR (qPCR) assay for Neoparamoeba perurans............................... 31 2.2.4. Statistical analysis .................................................................................................................. 33 2.3. RESULTS ...................................................................................................................................... 33 2.3.1. Isopods on salmon ................................................................................................................. 33 2.3.2. Neoparamoeba perurans in gill isopods ............................................................................... 35 2.4. DISCUSSION ................................................................................................................................ 37 CHAPTER 3 SPATIAL AND TEMPORAL DISTRIBUTION OF Neoparamoeba perurans IN A TANK RECIRCULATION SYSTEM DURING EXPERIMENTAL AGD CHALLENGE ................................ 42 3.1. INTRODUCTION ......................................................................................................................... 42 3.2. MATERIAL AND METHODS ..................................................................................................... 43 3.2.1. Experimental recirculation systems and inoculation with amoebae ...................................... 43 3.2.2. Collection samples and processing ........................................................................................ 44 7 3.2.3. Sample analyses ..................................................................................................................... 47 3.2.4. Quantitative real-time PCR evaluation for N. perurans ......................................................... 48 3.2.5. Quantification of 18S rRNA gene copy numbers of N. perurans in samples ........................ 48 3.2.6. Statistical analysis .................................................................................................................
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