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Diagnostic Investigation Into Summer Mortality Events of Farmed Chinook Salmon (Oncorhynchus Tshawytscha) in New Zealand

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Diagnostic Investigation Into Summer Mortality Events of Farmed Chinook Salmon (Oncorhynchus Tshawytscha) in New Zealand Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Diagnostic investigation into summer mortality events of farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand. A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science Massey University, Manawatū, New Zealand. Cara Brosnahan 2020 1 Abstract Salmon farming is the second highest value aquaculture species in New Zealand and produces approximately 88% of the global market of farmed Chinook salmon, Oncorhynchus tshawytscha (Tucker, 2014). New Zealand salmon are free of many significant diseases affecting salmonids globally (Diggles, 2016). Therefore, disease is one of the greatest threats to this New Zealand aquaculture species. Biosecurity, early detection, and characterisation of new or emerging diseases is vital for management and sustainability of the aquaculture industry. Elevated mortalities termed ‘summer mortalities’ with no cause identified have occurred in certain farmed Chinook salmon populations in the Marlborough Sounds since 2012. This study identified two potential bacterial pathogens involved in summer mortalities; New Zealand rickettsia-like organism (NZ-RLO) and Tenacibaculum maritimum. Distribution of NZ-RLO and T. maritimum within farmed Chinook salmon populations, phylogenetic analysis of these pathogens and the pathogenicity of two strains of NZ-RLO were assessed to provide an understanding of the role of NZ-RLO and T. maritimum in summer mortalities. Additionally, new diagnostic tests were developed to efficiently detect these pathogens. Identification of NZ-RLO in the summer mortalities was the first detection in New Zealand. Tenacibaculum maritimum had been reported in New Zealand previously, however it had not been associated with mortalities. This study confirmed three strains of NZ-RLO with restricted geographical distribution. Two strains of NZ-RLO were found exclusively in areas where fish experienced summer mortalities and were associated with clinical signs of disease, indicating certain strains of NZ-RLO were likely primary pathogens. Widespread distribution of T. maritimum was detected within farmed salmon and no association was found with T. maritimum and clinical signs of disease in areas experiencing summer mortalities, indicating T. maritimum was unlikely to be a primary pathogen. This study proves that laboratory exposure of salmon to two strains of NZ-RLO caused disease and mortalities however, the differences between the two strains suggest NZ- 2 RLO2 may be more pathogenic. This study suggests NZ-RLOs are likely to be involved in summer mortalities as primary pathogens however, the interaction between the pathogens and environment is likely to have amplified the levels of mortalities during these events. 3 Acknowledgements The work presented in this thesis was performed at the Animal Health Laboratory, Wallaceville, New Zealand from 2015 to 2019 under the supervision of Professors John Munday, Brian Jones, and Peter Davie. I could not have completed this thesis without the help of many people for whom I am very grateful. I would like to thank my supervisors Brian Jones, John Munday, and Peter Davie. Brian, I am grateful for the encouragement you gave me to start this thesis and for your guidance, discussions, anecdotes, and mentoring throughout. Your input has been crucial to the result. John, thank you for your support as well as the in- depth reviews of manuscripts and chapters, the skills from which I will take throughout my career. Peter, your expert knowledge on fish physiology, biology, and handling was crucial in achieving the results from my challenge trials. The work carried out during this thesis would not have been possible without many wonderful colleagues at the AHL. Special thanks goes to Wendy McDonald, Henry Lane, Hye Jeong Ha, Della Orr, Yen Yen Yuen, Katie Booth, Courtenay O’Sullivan, David Burr, Edna Gias, Joanne Howells, Laura Kennedy, and Taryrn Haydon for your advice, people-power, and support during this project. Also, a big thanks to Eugene Georgiades, Claire McDonald, and Suzanne Keeling particularly for your help in Chapter 8 but also throughout the project. I would also like to thank the following people for their technical help: Jordan Taylor from the Manawatū microscopy and imaging centre for introducing me to electron microscopy and analysing my samples, Duncan Colquhoun from the Norwegian Veterinary Institute for providing the Piscirickettsia salmonis infected tissue slides for the in-situ hybridization protocol, Mark Preece, Stuart Barnes, and Cesar Lopez from New Zealand King Salmon for providing fish and expertise for the challenge trials, and Naomi Cogger at Massey University for her valuable help with the experimental design of the challenge trials. 4 To my wonderful family and friends, thank you for all the support and interest you have offered me (and the salmon) over the past four years. Finally, and most importantly, the biggest thanks goes to my amazing husband for his endless love and support without question, for being my sounding board, sense checker, editor, and for always making me laugh. I couldn’t have done this without you and I am so grateful to have you in my life every day. Cara Lee Brosnahan New Zealand, 2020. 5 Table of Contents Abstract ............................................................................................................................... 2 Acknowledgements ............................................................................................................ 4 Table of Contents ................................................................................................................ 6 List of Figures ................................................................................................................... 11 List of Tables ..................................................................................................................... 14 Chapter 1 : General Introduction ..................................................................................... 16 1.1 Global seafood production ......................................................................................... 16 1.2 New Zealand seafood sector ..................................................................................... 17 1.3 History of salmonids in New Zealand ......................................................................... 17 1.4 New Zealand Chinook salmon aquaculture ................................................................ 22 1.4.1 Chinook salmon life-cycle in New Zealand – natural and farmed ......................... 25 1.4.2 Diseases status of Chinook salmon in New Zealand ........................................... 25 1.5 Piscirickettsia salmonis and NZ-RLO ......................................................................... 30 1.5.1 Detection of Piscirickettsia salmonis .................................................................... 32 1.6 Tenacibaculum maritimum ......................................................................................... 33 1.6.1 Detection of Tenacibaculum maritimum .............................................................. 34 1.7 Teleost Immune system ............................................................................................. 35 1.7.1 Innate immune system ........................................................................................ 36 1.7.1.1 Skin and mucus ............................................................................................ 36 1.7.1.2 Cellular ......................................................................................................... 37 1.7.1.3 Humoral ........................................................................................................ 37 1.7.2 Acquired immune system .................................................................................... 39 1.7.2.1 Cellular ......................................................................................................... 39 1.7.2.2 Humoral ........................................................................................................ 40 1.8 Conclusion ................................................................................................................. 44 Chapter 2 : First report of a rickettsia-like organism from farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), in New Zealand .............................................. 46 2.1 Introduction ................................................................................................................ 46 2.2 Materials and methods .............................................................................................. 48 2.2.1 Post mortem examination and sample collection. ................................................ 48 2.2.2 Histopathology .................................................................................................... 49 2.2.3 Bacteriology ........................................................................................................ 49 2.2.4 DNA extraction .................................................................................................... 50 2.2.5 PCR ...................................................................................................................
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