Final Report ATLANTIC SALMON AQUACULTURE SUBPROGRAM: COMMERCIAL AGD AND SALMON HEALTH Mark D. Powell, Joy A. Becker, J.A., Julie Ransome, Renee L. Florent, and Matthew Jones December 2007 Aquafin CRC Project 3.4.1(2) (FRDC Project No. 2004/213) Atlantic Salmon Aquaculture Subprogram: Commercial AGD and Salmon Health Mark D. Powell, Joy A. Becker, J.A., Julie Ransome, Renee L. Florent, and Matthew Jones. ISBN 978-1-86295-378-9 © University of Tasmania, Fisheries R&D Corporation, Aquafin CRC This work is copyright. Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owners. Neither may information be stored electronically in any form whatsoever without such permission. Every attempt has been made to provide accurate information in this document. However, no liability attaches to Aquafin CRC, its Participant organisations or any other organisation or individual concerned with the supply of information or preparation of this document for any consequences of using the information contained in the document. Published by The University of Tasmania, Launceston, 2007. ATLANTIC SALMON AQUACULTURE SUBPROGRAM: COMMERCIAL AGD AND SALMON HEALTH Mark D. Powell, Joy A. Becker, J.A., Julie Ransome, Renee L. Florent, and Matthew Jones December 2007 Aquafin CRC Project 3.4.1(2) (FRDC Project No. 2004/213) Aquafin CRC is established and supported under the Australian Government’s Cooperative Research Centres Program 1 Table of Contents Table of contents 2 Non-technical summary 3 Acknowledgments 5 Background 6 Need 9 Objectives 10 Methods 11 Lab infection and maintenance of AGD affected fish 11 Research approach 11 In vitro assessment of candidate amoebicides 13 Laboratory investigation of potential efficacy of N-acetyl cysteine 15 Laboratory assessment of the potential efficacy of bithionol 16 Laboratory investigation of the potential efficacy of salinomycin, lasalocid acid and maduramycin ionophores 18 Laboratory investigation of the potential efficacy of bithionol sulphoxide or garlic powder 21 Semi-commercial experimental assessment of the efficacy of AquaciteTM and BetabecTM. 23 Metabolic cost of gill disease 25 Results and Discussion 30 In vitro assessment of candidate amoebicides 30 Laboratory investigation of potential efficacy of N-acetyl cysteine 40 Laboratory assessment of the potential efficacy of bithionol 40 Laboratory investigation of the potential efficacy of salinomycin, lasalocid acid and maduramycin ionophores 44 Laboratory investigation of the potential efficacy of bithionol sulphoxide or garlic powder 51 Semi-commercial experimental assessment of the efficacy of AquaciteTM and BetabecTM. 53 Metabolic cost of gill disease 57 An adverse reaction to chloramine-T bathing 60 Benefits and adoption 61 Further development 62 Planned outcomes 63 Conclusion 64 References 65 Appendix 1: Intellectual property 73 Appendix 2: Staff 73 Appendix 3: Publications and presentations from this project (at time of going to press) 74 2 2004/213 Atlantic salmon aquaculture subprogram: Commercial AGD and Salmon Health PRINCIPAL INVESTIGATOR Dr M. D. Powell ADDRESS: School of Aquaculture University of Tasmania Locked Bag 1370 Launceston TAS 7250 Telephone 03 6324 3813 Fax 03 6324 3804 OBJECTIVES 1. To undertake commercial scale investigations into the potential use of seawater bath treatments (eg chloramine-T, artificially softened freshwater or hydrogen peroxide) as a strategy for AGD control. 2. To investigate the efficacy of in feed treatments such as: Parasiticides (eg, bithionol), Nutritional supplements (eg AquaciteTM and BetabecTM), Mucolytic agents (eg L cysteine ethyl ester) 3. To test new and novel anti-parasitic compounds for potential use in bath or in- feed treatments for AGD. 3 NON-TECHNICAL SUMMARY OUTCOMES ACHIEVED There have been commercial trials by the industry for the adoption of some of the results from this project, including the use of chloramine-T as a bathing additive in seawater and the inclusion in the feed of Aquacite and Betabec. While these treatments have been tested under commercial conditions, the results have not been sufficiently successful to warrant commercial development of these products at this stage. The research on in-feed additives such as bithionol as an amoebicidal treatment for AGD suggest that this remains an option for further research and development for the aquaculture industry in Tasmania. The determination of a metabolic cost for AGD provides a quantitative measure of the impact of disease management on the performance of the fish, suggesting new approaches to the development of health management strategies. Two PhD candidates and a post-doctoral fellow were involved and trained as a part of this project. Prior to this project there had been investigations into some potential candidate amoebicides, with little success except for the possibility of oxidative disinfectants such as chloramine-T. This project has since tested a number of amoebicides using a progressive approach of in vitro toxicity, in vivo efficacy in the laboratory through to in vivo efficacy under field conditions in either semi-commercial or under full commercialised field trials. Although the practical delivery of some of these as treatments of amoebic gill disease (AGD), such as chloramine-T bathing, appear not to be practicable, other avenues may have potential for further commercial development, such as the dietary inclusion of potential amoebicidal compounds, including bithionol and ionophore-based amoebicides. The project has explored the potential of bithionol, a registered 4 amoebicidal drug, as an in-feed treatment, showing that AGD severity can be reduced by approximately 50%. Similarly, the project has examined the efficacy of an immunostimulant-based feed additive, Aquacite and Betabec which reduced mortality in Atlantic salmon with AGD but did not affect the intensity of infection. This project has further characterised the effects of gill disease, in particular AGD, with respect to the metabolic cost of disease to the fish. This work has estimated that in excess of 17% of the ingested energy is likely to go to service the cost of AGD. This approach provides a useful tool to incorporate into bioeconomic models for assessing the efficacy of AGD treatments in the future. KEYWORDS: Amoebic gill disease, Atlantic salmon, disease treatment Acknowledgments We are most thankful to the following scientific collaborators without whose involvement this project could not have been completed: Dr Huub Bowers, Dr Rahim Peyghan, Professor Robert Raison, Professor Kevin Broady, Dr. Margarita Villavedra, Raymond Duijf, Christine Paetzold, Megan Barney, Scott Schilg, Heather Mlynarski, Jan Lovy, Dr Rick Butler and Dr Peter Thompson. Some of you also have contributed above and beyond the call of duty and provided hours of enjoyable discussion, usually over a beer or two, to keep the research team sane, focussed and productive – thank you. We are also thankful to Jeremy Lancaster, Jason Stalker, Mark Asman from Tassal Pty Ltd and Dr Steve Percival and Richard Taylor (formerly of Tassal Pty Ltd) for their individual and outstanding contributions to the project. Similarly, we are thankful to David Mitchell and Dr Dominic O’Brien of Huon Aquaculture Company for their input and ideas and excellent and lively discussions of AGD. In addition we would like to acknowledge the assistance offered by James Mackie (James Mackie Agricultural Ltd), Jean de Barbeyrac (Axentive), Sam Ludbrook (Mulitchem) and Oxbiopharm Pty Ltd for their advice or supply of compounds for investigation. Also we would like to thank Pheroze Jungalwalla (Tasmanian Salmon Growers Association), Greg Dowson and Warren Jones (DPIW environment management) and Dr Roger Meiske (Australian Pesticides and Veterinary Medicines Authority) for their insightful and valuable advice and discussion. Additional funding for parts of this work were provided by the CASS foundation and a Natural Sciences and Engineering Research Council Post-doctoral fellowship to Dr. Joy Becker. 5 Background The parasitic gill amoeba Neoparamoeba spp. is believed to be the primary causative agent of amoebic gill disease of cultured Atlantic salmon in the south-east of Tasmania. The amoeba attaches to the gills of susceptible salmon and the response to the irritation by the amoeba is an acute multifocal hyperplasia of the gill filamental epithelium. This response, coupled with a mucous cell hyperplasia, results in an impediment to carbon dioxide excretion (Powell et al. 2000). Also associated with AGD is an acute vascular hypertension (Powell et al 2002), caused in part by an increase in systemic vascular resistance leading to reduced cardiac output (Leef et al. 2005). The treatment of choice for AGD is currently a 2-4 h freshwater bath. However, it has been shown that the concentrations of Ca2+ and Mg2+ in the freshwater affect the ability of the amoeba to survive the freshwater bath (Powell and Clark 2003) and that bathing fish in artificially softened water improves the efficacy of the freshwater bath (Roberts and Powell 2003). Furthermore, research has shown that chloramine-T, a disinfectant used for the treatment of bacterial and other parasitic gill diseases in aquaculture, is effective at reducing amoeba numbers on the gills of AGD-affected salmon at a treatment concentration of 10 mg L-1 in seawater (Harris et al. 2004; 2005). Preliminary investigations