Final Report

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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

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

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

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.

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

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.

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 into the use of chloramine-T in seawater suggested that a 1 h bath of chloramine-T (10 mg.L-1) is as effective as a 1 h freshwater bath at reducing gill amoeba numbers, even though the resolution of gill lesions was more rapid in freshwater-treated fish (Harris et al. 2004; 2005). Nevertheless, a 1 h seawater treatment could realise a significant cost benefit to the aquaculture industry particularly in areas where freshwater resources may be limited and open opportunities for alternative use pattern strategies for gill parasite control. Alternative oxidative treatment compounds that work similarly to chloramine-T, such as hydrogen peroxide, have also been examined, although the toxicity of such treatments especially to fish with gills already compromised due to disease make these approaches impracticable (Powell et al. 2005).

Investigations into other AGD treatments have indicated possible agents that could be used either as disinfectants or in-feed treatments. Neoparamoeba spp. appears to be highly resilient with regards to its susceptibility to potential amoebicidal treatments. Powell et al. (2005) report the testing of a range of compounds of which the drug bithionol appeared to have some potential in in vitro tests (Powell et al 2003). Bithionol has low solubility, appears to be effective at killing Neoparamoeba spp and is registered for use in food animals, humans and even some fish species ((Bacq, et al., 1991; Enzie and Colglazier, 1960; Kim and Choi, 1998; Madsen, et al., 2000; McChesney, et al., 1961; Prasittirat, et al., 1997; Tojo, et al., 1994). As an antiprotozoal agent, bithionol works through a number of potential mechanisms including stimulating lactic acid production, inhibiting oxygen consumption and decreasing glycolytic and oxidative metabolism in the human lung fluke, Paragonimus westermani, in vitro (Hamajima, 1973). Furthermore, it was postulated to be linked to a variety of processes, including the inhibition of reduced nicotinamide adenine dinucleotide (NADH)-fumarate reductase, and to be involved in protein phosphorylation (Reid, et al., 2001). Bithionol has also been found to inhibit endogenous and 2-propanol-supported respiration (Takeuchi, et al., 1984) and is known to be a metabolic disruptor, uncoupling electron transport in fish ciliates including Tetrahymena pyriformis (Griffin, 1989) and Trichodina jadranica (Madsen, et al., 2000). Takeuchi et al. (1984) postulated that bithionol functioned as an uncoupler of mitochondrial oxidative phosphorylation in the trophozoite stage of the Entamoeba histolytica, which is the main amoeba pathogenic to humans. In contrast to mammalian therapeutics, the use of chemotherapeutic agents in fish, particularly antiparasitic drugs, is limited (Athanassopoulou et al. 2004a). Moreover, due to the limited efficacy of freshwater or chemical bathing as a treatment for AGD, there is a real need to identify efficacious in-feed chemotherapeutics. The major advantage of in-feed therapies is that before reaching their target, they are not compromised by environmental conditions (i.e. water solubility, activity diminished by organic loading, pH, O2 etc) to the same degree as bath treatments, they can have a longer window of activity, are less stressful to administer and the by-products generated by the host tissues can in some cases be more efficacious than the parent compound (Shinn et al. 2003). However, in-feed therapies are dependent on the fish host being hungry enough to ingest the medicated feed. More recently, members of the ionophore family of drugs, including monensin, salinomycin and others, have

been investigated with regards to their potential development as an in-feed treatment for AGD.

Ionophores are lipophilic compounds, usually synthesized by microorganisms, that are toxic to many bacteria, protozoa, fungi, and higher organisms (Russell and Strobel, 1989). The toxicity of these compounds arises from their capacity to penetrate into biological membranes and subsequently alter the flux of ions from and into the cell (Ipharraguerre and Clark 2003). An ionophore can interfere with the flux of ions either by forming cyclic-ion-ionophore complexes that function as ion-selective mobile carriers (e.g. monensin) or by creating pores that promote less specific influx and efflux of ions (Russell and Strobel 1989). During an ionophore exposure in bacteria, as the ion fluxes progress, Na/K and H+ ATPase systems of the bacteria cells increase significantly in order to maintain an ion balance and intracellular pH (Ipharraguerre and Clark 2003, Russell and Strobel 1989). As a result, there is a large energy expenditure for maintenance functions and this use of energy for non-growth purposes persists to the point where bacterial cells are virtually depleted of ATP, compromising their capacity to grow and reproduce (Bergen and Bates, 1984). The severity of these detrimental effects is dictated by the sensitivity of the pathogen to ionophores, which in turn has been shown to depend on the permeability of their membranes to macromolecules (Russell and Strobel 1989).

Ionophores, (in particular monensin and lasalocid acid) are commonly used in the poultry industry as coccidiostats to control Eimeria infections and in the bovine industry to improve ruminant fermentation (Russell and Strobel 1989). To date, the use of ionophores in aquaculture has been mainly limited to experimental treatments, and there are several advances of note. Monensin, at an inclusion dose of 1000 ppm and a daily feeding rate of 2% of fish biomass, was effective at reducing the formation of branchial xenomas during an experimental infection with the protozoan parasite, Loma salmonae (Becker et al. 2002). Monensin was the first compound to significantly reduce or block the inflammation-inducing development of xenomas during this parasitemia. Additionally, experimental combination salinomycin therapy has been effective at treating natural and induced myxosporean infections in cultured sharpsnout sea bream (Diplodus puntazzo) and sea bream (Sparus aurata) (Athanassopoulou et al. 2004a, 2004b, Karagouni et al. 2005). Oral administration of

salinomycin in the tapir fish, Gnathonemus petersii, naturally infected with Henneguya sp., a gill parasite, caused irreversible damage to plasmodial developmental stages of the parasite (Dohle et al., 2002). More recently, preliminary results have indicated that both lasalocid and maduramycin in-feed treatments were effective at preventing an experimental coccidial infection with Goussia carpelli in the common carp (Cyprinus carpio) (Molnar and Ostoros 2007).

Another approach to the management of AGD is to maintain fish growth and production despite infestation with amoebae. This approach focuses upon optimising fish health rather than on killing the parasite. Two commercially available β-glucan- based compounds (AquaciteTM and BetabecTM) have been identified as having potential to maintain fish growth and feeding during a Neoparamoeba spp. challenge in the laboratory, despite the presence of AGD (Powell et al. 2005; Mlynarski et al. submitted). Both Aquacite and Betabec have been used successfully in maintaining fish production and were associated with reduced mortality in Red sea bream (Diplodus punctatus), Atlantic cod (Gadus morhua) infected with Loma morhua, and Atlantic salmon and rainbow trout infected with Aeromonas salmonicida or Ichthyophirius multifiliis (J.Mackie pers. comm.)

Need There is an urgent need to develop and commercialise treatments for the control of amoebic gill disease in the Atlantic salmon aquaculture industry in Tasmania. The cost-benefit analysis undertaken in 2000 by the Aquafin CRC suggested a net present value of economic benefit of $21.6M AUD and a benefit/cost ratio of 5.3. The need for short term (even interim) solutions for the control of AGD is paramount.

This project complements and continues the advances made by previous research (FRDC 2000/266 and 2001/205) that identified potential treatments. The project has investigated the commercial feasibility of treatments previously identified, while providing an opportunity for examining potential new AGD treatments, on behalf of the salmon aquaculture industry. Industry representatives, in a meeting on January 15, 2004 at Marine Research Laboratories, Taroona TAS, iterated the need for a

flexible program that would allow potential treatments and control measures to be tested in the laboratory as well as in the field. This project (within budgetary constraints) was developed to provide that flexibility with clearly identified treatments to be investigated in the first instance. The road to commercialisation of any disease control treatment is a long and complex one, and this project was designed to provide the information that might lead to a change of commercial practice in the control of AGD.

This program for the development and commercialisation of disease treatments was grounded in the identification of potential treatments to be tested both in vitro and in vivo in the laboratory. Once suitable laboratory testing was complete, then small- scale field trials were required and finally the scaling up of trials for commercialisation.

Objectives

2. To investigate the efficacy of in feed treatments such as: Parasiciticides (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.

Methods This project was designed to provide the opportunity to test potential candidate treatments firstly using an in vitro toxicity assay, then if successful, in a tank challenge system. If treatments show potential for success from laboratory studies, then short–term cage trials on farms (using replicated experimental cages and 1 AGD- treatment cycle) are followed by replicated experimental cage repeated treatment trials, and finally by full scale replicated commercial trials.

Laboratory infection and maintenance of AGD affected fish It was essential for this project that a reliable supply of the same source of infective Neoparamoeba spp. was available for challenge experiments. The source of amoebae for all laboratory experiments was a live infection maintained by co-habitation at the School of Aquaculture, University of Tasmania. An active Neoparamoeba spp. infection has been maintained by continuous passage since February 2001. This laboratory infection was determined to include Neoparamoeba pemaquidensis and Neoparamoeba branchiphila using immunohistochemistry with polyclonal antibodies to the PA027 and morphology (Dyková et al. 2005). Naïve salmon (approximately 100-300g) were acclimated to full strength seawater over a 7-14 day period and then transferred at intervals into the tank to maintain the infection. Moribund fish were periodically removed and the amoebae harvested from the gills and used to inoculate experimental challenges as described by Morrison et al. (2004) or used for in vitro toxicity assays as described by Powell et al. (2003). More recently, it has been shown that the amoeba closely associated with the lesions is a newly-described species, Neoparamoeba perurans, which is distinguishable genetically but not morphologically, and that this species is present in the amoeba population harvested as above (Young, N., Adams, M., Crosbie, P., Nowak, B., Morrison, R, International Journal for Parasitology (in press)

Research approach Laboratory testing of potential AGD control agents The in vitro toxicity of potential compounds to Neoparamoeba spp. was assayed according to Powell et al. (2003). In brief, isolated amoebae were incubated in 96 well microtitre plates in seawater containing the chemical treatment under

investigation for up to 4 h (short term assay) as potential bath disinfectants, or for up to 7 days as potential in-feed treatment compounds. A total of at least 8 replicates was tested over 2 assays. Viable amoeba numbers were determined using a haemocytometer, where amoeba suspensions were stained with 0.5 % trypan blue. Stained cells represented cells that could no longer exclude the dye. Only unstained, viable, amoebae were counted. Controls always consisted of seawater only, de- ionised freshwater (positive control), and, if required, an alumina control in seawater to account for insoluble compounds exerting a surface effect on the amoebae (Powell et al. 2003).

Standardized laboratory in-feed treatment Seawater-acclimated Atlantic salmon smolt were held in one of three replicated modules containing 3 tanks each (n = 3 per treatment for 3 treatments). Fish were fed, either to satiation or 1% body weight per day, on a commercial pelleted diet that was top-dressed with the treatment being tested. Top dressing of feed consisted of mixing the compound of interest with moistened pelleted feed and coating the mixture with fish oil. Control fish received a coated (top dressed) pellet but no treatment; oil only). Fish were fed the medicated or control diet for up to 14 days prior to infection (depending upon experiment see below). After the specified pre-feed period, each tank module was inoculated with 250-300 Neoparamoeba spp. cells.L-1 (see below). Fish (2-5 per tank, n=6-15 per treatment; see below) were sampled at day 0 and then at specified periods during the Neoparamoeba spp. exposure. The progression of gill disease was determined histologically for the development of AGD gill lesions, and the proportion of AGD lesioned filaments was determined.

Assessment of field treatment efficacy The goals of this research are ultimately the development of treatments suitable for commercialisation. In order to ensure that field trial research did not pursue research “dead ends”, field trials were progressive and undertaken only when industry partners were willing to commit to the research. Experimental repeated-treatment trials were conducted whereby fish were treated in replicate experimental cages over an extended period (14 weeks) and ideally 3 treatment cycles, to assess a reduction in the time-to- next-bath, although this was not always possible given the timing of potential field trials and the resources committed by industry partners. Finally where treatments

were considered sufficiently promising to be tested on commercial-sized cages over at least 3 treatment cycles, industry partners undertook such treatments with the support of the project team. To assess the efficacy of bathing treatments a number of indicators were used. The gross gill score is the method favoured by farms for determining AGD severity and time to bathe (Powell et al. 2005); nevertheless gill histology remains the gold standard for determining AGD severity. Gills from AGD affected fish were fixed in seawater Davidson’s solution for 24 h, then transferred to 70% ethanol, paraffin embedded, sectioned at 5 μm, and stained with haematoxylin and eosin. Histology offers the advantage of determining the presence of amoebae associated with gill lesions but also provides an indication of the severity of disease based upon the number of lesioned filaments per fish.

Assessment of AGD severity In both types of trials (laboratory and field), excised gill baskets were fixed in seawater Davidson’s solution for 24 h and transferred into 70% ethanol. The second left gill arch was removed, dehydrated in a series of ethanol concentrations, embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin using standard methods. Gill sections were examined with a light microscope (Olympus BH2, Hamburg, Germany) at x100 to x400 magnification.

For both types of trial, the number of gill filaments with AGD lesions (Kent et al. 1988; Munday et al. 1990) was counted and expressed as the proportion of the total number of filaments in each section (Parsons et al., 2001b). A filament was counted only when the central venous sinus was visible in at least two-thirds of the filament and lamellae were of equal length bilaterally present to near the tip of the filament (Speare et al., 1997).

In vitro assessment of candidate amoebicides The assessment of potential therapeutic treatments has focussed upon the identification of chemicals and treatments that were either registered for use in other applications (eg: chicken industry) or known biocides of general application as disinfectants. In this latter case, the treatments were tested to ensure that even though treatment of AGD-affected salmon might not be a practicable option, the information

gained would at least highlight the value of these compounds as biocides for maintaining biosecurity with regard to Neoparamoeba spp.

The toxicity of the potential therapeutic treatment to isolated wild-type (or cultured) Neoparamoeba spp. was determined using the toxicity assay developed by Powell et al. (2003), modified slightly to facilitate assessment of toxicity to Neoparamoeba spp. over a short term exposure (3 hours) as an alternative to the 96 h exposure of the original assay (Powell et al. 2003).

Table 1. Potential amoebicides tested for toxicity against isolated Neoparamoeba spp.

Compound Active ingredient Concentration Acute (3h) 96h testing testing Biocides PycezeTM Bronopol 0, 1, 10, 100 mg L-1 Samaki BiocideTMBronopol 0, 1, 10, 100 mg L-1 Acticide DDQTM Formalin 0, 1, 10, 100 mg L-1 Acticide F(N)TM Formalin 0, 1, 10, 100 mg L-1 VirkonTM Potassium 0, 0.1, 10 mg L-1 a peroxymonosulphate 0, 25, 50, 100 mg L-1 b Ionophores Monensin Monensin 0, 0.1, 1, 10 mg L-1 Bio-CoxTM Salinomycin 0, 0.1, 1, 10 mg L-1 BovatecTM Lasalocid acid 0, 0.1, 1, 10 mg L-1 CygroTM Maduramycin 0, 0.1, 1, 10 mg L-1

Metabolic inhibitors Metronidazole Metronidazole 0, 0.1, 1, 10, 50, 100 mg L-1 a tested at 1, 4, 24 and 48 h exposure b tested at 1 and 2 h exposure

The mean efficacy of a given candidate compound was determined by assessing the reduction in viable amoeba numbers relative to a seawater control. The mean percent survival of amoebae was statistically assessed using a two factor analysis of variance with treatment and exposure duration as factors after checking for normality and homoscedasticity of the data from residual plots. Where the data were not considered

normal nor could equal variance be assumed, log or square root transforms were conducted to maintain the assumptions of the analysis of variance.

Laboratory investigation of the potential efficacy of N-acetyl cysteine N-acetyl cysteine is an analogue of L-cysteine ethyl ester. The latter has been previously demonstrated to show some protection against amoebic gill disease (Roberts 2005; Roberts and Powell 2005). N-acetyl cysteine is the active ingredient of many commercially-available mucolytic drugs (Schrier et al. 2002) whereas LCEE is not commonly available. Mucolytic drugs work by having free sulfhydryl groups capable of reducing intermolecular disulfide bonds in mucin, thereby disrupting the mucus glycoprotein polymer and reducing mucus viscosity (Puchelle et al. 1980; Majima et al. 1990; Tomkiewicz et al. 1995; Schrier et al. 2002).

It has been previously demonstrated that although both Atlantic salmon and rainbow trout are susceptible to AGD, the relative viscosity of their cutaneous mucus is different, with rainbow trout having an inherently lower mucus viscosity than that of Atlantic salmon even in seawater (Roberts and Powell 2005). As a consequence, assessment of treatments that may affect the viscosity of the mucous coat and thus susceptibility of the fish to AGD should take into account this species difference. This species comparison was undertaken in the context of the in vitro effects of N- acetyl cysteine addition to mucus (Powell et al 2007 in press). In addition, the effects of feeding N-acetyl cysteine to rainbow trout and Atlantic salmon prior to infection with Neoparamoeba spp. and subsequent AGD were also studied (Powell et al. 2007 in press). In brief, salt water acclimated rainbow trout (mean mass ± SEM of 124.4 g ± 3.5 g) and Atlantic salmon smolts (mean mass ± SEM of 78.5 g ± 19.1 g) were fed diets supplemented by top-dressing N-acetyl cysteine in fish oil at 8 g NAC kg-1 feed. Fish were fed to satiation for 4 or 2 days (rainbow trout) or 5 or 3 days (Atlantic salmon) prior to challenge with Neoparamoeba spp. and continued to be fed with the medicated feed throughout the subsequent infection. NAC intake for rainbow trout was 0.039-0.038 g NAC kg-1 fish d-1 and for Atlantic salmon 0.079-0.075 g NAC kg-1 fish d-1. The severity of the resultant AGD was assessed histologically as described above.

Laboratory investigation of the potential efficacy of bithionol Bath toxicity and efficacy In order to examine initial in vivo toxicity and efficacy a bath administration of bithionol was used. Briefly, both fresh- and seawater Atlantic salmon Salmo salar (L.) and rainbow trout Oncorhynchus mykiss were bathed for a maximum 3 h bath duration at bithionol concentrations ranging from 0-35 mg L-1. Baths were prepared following the method described in Florent et al., (2007) and time to morbidity, chloride, Na+/K+ ATPase and succinic dehydrogenase (SDH) activity in plasma and gill along with histopathology of gill, liver and kidney were examined.

Bithionol palatability Following initial toxicity and efficacy studies, palatability of bithionol was examined using four different diets at a bithionol concentration of 25 mg kg feed-1. Atlantic salmon, Salmo salar L. of 64.30g (± 1.50g) and 20.20cm (± 0.20cm) (n=20), were acclimatised to seawater (35‰) over a 14 day period after which they were randomly allocated into four 400L recirculation systems consisting of a tank and a sump. Fish were housed for one week to allow acclimatizing to the system conditions of 12L:12D, 16.6°C (± 0.1°C), 34.6‰ (± 0.1‰), DO 98.3%sat (± 0.2%sat), ph 8.1 and total ammonia of 0.4mgL-1. Following this, systems were randomly allocated one of the following top coated diets: control; bithionol and gelatin; bithionol and fish oil combined, and bithionol; fish oil and gelatin.

Fish were fed to satiation twice daily at 0900hr and 1600hr for 14 days, with feed intake, water quality and mortality data recorded daily. Following the 14 days, diets were re-allocated to different tanks and the process repeated in order to assess if there was a tank difference in feed intake and how quickly fish would change from one diet to another. Water changes of 20% were conducted on a weekly basis to maintain optimal conditions.

Diets were prepared using a commercially available 3mm Atlantic Salmon Grower LE pellet obtained from Skretting™, Hobart, Australia. Each diet consisted of 100g commercial feed which was then coated in the appropriate ingredients seen in Table 2.

Table 2. List of ingredients and quantities used to create coatings for the different diets Ingredients Bithionol and Bithionol, fish oil and Bithionol gelatin coat gelatin coat combined with fish oil coat Bithionol 2.5mg 2.5mg 2.5mg Gelatin 4 mL 4 mL Fish oil 3 mL 3 mL

For each diet with the exception of bithionol combined with fish oil the feed was placed into a bag and each ingredient was added separately and shaken for 15mins to ensure adequate and even coating. For the bithionol combined with fish oil diet the feed was first moistened with 6 mL of water to allow for greater absorption of the oil mix, it was then coated following the same procedure used on previous diets. Prepared diets were placed onto trays and allowed to air dry in the shade for 48 h, then stored in the fridge during the experiment.

Oral efficacy of bithionol The most palatable diet of bithionol combined with fish oil coating a water moistened pellet was then taken and used to examine oral efficacy of bithionol when treating AGD in two separate studies, which are outlined below.

Study One Briefly, Atlantic salmon smolts with a mean (± SEM) mass of 90.4 g (± 5.2) were fed to satiation on either a commercial control, oil coated commercial control or the bithionol diet for a period of 2 weeks, and then exposed to Neoparamoeba spp. Following the protocol described in Florent et al., (2007, in press), feeding was continued for 28 days post Neoparamoeba spp. exposure. Sampling occurred twice weekly with weight, length, plasma, gill, liver and kidney taken for analysis and examination. For details regarding examination and analysis please refer to Florent et al., (2007, in press).

Study Two This study examined the efficacy of bithionol both as a prophylactic and as a therapeutic diet. The prophylactic diet consisted of a 2 week feed prior to exposure with Neoparamoeba spp. and the therapeutic diet commenced upon the presentation

of gross gill lesions. Atlantic salmon smolts were fed at 1% BW d-1 with commercial control or prophylactic bithionol at a concentration of 25 mg kg feed-1 diet 14 d prior to challenge with Neoparamoeba spp. following which a therapeutic bithionol diet was added upon gross signs of gill lesions. Following 4 wks Neoparamoeba spp. exposure, fish were administered a 3 h freshwater bath and then disease progression was monitored for a further 3 wks. Feed intake was assessed weekly and the severity of AGD was assessed both grossly and histologically on a weekly basis.

Laboratory investigation of the potential efficacy of salinomycin, lasalocid acid and maduramycin ionophore. Pilot Study As previously described, the pilot study used two re-circulation systems, each containing three tanks for a total system volume of 2700 L. Water quality was measured daily with water temperatures ranging from 14 to 17°C, a minimum dissolved oxygen greater than 7.0 mg L-1 and salinity between 33 to 35 ppt. Twelve sea-water acclimated Atlantic salmon, with a mean (± SEM) mass of 156 (± 4) g were transferred into each of six tanks and fed to satiation daily for 17 days with a commercial diet. For the following seven days, salmon were offered a diet top-coated with fish oil (2%) with a daily ration equal to 1% of the tank biomass (TB). This 24 day period was required to establish a reliable and consistent feeding response in the experimental animals. The four treatment groups (3 drugs and 1 control) were randomly allocated to the four tanks with the highest specific growth rates over the 24 day habituation period. The other two tanks were designated as control tanks, resulting in one tank per drug and three control tanks.

Salmon were offered a daily ration at 1% TB, which consisted of a commercial pellet top-coated with fish oil only (control) or with fish oil and salinomycin, lasalocid acid or maduramycin. The inclusion rates for both salinomycin and lasalocid acid were 100 mg kg-1 of feed, which was given for 12 days prior to Neoparamoeba exposure. Maduramycin was initially included at 100 mg kg-1, however due to an apparent palatability issue, the dose was reduced to 50 mg kg-1 7 days prior to amoeba exposure. Each tank was fed daily with the assigned diet and feed was withheld on each weekly sampling day.

Neoparamoeba spp. were isolated from the gills of experimentally challenged Atlantic salmon, based on the method from Morrison et al. 2004. Amoebae were isolated on two consecutive days giving an inoculating concentration of 275 and 185 cells L-1, for a total concentration of 460 cells L-1. This dose is approximately 1.5 times the usual inoculating concentration used in our laboratory and was chosen because we wanted the fish to experience a strong infection pressure as an initial test of the ionophore efficacy.

Prior to amoeba exposure, tank weights were determined on a fortnightly basis and feeding rations were adjusted accordingly. Following parasite exposure, daily feed rations were adjusted based on weekly sampling of three fish from each tank. Additionally, if a mortality occurred, the ration was adjusted on the following day. Beginning at day 7 post exposure (PE) and continuing on a weekly basis, three fish from each tank were weighed, measured and the gills were assessed grossly for the white patches indicative of AGD lesions (scaling was from zero to three). The branchial basket was excised and placed in Davidson’s fixative for further H & E histology processing. As usual, the second left gill arch was examined under a light microscope to determine the proportion of filaments exhibiting AGD-associated lesions relative to the number of well oriented filaments. The ventral aspect of the salmon was incised and the organs inspected grossly for pathological changes. Specific growth rates (SGR) and condition index (K) were calculated based on:

SGR = [Ln (final weight) – Ln (initial weight)] * 100 (1) days elapsed

K = weight (2) L3

Significant differences were investigated using non-parametric Wilcoxon rank sum tests (also known as Mann-Whitney) to determine differences between the control and each ionophore individually for each parameter (e.g. percent lesioned filaments, SGR, K etc). Like the t-test (the parametric equivalent), the Wilcoxon test involves comparisons of differences between measurements (e.g. control vs. drug), however it does not require assumptions about the form of the distribution of the measurements.

Therefore it was used because the distributional assumptions that underlie the t-test cannot be satisfied due to the low sample number. P-values below 0.05 were considered statistically significant, unless noted otherwise.

Full Study This study used three re-circulation systems, each containing three tanks for a total system volume of 2700 L. Water quality was measured daily with water temperatures ranging from 14 to 17°C, a minimum dissolved oxygen greater than 7.0 mg L-1 and salinity between 33 to 35 ppt. Water changes were completed on a weekly basis or as needed (e.g. if the ammonia level was >0.75 mg L-1). Since the pilot study, individual UV light sterilizing systems were installed on each recirculating system. The UV systems were activated throughout the entire trial except from day 0 to day 8 post exposure (PE).

Forty-five sea-water acclimated Atlantic salmon, with a mean mass of 100 g were transferred into each of nine tanks and monitored for 48 hours. All salmon were offered a commercial diet top-coated with fish oil (2%) for seven days at a daily ration equal to 1% tank biomass (TB). The three treatment groups were (1) salinomycin, (2) lasalocid acid and (3) control with three replicate tanks for each group. Within each recirculating system, the three experimental tanks were identified based on the distance from the sump (near, middle and far), because this may influence flushing times, water turnover, horizontal transmission of an external parasite, etc. Therefore, treatments were assigned to each tank so that one replicate was in each of the positions from the sump and there was only one of each treatment group in each system. For example, there was one salinomycin tank in each system at the position nearest, middle and furthest from the sump.

Salmon were offered a daily ration at 1% TB, which consisted of a commercial pellet top-coated with fish oil only (control) or with fish oil and salinomycin or lasalocid acid. Both ionophores were included at a rate of 100 mg kg-1 of feed, which was given for 14 days prior to Neoparamoeba exposure. Each tank was fed daily with the assigned diet and feed was withheld the day prior to each weekly sampling (resulting in 6 daily rations per week).

Neoparamoeba spp. were isolated from the gills of experimentally challenged Atlantic salmon based on the method from Morrison et al. 2004. Amoebae were isolated on two consecutive days giving an inoculating concentration of 165 and 123 cells L-1, for a total concentration of 288 cells L-1.

Beginning four days prior to infection, tank weights were determined on a weekly basis and feeding rations were adjusted accordingly. Additionally, if a mortality occurred, the ration was adjusted on the following day. Three fish were sampled for gills, organs and plasma (as described below) four days prior to exposure to ensure the animals did not exhibit clinical signs of AGD.

Beginning at day 3 PE and continuing on day 7, 10, 14, 21, 28 and 35, five fish from each tank were weighed, measured and the gills were assessed grossly for the white patches indicative of AGD-associated lesions (scaling was from zero to three). The branchial basket was excised and placed in Davidson’s fixative for further H & E histology processing. On day 10 and 28, each fish was bled and the plasma decanted and frozen (-80°C) and the internal organs (including heart, liver, posterior kidney and spleen) were placed into formalin for histology processing (H & E).

The second left gill arch was examined under a light microscope to determine the proportion of filaments exhibiting AGD-associated lesions relative to the number of well oriented filaments. Statistical differences were investigated using analysis of variance (nested and one way) with P < 0.10 considered significant.

Laboratory investigation of the potential efficacy of bithionol sulphoxide or garlic powder One of the primary metabolites of bithionol is believed to be the sulphoxide derivative. Sulphoxidation is a common phase II biotransformation that facilitates excretion of compounds typically across the kidney (a known excretion pathway for bithionol in vertebrates) and potentially, the gill in fishes. Bithionol sulphoxide is also a less expensive form of the bithionol drug and since metabolites are often the effective form of a given drug or at least have some efficacy compared to the parent compound, the preliminary testing of bithionol sulphoxide as a potential in feed amoebicide was justified.

Garlic Seawater-acclimated Atlantic salmon smolts of mean mass127.0 g (± SE 0.4g) and fork length 18.5 cm (± SE 0.1cm) were randomly allocated to a tank in one of three replicate tank systems (3 tanks per system) as described by Powell et al. 2005. To one tank in each system fish were fed a control diet, bithionol sulphoxide-coated diet, or a diet coated with garlic powder. For all diets the compound inclusion consisted of top dressing the feed (Skretting Atlantic HP 3mm pellets) with fish oil (including control diet). Bithionol sulphoxide was included in the feed at a concentration of 25 mg kg-1 feed (after Florent et al. 2007 in press) and garlic at 100 mg kg–1 feed. Feed was made up three times weekly and kept refrigerated until use. All fish were fed to satiation a target ration of 1% body weight per day over 2 feeding periods 6 days out of 7 (Sunday excepted). Any uneaten feed was weighed and the feed intake of the tank recorded.

Following a period of 2 weeks of feeding the respective diets, a sub sample of 3 fish from each tank was randomly removed (designated the Pre, day 0 sample) as the tank weight was measured, then all systems were inoculated with 300 Neoparamoeba spp. cells L-1 (isolated by standard methods Morrison et al. 2004). Sub-samples of 4 fish per tank were randomly removed from each tank (as tank weights were determined) 5, 10, 17 and 24 days post-inoculation with amoebae. The removed fish were weighed and length recorded, a gross gill score determined (see above) and the gill basket removed and fixed in Davidson’s seawater fixative for histological examination and evaluation of the number of lesioned gill filaments by standard methods (Powell et al. 2005).

All variables were statistically tested using a nested two factor analysis of variance with time and treatment as the fixed variables. Where significant differences were found, a Tukey’s HSD test was used to discriminate differences between means. P values of less than 0.05 were considered statistically significant.

Semi-commercial experimental assessment of the efficacy of AquaciteTM and BetabecTM AquaciteTM and BetabecTM are two products produced by James Mackie Agricultural Ltd that contain β-glucans, bioflavenoids and vitamin mixes in proprietary formulations. Previous lab studies have indicated that when these compounds were included in the diet of Atlantic salmon smolts inoculated with Neoparamoeba spp., growth rates were maintained even though AGD as determined from gross and histological gill lesions was not affected. On the basis of these data (Powell et al. 2005), a semi-commercial scale assessment of efficacy was undertaken by Tassal Group Ltd with support from the project team.

A semi-commercial field trial was undertaken from January to April 2005 at Killala Bay, Tasmania, Australia (43°13’07.42 S, 147°01’25.84 E) to determine the growth and health effects of AquaciteTM and BetabecTM with a natural exposure to Neoparamoeba spp. It should be noted that this site often has lower salinity and is not always typical of sites constantly affected by AGD. Due to the endemic nature of this protozoan, salmon from a commercial-sized pen were exposed to a 2 h freshwater bath to terminate pre-existing infections (Powell et al., 2001). Experimental design was as described by Mlynarski et al. (submitted). In brief, 2000 diploid all female Atlantic salmon, with a mean (± SEM) mass equal to 581.7 g (± 23.4), were removed from a commercial pen using an air lift pump and 500 fish were placed into each of four 125 m3 square experimental pens for a stocking density of 2.1 kg m-3. The experimental pens were set in a 10 x10 m polyethylene cage collar approximately 500 m offshore and with an approximate water depth of 23 m. The pens were surrounded by predator netting in order to minimize threats from birds and seals.

Two pens were designated as controls and two were designated as Aquacite™ and Betabec™ treatment pens. The treatment pens were fed a commercial diet with pellets top-dressed with a mixture of Aquacite™ (2.5 g kg-1), Betabec™ (8 g kg-1), water (60 mL kg-1) and fish oil (3%). Aquacite™ was fed throughout the duration of the 90 day experiment with the inclusion of Betabec™ beginning on days 0 and 60 for two weeks, according to the manufacturer’s recommendation. The control pens were fed pellets top-dressed with only water and oil. All pens were fed to satiation two or

three times daily, with the third feeding discontinued at day 35 because all pens were consuming a commercial ration level.

Fish were sampled fortnightly, with day 0 PE being the day following the freshwater bath, and continuing on days 18, 32, 46, 60, 74 and 91 PE. At each sample period, 30 salmon were captured from each experimental pen using a 5 m2 box net and individuals were dip-netted from the crowd, anesthetized (n = 20) or euthanized (n = 10) with either AQUI-S® (Aqui-S NZ, Lower Hutt, New Zealand) (0.05%) or clove oil (0.02%). The twenty fish were non-lethally sampled for mass, fork length and gross pathology and returned to their respective pens, whereas the remaining 10 fish were lethally sampled as described and the gill basket was excised and placed in Davidson’s seawater fixative for histology. Mortalities were collected by divers from each pen on a weekly basis.

Growth parameters, including condition factor (K), specific growth rate (SGR), feed conversion rate (FCR) and the daily feed intake were calculated over the duration of each trial (Mlynarski et al. submitted). The formulas used were:

K (g cm-3) = mass * (100) length3

FCR (kg feed kg-1 fish) = amount of dry food eaten (kg) change in mass (kg)

SGR (Ln kg day-1) = Ln final mass (kg) – Ln initial mass (kg) period of time elapsed (days)

Feed Intake (% population mass) = daily feed intake (kg) * 100_____ total estimated pen biomass (kg)

Cumulative mortality and relative percent survival (RPS) were calculated for the field trial based on the numbers of mortalities from weekly dives, based on:

RPS = [1–(% mortality in treatment pens / % mortality in control pens)] *100

During the first 46 days of the field experiment, there were some escaped salmon. The losses were accounted for based on a linear regression equation (the rate of fish lost) using the known numbers of fish for each pen at the start and end of the trial. All data presented are corrected for the lost fish.

Condition factor and percent lesioned filaments were separately analyzed using a nested ANOVA comparing treatment and control diets with time and pen as fixed factors, followed by Tukey’s post-hoc tests. Homogeneity was assessed using a residual plot. Also, in the field trial, FCR, SGR and feed intake were analyzed using t-tests assuming equal variance to identify changes from the start (day 0) and end of the study (day 91). All statistical analyses were performed using SPSS® (version 11.5, SPSS Science) and p values ≤ 0.05 were regarded as statistically significant (Mlynarski et al. submitted).

Metabolic cost of gill disease Metabolic rate Measurements of oxygen consumption rates, which are essentially an indirect measurement of metabolic rates (Mo2), are used to evaluate the amount of energy an organism is using at any given moment (Jobling, 1994). The metabolic rate of most animals fluctuates between two extremes, with the lower limit typically referred to as standard or basal metabolic rate (Mo2 basal) and is the metabolic rate of a quiescent animal in a post-absorptive nutritional state, below which physiological function is impaired (Brett and Groves, 1979; Jobling, 1994). However, fish ordinarily expend energy above this level, due to activities such as feeding and locomotion, and this intermediate level is referred to as routine metabolic rate (Mo2 rout). The upper extreme is referred to as active or maximum metabolic rate (Mo2 max) and the range through which the aerobic metabolic rate can vary is regarded as the scope for activity or metabolic scope (Mo2 max – Mo2 basal) (Fry, 1947). Alternatively, metabolic scope can be expressed as relative metabolic scope, which is the difference between the routine and maximum metabolic rate (Wieser, 1985). Stressors, including disease, impose a metabolic cost on fish that consists of two mechanisms; (1) an energy requirement to manage the disturbances associated with the stressor and (2) an energy cost related to correcting the associated ionoregulatory imbalance (Barton and Iwama, 1991).

Recent studies have begun to highlight the effectiveness of metabolic rate measurements as a non-destructive means of assessing the physiological impact that disease has on a fish (Wagner et al., 2003; Tierney and Farrell, 2004; Powell et al., 2005a; Wagner et al., 2005).

Tenacibaculosis Tenacibaculum maritimum (formerly Flexibacter maritimus) causes a skin infection resulting in ulcerative dermatitis, although gill infections with a necrotizing branchitis are not unusual, with experimental infections progressing similarly to natural infections (Handlinger et al. 1997). This disease is prevalent in a number of species worldwide including Atlantic salmon (Salmo salar), greenback flounder (Rhombosolea tapirina), striped trumpeter (Latris lineata), red sea bream (Pagrus major), black seabream (Acanthopagrus schegeli), Japanese flounder (Paralichthys olivaceus) and rock bream (Oplegnathus fasciatus) (Baxa et al., 1986, Wakabayashi et al., 1986; Handlinger et al., 1997). Experimentally infected Atlantic salmon have been shown to be unable to maintain homeostatic regulation of blood plasma osmolality (Powell et al., 2004). Osmoregulation makes up a large percentage of the daily energy costs of marine teleosts, thus any disturbance may significantly impact the energy requirements of a fish, which would essentially manifest as an increase in oxygen consumption rates.

Food deprivation has been shown to significantly affect the metabolic rate of teleosts, furthermore, it has also been shown to affect the response to disease (O’Connor et al., 2000; Shoemaker et al., 2003). However little work has been published examining the synergy between feed deprivation and disease with regard to metabolic rate.

All the fish used in the experiments were fully acclimated to seawater (35ppt) two weeks prior to use. All experiments were conducted in a temperature-controlled room (17°C) that housed four individual 400 L recirculation systems. Each system consisted of a 200 L conical bottom tank and a 200 L sump, which contained a biofilter and mesh to remove solids and catch uneaten feed. Fish were stocked at 25 fish per tank, with each fish being individually identified by a subdermal injection of Alcian blue dye. Fish were fed to satiation or had their feed withheld for a two-week period prior to metabolic rate measurements.

Metabolic rate measurements were made prior to infection and 30 hours post infection. Food was withheld for a 24 hour period prior to measurements and fish were placed into boxes 8 hours prior to measurement. The respirometry boxes were 1L black acrylic boxes, with each box having its own water and air supply. Routine metabolic rate measurements were made by stopping the air and water supply to the box, immediately a water sample was taken and the oxygen content determined by running the water sample through a thermostatically controlled oxygen electrode (1302 electrode, Strathkelvin instruments Ltd Glasgow, UK) connected to a

Strathkelvin instruments model 782 O2 meter. A second water sample was taken 10 minutes after the first, metabolic rate was then determined by using the following formula:

(( 2i − POPO 2e α *)*) V Mo2 = [1] T * M

Where PO2i and PO2e are the initial and final oxygen tensions respectively (mm Hg), -1 -1 α is the molar O2 solubility in water (µM O2.L mm Hg ), V is the respirometer box volume (L), T is the time between the initial and the final oxygen measurements (s) and M is the mass (g) of the fish (Cameron, 1986; Cech, 1990).

Immediately after the routine metabolic rate measurements were completed the maximum metabolic rate was determined. Fish were removed from the acrylic boxes and placed into a 50L round container with 120% oxygenated seawater. They were then chased for a 10 min period, after which their metabolic rates were determined using the aforementioned methods. Metabolic scope was determined by subtracting routine metabolic rate from maximum metabolic rate. All measurements were repeated 30 hours following infection.

Cultures of T. maritimum strain 00/3280 were obtained from cultures held at the Tasmanian Aquaculture and Fisheries Institute (Fish Health Unit, Department of Primary Industries and Water, Tasmania, Australia. Following the pre-infection 12 -1 Mo2max measurement, a 200 µL suspension of T. maritimum (5 x 10 cells mL ) was applied evenly over all of the eight gill arches of the anaesthetised Atlantic salmon.

Control fish received a saline solution and all fish were returned to their respective tanks.

Following the post-infection metabolic rate measurements, fish were given a lethal overdose of clove oil, weighed and fork length measured. A sterile plastic loop was used to sample mucus from the gills and plated on Sheih’s marine agar to determine the presence of T. maritimum. Histology of the gill was used to confirm the presence of T. maritimum and to assess the extent of necrosis.

Due to morbidities of tagged fish in all groups the first experiment was completed three times in order to gain enough viable replicates to conduct statistical analyses. Statistical analyses were conducted using the statistical package SPSS for Windows (Version 11.5). A paired sample t-test was used to determine the difference between

Mo2 rout pre- and post-infection for each individual group (referred to as fed infected, fed uninfected, unfed infected, unfed uninfected respectively). A similar analysis was used for Mo2 max and metabolic scope. An one-way analysis of variance (ANOVA) was used to determine differences in blood plasma osmolality among the four groups and significant differences were investigated using a Tukey’s post-hoc test. Furthermore, two one-way ANOVA’s were used to determine whether there was a significant difference between the four treatments pre-infection and post-infection.

Amoebic gill disease The hyperplasia commonly associated with AGD effectively reduces the surface area available for respiration. It was initially believed that the reduction in surface area was the primary cause of mortality, as affected fish have been shown to exhibit typical signs of respiratory distress such as lethargy and ‘coughing’. However, fish showing AGD lesions do not suffer from an overt hypoxemia (Kent et al., 1988; Powell et al., 2000), and recent evidence suggests that mortality may in fact be related to cardiovascular dysfunction (Powell et al., 2002; Leef et al., 2005a). Atlantic salmon with AGD have significantly elevated systemic vascular resistance and reduced cardiac output, which under stressful conditions could become exacerbated and result in cardiac failure (Leef et al., 2005a). However it was unknown just what would be

the specific effect on routine metabolic rate of a reduction in gill surface area coupled with the gill cell proliferation typically seen with AGD.

Methods used are similar to those described for the T. maritimum experiments above with the following differences

Metabolic rate measurements were taken prior to infection and 10 and 20 days post infection. 3-litre respirometry boxes were used as the fish were slightly larger than in the previous experiment. Fish were challenged with amoebae at 300 cells L-1, at the end of the experiment gills were excised and the extent of infection was determined using histology (methods previously described). Satellite fish where removed from the tank at each sampling period (day 0, 10 and 20) to give an indication of the extent of the level of infection in the experimental animals.

Results and discussion In vitro assessment of candidate amoebicides Biocides: Bronopol Bronopol was tested as either the commercial preparation PycezeTM (Novartis Ltd) and as Samaki BiocideTM (J. Mackie Trading Ltd). There were no significant reductions in amoeba numbers for Pyceze (P = 0.3320) although a small effect of time of exposure was seen (P = 0.0034). However, this result was marginal in degree (Fig. 1). There were significant effects of concentration (P = 0.0009) for Samaki Biocide although the reduction in amoeba numbers was marginal in degree (Fig.1).

PycezeTM

250 1 h 2 h 3 h 200

150

100

50 Amoeba number (% seawater control) seawater (% number Amoeba

0 0 1 10 100 Concentration (mg L-1)

Samaki BiocideTM

250 1 h 2 h 3 h 200

150

100

50 Amoeba number (% seawater control) seawater (% number Amoeba

0 0 1 10 100 Figure 1. Effects of acute (1-3 h) exposure of isolated Neoparamoeba spp. to 1 , 10 and 100 mg L-1 of two commercially available bronopol-containing biocides relative to seawater exposed controls. Error bars represent ± 1 SEM.

When both Pyceze and Samaki Biocide were tested over 5 days of continuous exposure to Neoparamoeba spp in primary culture both compounds were toxic to amoebae. Pyceze had a significant effect of concentration ( P = 0.0011) and time (P = 0.0210) but no significant interaction. Similarly Samaki biocide had a significant

effect of concentration (P < 0.0001) although the effect of time was not significant at the 0.05 level (P = 0.0602), nevertheless there was a significant interaction of time and concentration (P < 0.0001). Despite the longer term exposure of amoebae to bronopol having significant toxic effects, a 96 h exposure is not likely to be considered feasible in a commercial operation (Fig. 2).

10000

8000 -1 Seawater control -1 6000 Pyceze 10 mg L Pyceze 100 mg L-1 Samaki Biocide 10 mg L-1 Samaki Biocide 100 mg L-1 4000 Amoeba numbers mL numbers Amoeba

2000

0 012345 Exposure (days) Figure 2. Effects of 96 h (4 day) exposure of isolated Neoparamoeba spp. to 10 and 100 mg L-1 of two bronopol containing biocide and seawater controls (no biocide added). Error bars represent ± 1 SEM.

Biocides: Acticide There was a significant effect of concentration upon decreased amoeba survival when exposed to both Acticide F(N) (P = 0.0020) and Acticide DDQ (P < 0.0001) over the 3 h duration of the toxicity assay (Fig. 3). It was apparent however that the duration of exposure for either Acticide F(N) and Acticide DDQ was not significant (P = 0.4658).

Acticide F(N)

2.0 1h 2h 3h

1.5

1.0

0.5 Amoeba number (proportion of control) of (proportion number Amoeba

0.0 0 1 10 100 Exposure concentration (mg L-1)

Acticide DDQ

2.0 1h 2h 3h

1.5

1.0

0.5 Amoeba number (proportion of control) of (proportion number Amoeba

0.0 0 1 10 100 Exposure concentration (mg L-1) Figure 3. Effects of acute (1-3 h) exposure of isolated Neoparamoeba spp. to 1 , 10 and 100 mg L-1 of two commercially available formaldehyde-containing biocides (Acticide F (N) and Acticide DDQ) relative to seawater exposed controls. Error bars represent ± 1 SEM.

Acticide DDQ appeared to show a consistent concentration response relationship with regard to its toxicity compared with Acticide F(N). Despite these results the use of either Acticide compound would not likely to be feasible as a bath treatment since both contain formaldehyde which raises issues of user safety but also environmental discharge with conventional bathing practices.

Biocides: VirkonTM Virkon® (DuPont) is the brand name of a disinfectant and is frequently used as a veterinary disinfectant, as well as in laboratories for cleaning up spills, soaking equipment or wiping benches. It has a remarkable spectrum of activity against viruses, fungi, and bacteria, including mycobacteria, such as tuberculosis. Virkon® is most often sold as a pink-coloured

powder, that is mixed with water to form a 1% or 2% solution (i.e. 10 g or 20 g L-1 ). The pink colour is useful in that it helps gauge the concentration when preparing the Virkon® and importantly, as the Virkon® ages it discolours, making it obvious when it needs to be replaced. The solution is stable for 7 days. Although stated to have a lemon scent, the smell of Virkon® is very faint but still considered unpleasant to many. It is relatively safe in terms of skin contact, but should not be used as a hand-washing liquid. A major disadvantage to using Virkon® is the price. As a cost saving measure, many laboratories make their own disinfectants with relatively low cost solvents that can be purchased in bulk.

For both the long term (48 h) and short term (2 h) assay, various concentrations of Virkon® were effective at reducing the numbers of amoebae, but the chemical treatment did not completely kill all of the cells. Over the 48 h assay, the 10 mg L-1 dose significant reduced the numbers of amoebae compared to 0 (P = 0.015) and 0.1 mg L-1 (P = 0.001) (Fig. 1). Additionally, a significant reduction was observed in the 1 mg L-1 treatment compared to the 0 mg L-1 (P = 0.002) (Fig. 4). During the short term assay, mean numbers of amoebae were significantly reduced at a concentration of Virkon® of 100 mg L-1 compared 0, 25 and 50 mg L-1 (all P = 0.001) (Fig. 5. ). As expected, Virkon® is an effective amoebicide and should be considered for use as a disinfectant (e.g. nets and equipment) and for increasing biosecurity measures (e.g. foot baths) in both laboratories and areas endemic to Neoparamoeba spp.

40000 Virkon mg/L 0 0.1 1 10 35000

30000

25000

20000

15000 numbers of amoeba per ml 10000

5000

0 0 4 24 48 Time (hr)

Figure 4. The toxicity to Neoparamoeba spp. of varying concentrations of Virkon® for up to 48 h (all n = 8 except at t = 48h; n = 4). Error bars represent + 1 SEM.

Virkon mg/L

30000 0 25 50 100

25000

20000

15000

10000 numbersamoeba per ml of

5000

0 12 Time (hr)

Figure 5. The toxicity to Neoparamoeba spp. of varying concentrations of Virkon® for up to 2 h. Error bars represent + 1 SEM.

Ionophores: Monensin Monensin is an ionophoric antibiotic that facilitates the transfer of Na+ across cell membranes and so disrupts membrane potentials of target organisms. The use of monensin in animal medicine is common and it is a registered drug in Australia for use in a number of livestock. The normal method of delivery of this drug is in-feed. It was tested in a 96 h toxicity assay only. The insolubility of monensin in seawater meant that two different tests were conducted. In the first, monensin was added directly to the amoeba cultures as a suspension and an additional control of 10 mg L-1 alumina suspension was included (Powell et al. 2003). In the second test, monensin was dissolved in a maximum concentration of 10 mg L-1 ethanol prior to adding to the test medium. An additional control consisting of 10 mg L-1 ethanol was included in these tests.

Monensin with alumina control

10 Seawater control Alumina control 0.1 mg L-1 8 1.0 mg L-1 10.0 mg L-1

Amoeba numbers (proportion of seawater control) seawater of (proportion numbers Amoeba 0 0 20 40 60 80 100 120 140 Exposure time (h) Monensin with ethanol control

2.5 Seawater control 10 mg L-1EtOH control 0.1 mg L-1 2.0 1.0 mg L-1 10.0 mg L-1

1.5

1.0

0.5

Amoeba numbers (proportion of seawater control) seawater of (proportion numbers Amoeba 0.0 0 20 40 60 80 100 120 140 Exposure time (h)

Figure 6. Survival of Neoparamoeba spp. during continuous exposure of to 0.1, 1 or 10 mg L-1 monensin in suspension (with alumina control) or pre-dissolved in 10 mg L- 1 ethanol (with ethanol control) relative to seawater controls. Error bars represent ± 1 SEM.

There was a significant effect of exposure time for both monensin as a suspension (with an alumina control P = 0.0050) and when pre-dissolved in ethanol (P = 0.0005). This indicates that there was an increase in the relative survival of amoebae compared to seawater controls for all concentrations of monensin and the alumina control (Fig. 6). There was an apparent decrease in amoeba survival compared to seawater controls for all concentrations of monensin and ethanol only controls when the monensin was

pre-dissolved prior to exposure of Neoparamoeba spp (Fig. 6). In both cases (alumina control and ethanol control experiments), there were no significant effects of concentration (P = 0.1115 and P = 0.8167 respectively). These data therefore suggest that monensin is not likely to have a toxic effect to Neoparamoeba spp. when continually exposed for several days. Therefore the likelihood of monensin alone being a viable option for further laboratory testing for amoeba toxicity or efficacy as a treatment for AGD is low. This is different from its reported efficacy at controlling internal protozoal infections of fish such as Loma salmonae infections in rainbow trout. The decreased amoeba survival in the ethanol controlled experiments reflects previous observations that ethanol (even at low concentrations) is toxic to Neoparamoeba spp. (Powell unpublished).

Salinomycin, lasalocid acid and maduramycin Over the 96 hour assay, the highest dose of salinomycin tested of 10 mg L-1 significantly reduced mean numbers of amoeba compared to 1 mg L-1 (P = 0.003), 0.1 mg L-1 (P = 0.057) and 0 mg L-1 (P = 0.034) (Fig. 7). No significant differences were observed between concentrations of 0, 0.1 and 1 mg L-1 of salinomycin (all P > 0.12). Similarly, a lasalocid acid dose of 10 mg L-1 significantly reduced the mean numbers of amoeba compared to 1 mg L-1 (P = 0.077), 0.1 mg L-1 (P = 0.001) and 0 mg L-1 (P = 0.001) (Fig. 8). No significant differences were observed amongst concentrations of 0, 0.1 and 1 mg L-1 of lasalocid acid (all P > 0.10). Finally, maduramycin at concentrations of 0.1 and 10 mg L-1 significantly reduced mean numbers of amoebae compared to the 0 mg L-1 treatment (P = 0.001 and 0.004, respectively) and no differences were observed between 1 mg L-1 and 0 mg L-1 doses for this ionophore (Fig. 9).

35000 Salinomycin (mg/L)

30000 0 0.1 1 10

25000

20000

15000

numbersamoeba of mL per 10000

5000

0 24 48 72 96 Time (hr) n = 8

Figure7. Mean (± SEM) numbers of amoeba with varying concentrations of salinomycin (in vitro).

Lasalocid Acid (mg/L)

50000 0 0.1 1 10 45000

40000

35000

30000

25000

20000

15000 numbers of amoeba per mL per of amoeba numbers 10000

5000

0 24 48 72 96

Time (hr) n = 8

Figure 8. Mean (± SEM) numbers of amoeba with varying concentrations of lasalocid acid (in vitro).

Maduramycin (mg/L)

35000 0 0.1 1 10

30000

25000

20000

15000

10000 numbersamoeba of mL per

5000

0 24 48 72 96 Time (hr) n = 8

Figure 9. Mean (± SEM) numbers of amoeba under varying concentrations of maduramycin (in vitro).

It is important to note that the ionophores tested are insoluble in water, which is a desirable characteristic for an in-feed drug for aquaculture, although it is troublesome for accurate in vitro testing, which relies on a homogenous ‘solution’. The reductions observed with the three ionophore treatments were similar to those found with bithionol, another antiprotozoal compound currently being investigated as an AGD treatment and sufficient evidence was provided to move all three ionophores into the next stage of testing with in-feed trials.

Metabolic inhibitors: Metronidazole. Metronidazole was identified as a potential candidate amoebicide since it is a potent treatment for amoebic dysentery caused by the facultative anaerobic amoeba Entamoeba histolytica (Best 2002).

16000 14000

12000 Control

10000 100 mg/l Amoeba 50 mg/l 8000 Number /ml 10 mg/l 6000 1 mg/l 4000 0.1 mg/l 2000 0 123456 Days

Figure 10. In vitro toxicity test of Neoparamoeba spp. (cultured strain NP251002) continuously exposed to metronidazole at different concentrations over 6 days. Error bars represent ± 1 SEM. (Peyghan and Powell submitted).

Metronidazole is used to treat a broad range of infections caused by anaerobic protists and bacteria. This drug is also effective against human amoebiasis (Land and Johnson, 1997). The selective cytotoxicity of metronidazole relies on biochemical properties of anaerobic organisms that are lacking in aerobic cells. For aerobic cells the inhibitory concentration of metronidazole is two to three orders of magnitude higher than for anaerobes (Land and Johnson, 1997). We tested up to 100 mg L-1 and concentrations above this, although they might show greater toxicity, would likely be impractical for the inclusion of this drug into feed. Moreover, the levels that would be required to achieve tissue concentrations that are toxic to Neoparamoeba spp. would likely be prohibitively costly, impractical or toxic to the fish.

Metronidazole had minimal effects on amoeba survival (Fig. 10). However, morphological changes due to metronidazole exposure (Fig. 11) may be the effect of DNA changes in the treated amoebae. Metronidazole appeared to have a role in inducing DNA damage and DNA breakage due to treatment in human lymphocytes through the futile cycle. The one-electron reduction of the drugs leads to the production of nitro radical anions. In the presence of oxygen, these radicals are re- oxidized and generate oxygen-activated species that can be responsible for the DNA and other cell component damages. (Re et al., 1997; Menéndez et al., 2001). This ability of metronidazole may inhibit DNA segregation or modify the amoeba genes involved in the completion of mitosis. This mechanism is shown by the ability of

metronidazole to arrest the cell cycle in the G2+M stage of Giardia intestinalis after 24 h in vitro treatment. (Hoyne et al., 1989). However, because the morphological changes in our study were not characterized by more specific staining and or ultra structural studies, further studies in this respect are recommended.

A B

Figure 11. A. Methylene blue stained Neoparamoeba spp. from the control group, ¹־B. Methylene blue stained Neoparamoeba spp. following exposure to 100 mg l Metronidazole for 4 days. Note the changes in intracellular and cell membrane morphology (C). (x1000 oil immersion) (From Peyghan and Powell submitted).

Laboratory investigations of the potential efficacy of N-Acetyl Cysteine Although N-Acetyl cysteine was effective at reducing cutaneous mucus viscosity significantly in both naïve and AGD affected rainbow trout and Atlantic salmon, it imparted no significant protection against AGD (Powell et al. 2007 in press). It therefore appears that unlike its analogue LCEE, NAC does not affect the mucous coat of the gill to a sufficient extent so as to prevent the attachment of Neoparamoeba spp. to the gill and subsequent infection.

Laboratory investigations of the potential efficacy of bithionol Bath toxicity and efficacy Bithionol when administered as a bath treatment was found to be toxic to fish, exhibiting high morbidity rates in both Atlantic salmon and rainbow trout fresh- and seawater at concentrations greater than 10 mg L-1., whilst AGD affected Atlantic salmon and rainbow trout exhibited a concentration dependant decrease in percent lesioned gill filaments, with 1 mg L-1 bithionol reducing percent lesioned gill filaments to similar levels as seen in the freshwater controls (Florent et al., 2007). Interestingly, no treatment differences were observed in plasma osmolality or chloride levels or in gill SDH and Na+/K+ ATPase activity, suggesting that these toxicity

markers are not acutely affected and the morbidity observed is most likely not related to gill function. There were no signs of abnormal histopathology in the liver or gill sections examined.

Bithionol palatability When comparing bithionol diets, the most palatable diet was obtained by combining bithionol at 25 mg kg feed-1 with 3% fish oil and using this mixture to coat pellets that had been moistened with 6% distilled water. High palatability was observed throughout the trial but more noticeably after changing tanks on day 15 (Fig. 12). This study was not continued any further as the aim was to determine if bithionol was palatable, and an achievable way to orally administer bithionol to fish had been identified.

2 Feed Intake (g)

0 0 5 10 15 20 Days Figure 12. Feed intake (g) for Atlantic salmon fed to satiation twice daily on either commercial feed (open squares), 25 mg kg -1 bithionol and gelatin feed (closed circles), 25 mg kg -1 bithionol, fish oil and gelatin feed (open circles) or 25 mg kg -1 bithionol combined with fish oil feed. Error bars represent ± 1 SEM.

Oral efficacy of bithionol Study One Bithionol exhibited potential as an oral treatment for AGD, at 25 mg kg feed-1 when fish were fed to satiation for 2 weeks prior to Neoparamoeba spp. exposure and 4 weeks following exposure. A delay and reduction in percent lesioned gill filaments and gross gill score (Fig. 13) was achieved over the trial period. Furthermore, bithionol exhibited higher feed consumption (Fig. 14) throughout the study compared to plain and oil coated commercial diet and a reduction in mortalities (Florent et al.,

2007, in press). Further investigation of bithionol as an in-feed treatment for AGD is warranted, including examining the effects of prophylactic and therapeutic treatments, running a similar experiment for a longer period of time to obtain more growth data, examining the effect of pulse feeding, or conducting trials under more realistic field conditions with lower exposure doses and freshwater baths.

2 a

1 Gross gill score gill Gross

0 -15 -10 -5 0 5 10 15 20 25 30 Days from Neoparamoeba spp. exposure Figure 13. Mean (± SEM) gross gill score for Atlantic salmon with amoebic gill disease (AGD) when fed either commercial feed (closed circles), oil-coated commercial feed (open circles) or 25 mg kg -1 bithionol feed (broken line and open squares). No SEM bars indicate that all replicates exhibited the same value. Letters denote significant differences within treatments. (From Florent et al 2007 in press)

2.2

2.0

1.8

1.6

1.4

1.2

1.0 a

0.8 b Feed intake (%TW) 0.6

0.4 c 0.2

0.0 -14 -7 0 7 14 21 28 Day from Neoparamoeba sp. exposure Figure 14. Mean (± SEM) weekly tank feed intake (n = 3) for Atlantic salmon exposed to Neoparamoeba spp. when fed to satiation on either commercial feed (closed circles), oil-coated commercial feed (open circles) or bithionol at 25 mg kg feed -1 (broken line and open squares). No SEM bars indicate that all replicates

exhibited the same value. Letters denote significant differences among treatments over the trial duration (p < 0.05) (From Florent et al 2007 in press).

Study Two When bithionol was administered as either prophylactic or therapeutic diets there appeared to be no differences between these treatments according to the gross gill score of AGD affected Atlantic salmon, but both bithionol treatments had gross gill scores that were generally one score lower than score exhibited in fish fed the control diet (Fig. 15). The higher feed consumption of bithionol diets compared to controls observed in study one was not observed in this study (Fig. 16), with feed intake being similar across all treatments. The differences seen in feed intake between studies may be due to the feeding regime: in Study One fish were fed to satiation and in most cases were eating over the 1% BW d-1 (Fig. 14), whereas in Study Two they were limited to 1% BW d-1, this was done in order to be consistent with other feed trials in the literature.

Gross Gill Score Gross Gill 1

0 5 10 15 20 25 30 35 40 45 50 Day from Neoparamoeba spp. exposure Figure 15. Mean (± SEM) gross gill score for Atlantic salmon with amoebic gill disease (AGD) when fed either commercial feed (closed circles), prophylactic bithionol at 25 mg kg feed-1 (closed squares) or 25 mg kg -1 bithionol therapeutic feed (dotted line and closed triangles). No SEM bars indicate that all replicates exhibited the same value. (From Florent et al in preparation).

1.1

1.0

0.9

0.8

0.7

Feed Intake (%BW) 0.6

0.5

0.4 -21 -14 -7 0 7 14 21 28 35 42 49 Time from Neoparamoeba spp. exposure Figure 16. Mean (± SEM) weekly feed intake (% BW) for Atlantic salmon with amoebic gill disease (AGD) when fed to a maximum 1% BW d-1 on either commercial feed (closed circles), prophylactic bithionol at 25 mg kg feed-1 (dashed line and closed squares) or 25 mg kg -1 bithionol therapeutic feed (dotted line and closed triangles). No SEM bars indicate that all replicates exhibited the same value. From Florent et al. in preparation.

Laboratory investigation of the potential efficacy of salinomycin, lasalocid acid and maduramycin ionophores Pilot Study There were no apparent acute toxic affects at these doses for all three ionophores. No mortalities were recorded in the ionophore tanks and there were four mortalities in the control tanks. Interestingly, the control mortalities came from the two tanks with the lowest growth rates during the initial 17 day feeding period. Additionally, no gross lesions were observed on the internal organs during necropsies.

No palatability problems were observed with salinomycin and lasalocid acid administered at 100 mg kg-1 feed, although there was a decrease in the daily feed intake for fish offered salinomycin from day 14 to 21 PE. There were apparent palatability and practicality issues with maduramycin incorporated at 100 mg kg-1 of feed during the prophylactic feeding period. Maduramycin is sold commercially as a premix and consists of 1% drug and 99% calcium carbonate and corn meal. The premix did not remain on the 3 mm feed pellet and there could have been an issue

with the amount of surface area on the pellets available for the premix to adhere. However, once the dose was decreased to 50 mg kg-1, there were no palatability issues and the premix adhered well to the pellets.

Efficacy as a prophylactic treatment for AGD At day 7 PE, salmon given an ionophore prophylactic treatment had a reduced proportion of lamellae exhibiting AGD lesions compared to the control fish (Table 5). Typically, an average of 8 to 14% lesioned filaments were observed on the ionophore treated salmon compared to over 30% in the control fish. At day 14 and 21 PE, Atlantic salmon in all groups exhibited similar proportions of lesioned filaments (Fig. 17).

Table 5. At day 7 post exposure to Neoparamoeba spp., mean (± SEM) percent lesioned filaments, mean condition index (K) and specific growth rate (SGR) in Atlantic salmon, Salmo salar, fed a pellet diet top-coated with either lasalocid acid, maduramycin, salinomycin or an oil only control diet.

Treatment Dose1 % Lesioned Filaments K SGR2

Control (n=9) 0 mg kg-1 32.1 ± 7.52 0.861 ± .0502 -1.14 Lasalocid acid (3) 100 13.6 ± 3.08†* 1.04 ± .00944† 2.85 Maduramycin (3) 50 8.2 ± 2.92† 1.10 ± .0695† 1.93 Salinomycin(3) 100 10.9 ± 5.09† 0.984 ± .0760 1.62 1daily ration equal to 1% of total tank biomass 2based on an average fish weight on May 22 (day - 4) and June 2 (day 14 PE) †statistical difference compared to the oil only control group using non-parametric Wilcoxon rank sum tests (Mann-Whitney) * p = 0.079

control lasalocid acid maduramycin salinomycin 70

percent lesioned filaments 20

0 123 week post exposure

Figure 17. Mean (± SEM) percent lesioned filaments observed in Atlantic salmon, Salmo salar with a prophylactic ionophore treatment during a Neoparamoeba spp. experimental challenge (pilot study).

Moreover, from four days before infection to day 14 PE, all three ionophores tested showed positive SGR and the three control tanks combined had a negative rate (Table 5). Growth rates were not assessed after day 14 PE due to low numbers of fish in each tank. On day 7 PE, fish offered maduramycin and lasalocid acid had significantly higher mean condition index compared to the control fish.

As mentioned above, the inoculating dose for this trial was almost 500 cells L-1, which created a continually increasing infection pressure due to recirculating nature of the experimental systems (even with normal weekly water changes). The design of

the pilot study had only a single tank for each ionophore tested so one cannot differentiate between treatment and tank effect. Thereby, the combination of a potent infection pressure and multiple control tanks provides strong evidence that the results are reflective of a treatment effect. We concluded that the evidence supported a reduction in the proportion of lamellae exhibiting AGD-associated lesions in salmon offered a prophylactic treatment with lasalocid acid, maduramycin or salinomycin compared to the control groups. A full challenge trial was recommended to further investigate these drugs.

Full Study Similar to the pilot study, the diets were palatable and there was no apparent host toxicity resulting from the oral ionophore therapy. Typical mortality levels, ranging from 0 to 8.8% were observed impartially across systems and treatments, as observed with previous Neoparamoeba trials. The mean percent lesioned filaments was not significantly different in either ionophore treatment compared to the control fish (P = 0.6263), however there was significant interaction between tank and treatment group (P = 0.0016). Large variability in the mean percent lesioned filaments was observed within the three tanks for each treatment group (Fig. 18). When the three systems were analyzed independently, salinomycin-fed fish in only system II showed a significant average reduction in lesioned filaments of 6% with 90% confidence intervals from 2 to 12% over the entire 35 day trial compared to the control fish (P = 0.24). As expected, in all three treatments the proportion of lesioned filaments significantly increased with time (P = 0.005) (Fig. 18). Mass increased similarly in all treatments with time (P < 0.05) and there were no differences observed in K across treatments and time (P > 0.10) (Fig. 19). The weekly average feed intake was similar across all treatments, which was at or near the maximum 1% TB until the final week of study beginning on day 28 PE (Fig. 20.) Specific growth rates from day 3 pr- exposure to day 28 post-exposure were 0.452 ± 0.26, 0.29 ± 0.36 and 0.713 ± 0.08 for the control, lasalocid acid and salinomycin treatments, respectively.

100 Control Lasalocid 75 Salinomycin

lesioned% filaments

0 0 3 7 1014212835 day post exposure

Figure18. Mean (±SEM) percent lesioned filaments observed in Atlantic salmon, Salmo salar with a prophylactic ionophore treatment during a Neoparamoeba spp. experimental challenge (full study).

Control weight k 160 1.2 140 1 120 0.8 100

80 0.6 k

(g) weight 60 0.4 40 0.2 20 0 0 0 3 7 10 14 21 28 35 day post exposure

Salinomycin weight k 160 1.2

140 1 120 0.8 100

80 0.6 k

weight (g) 60 0.4 40 0.2 20

0 0 0371014212835 day post exposure

Lasalocid weight k 160 1.2 140 1 120 0.8 100

80 0.6 k

(g) weight 60 0.4 40 0.2 20 0 0 0371014212835 day post exposure Figure19. Mean (± SEM) mass and condition factor (K) in Atlantic salmon given an oral prophylactic ionophore therapy, followed by a challenged with Neoparamoeba spp (full study).

Control 1.00 Lasalocid Salinomycin

0.75

0.50

0.25 weight) tank (% Intake Feed

0.00 -14 -7 0 7 14 21 28 day post exposure

Figure 20. Mean (± SEM) feed intake (as percent tank mass) in Atlantic salmon given an oral prophylactic ionophore treatment during an experimental challenge with Neoparamoeba spp (full study).

Shinn et al. (2003) reported salinomycin as efficacious against experimental infections with Ichthyophthirius multifiliis, although efficacy was highly dependent on duration of treatment and dose of the drug. Salinomycin offered at 50 ppm for 10 days or at 100 ppm for 5 days following I. multifiliis infection did not reduce trophonts numbers, although treatments at 100 ppm for 10 days showed a reduction in parasitemia (Shinn et al. 2003). Similarly, a combined salinomycin and amprolium treatment was

declared effective at reducing the prevalence of Myxobolus infections in Mediterranean farmed sea bream (Athanassopoulou et al. 2004a, 2004b, Karagouni et al. 2005), however, actual dosages at a fish level are not reported and repeatability of efficacy is not consistent across trials and challenge method. Likewise, our results revealed that a salinomycin treatment for AGD was marginally beneficial at reducing percent lesioned filaments in two (one tank each from the pilot & full study) of the four tanks the drug was offered compared to the non-treated control. The inconsistencies in the repeatability may be related to the high degree of variability observed on a fish-to-fish basis, which was mostly likely to be directly proportional to the actual amount of drug ingested. Based on poultry research, a single dose of salinomycin fed to chickens at 20 mg kg-1 body weight had a half-life of less than four hours and residues were only detected in the liver at 48 hours and completely disappeared by 72 hours (Atef et al. 1993). The presumed short life span of salinomycin in body tissues, combined with reduced feeding intake of parasitized Atlantic salmon could have resulted in the perplexing results for this drug.

Recently, several ionophores including salinomycin, lasalocid acid and maduramycin were found to have moderate to high levels of efficacy at reducing/eliminating a coccidial infection in the gastro-intestinal tract of the common carp, Cyprinus carpio (Molnar and Ostoros 2007). Interestingly, the ionophores were incorporated with the feed at 200 mg kg-1 of feed and the fingerlings consumed a minimum of 5% of their body weight per day (Molnar and Ostoros 2007). Compared with our studies, this was twice the dose incorporated in the feed and more than five times the amount of feed consumed on any day, which may be indicative of the concentration of ionophore required in the body tissues to confer parasite efficacy.

Lasalocid acid appears to have minimal if any efficacy for preventing AGD- associated lesions in Atlantic salmon and should not be considered further. At this time, we continue to consider salinomycin as a possible therapeutic, which warrants an additional in feed trial to further investigate efficacy. Preferably, the next study would investigate salinomycin and maduramycin (monovalent monoglycoside) oral therapy as a prophylactic treatment for AGD.

Laboratory investigation of the potential efficacy of bithionol sulphoxide or garlic powder There were no significant effects of treatment (P = 0.514) nor time (P = 0.651) on condition factor for any of the diets tested. This was also reflected in the fact that there were no significant effects of treatment (P = 0.713) nor time (P = 0.570) on fish weight over the duration of the study. Not surprisingly, there was a significant effect of time on the percentage of lesioned filaments (P < 0.0001) although there was no effect of treatment (Fig. 21). Thus it was apparent that none of the treatments tested retarded the rate of gill disease development.

100 Bithionol sulphoxide Control 80 Garlic powder

20 % Lesioned filaments Lesioned %

0 5 10 15 20 25 30 Days post-exposure

Figure. 21. Effects of feeding bihionol sulphoxide or garlic powder on the progression of AGD type lesions following challenge exposure of Atlantic salmon smolts to Neoparamoeba spp. Error bars represent ±1 SEM.

There was a marginally reduced percent cumulative mortality rate (as shown by improved survival) for the bithionol sulphoxide-treated group compared with either controls or garlic-treated fish (Fig. 22). There was an elevated mortality in one of the systems of 3 tanks that affected all of the treatments. The cause of this increased mortality remains unknown, however, if that system of tanks is removed from the analysis, it is clear that there was a reduced mortality rate in bithionol sulphoxide treated fish compared with control or garlic treated fish (Fig. 23.).

Bithionol sulphoxide 100 Control Garlicpowder

40 Percent cumulative survival cumulative Percent 20

0 -20 -10 0 10 20 30 Days post-exposure Figure 22. Percent cumulative survival of Atlantic salmon smolts fed a diet including bithionol sulphoxide, garlic powder or control (oil only) coated pellets for 14 days then challenged with Neoparamoeba spp. Results of 3 replicate systems combined.

110 Bithionol sulphoxide Control Garlic powder 100

80 % cumulative survival

60 -20-100 102030 Day post-exposure Figure 23. Percent cumulative survival of Atlantic salmon smolts fed a diet including bithionol sulphoxide, garlic powder or control (oil only) coated pellets for 14 days then challenged with Neoparamoeba spp. Results of 2 replicate systems combined, 1 replicate system removed due to high non-specific mortality.

Although there were no differences in the severity of disease as evidenced by the number of lesioned filaments on the gills, the difference in mortality rate between the treatments is interesting. A similar phenomenon has also been described with immunostimulant diets and AGD (Mlynarski et al. submitted, see below). It is possible that in the present study, bithionol and its metabolites may exert other physiological effects upon the fish, perhaps causing a vasodilation and mitigating the vascular hypertension and increase vascular resistance as has been described for this

disease (Powell et al. 2002; Leef et al. 2005, 2007 in press). However, this remains speculative and should be investigated further.

Semi-commercial experimental assessment of the efficacy of AquaciteTM and BetabecTM

Aquacite™ and Betabec™ diet showed increases in the growth parameters compared to the control fish (Table 8) but these were not statistically significant due to the low level of replication (n = 2). The two treatment pens had significantly increased SGR of 0.0091 and the growth rate for control pens was 0.0070 and 0.0077 (P = 0.037). Typically, the FCR for salmon fed the control diet was higher compared to fish fed the supplemented diet (P = 0.061) and on average, the control fish consumed more feed per day (P = 0.064). The condition factor increased similarly across all four pens (treatment and non-treatment) (P > 0.283) (Mlynarski et al submitted).

Table 8: Mean specific growth rate (SGR), feed conversion ratio (FCR), daily feed intake and condition factor (K) for Atlantic salmon fed a control or beta-glucan supplemented diet during a field study in a Neoparamoeba-endemic area (n = 30). (From Mlynarski et al submitted)

Treatment Pen SGR† FCR Feed K*± SEM Intake T18 T91 Control 1 0.0070a 4.00 1.03 1.10±0.065 1.24±0.052 2 0.0077a 1.87 0.93 1.05±0.100 1.24±0.033

Aquacite™ b 1 0.0091 1.44 0.72 1.13±0.091 1.21±0.028 & 2 0.0091b 1.22 0.72 1.09±0.080 1.29±0.018 Betabec™ † n=10 *assessed from day 18 (T18) until day 91 (T91)

The background levels of AGD were approximately 7.2 and 4.5% of gill filaments showing lesions in pens fed the control or supplemented diet (P = 0.204) (Fig. 26). However, over the study, all pens showed low levels of mortality, with notable increases coinciding with increases in the numbers of AGD lesions (Fig. 27). By the end of the treatment period, the relative percent survival of the Aquacite™ and Betabec™ supplemented pens was 37% compared to the control pens (Mlynarski et al. submitted).

50 Control Aquacite + Betabec

20 AGD lesioned (%) gill filaments 10

0 0 46607491 Day post exposure Figure 26. Mean (± SEM) numbers of gill filaments exhibiting AGD lesions over time following a freshwater bath in Atlantic salmon held at a Neoparamoeba-endemic aquaculture site (From Mlynarski et al submitted).

10 Control Aquacite + Betabec

(%) mortality Cumulative

0 0 4 7 2536414853606368778891 Day post exposure Figure 27. Mean (± SEM) cumulative mortality for Atlantic salmon fed either a control or beta-glucan and vitamin/bioflavenoid supplemented diet following a freshwater bath and held at a Neoparamoeba-endemic aquaculture site (From Mlynarski et al. submitted).

The overall objective for the study was to determine the impact of feeding beta-glucan and vitamin/bioflavenoid based Aquacite™ and Betabec™ on the growth, condition and AGD susceptibility of Atlantic salmon.. Salmon were supplemented with beta- glucans and vitamins/bioflavenoids for seven weeks in total. (It is possible that this may have led to a hypersensitivity, such as has been occasionally observed with over- dosing of immunostimulants, particularly beta-glucans (Bricknell et al., 2005), possibly compromising any effect on gill lesions.

The field trial showed increasing trends in growth for salmon fed the beta-glucan and vitamin/bioflavenoid supplemented diet compared to their control counterparts. Interestingly, the specific growth rate for salmon fed the supplemented diet was significantly greater compared to the controls, resulting in an average final weight 200g greater than fish fed the control diet. Overall, the results indicate that Aquacite™ and Betabec™ fed salmon had increased growth rates and lower feed intake and conversion ratios compared to the controls. This suggested that fish fed the supplemented diet consumed less feed, were more efficient at converting feed to mass and out-grew the fish fed the control diet. Additionally, fish fed the supplemented diet showed a delay in the onset of AGD lesions of at least 14 days and a reduction in the observed numbers of mortalities (Mlynarski et al. submitted).

The main immunostimulants in both Aquacite™ and Betabec™ are beta-glucans, which have been shown to increase phagocyte-macrophage activity in fish and stimulate the innate immune system when administered via an intraperitoneal (i.p.) injection or through the diet (Bridle et al., 2005). Atlantic salmon i.p. injected with beta-glucans had increased lysozyme activity, which may be due to increasing numbers of phagocytes secreting lysozymes and an incremental increase in the amount of lysozyme synthesized by these cells (Santaren et al., 1997). In vitro testing has revealed that commercially available beta-glucans can stimulate head kidney macrophage respiratory burst activity (Bridle et al., 2005). Moreover, the other ingredients of these two nutritional supplements including, bioflavonoids and vitamins, are known to stimulate the non-specific immune response (Hardie et al., 1990; Robertsen et al., 1990; Engstad et al., 1992; Lee et al., 1994), facilitate respiratory burst and growth activation during repairing of lesions (Erdal et al., 1991;

Hardie et al., 1991) and increase the immunocompetance of both humans and fish (Albrektsen and Sandnes, 1995).

Atlantic salmon, offered a commercial diet that was hammer-milled to incorporate commercially available beta-glucans, were exposed to an experimental challenge with Neoparamoeba spp. (Bridle et al., 2005). The resulting prevalence of AGD lesions was similar to that in control fish (Bridle et al., 2005). Rainbow trout experimentally challenged with another protozoan gill parasite, Loma salmonae showed both delayed onset and reduced intensity of branchial xenomas in fish i.p. injected with 100 µg of beta-glucans (Guselle et al., 2006). Interestingly, doses above (e.g. 500 and 1000 µg) and below (50 µg) produced inconsistent reductions in xenoma numbers and there was evidence of a hypersensitivity with the highest concentrations (Guselle et al. 2006). Similar results were also observed with dietary administration of beta-glucans resulting in a decreased prevalence of Loma morhua in Atlantic cod by 38-100% (Mackie, unpublished data).

This study revealed positive effects on growth and condition, as well as reducing mortalities in Atlantic salmon using dietary supplementation of beta-glucans, vitamins and bioflavenoids; however, there may have been a hypersensitivity with continual feeding. Disease due to Neoparamoeba spp. follows an annual cycle with outbreaks beginning in Spring and continuing through Summer, coinciding with the warmest water temperatures (Munday et al. 1988). Feeding a beta-glucan, vitamin and bioflavenoid supplemented diet, in anticipation of the predicted outbreaks, may reduce the numbers of mortalities and maintain commercially viable growth rates.

Metabolic cost of gill disease Tenacibaculosis Metabolic scope and metabolic rate Metabolic scope decreased significantly for fed infected fish from pre- to post- infection, the reduction represented an overall decrease of 40% from pre-infection levels (P = 0.036, Fig. 28). Also, metabolic scope decreased significantly in the unfed infected group with an overall reduction of 49% from pre-infection levels (P = 0.04, Fig. 20). Uninfected groups had no significant change in metabolic scope. The results suggested that fish with a bacterial infection have reduced capacity to perform work (Lankford et al., 2005). A decrease in metabolic scope has implications concerning energy allocation by individuals, fish with smaller metabolic scope will inevitably have less energy to allocate to functions such as the replenishment of energy stores growth, reproduction and immune response (Cutts et al., 2002; Lankford et al., 2005).

The reduction in metabolic scope was brought about due to a significant increase in routine metabolic rate in both fed and unfed treatments. Fed infected fish had an -1 -1 average increase in Mo2 rout of 1.86 µM O2 g h (± 0.66, mean ± SEM) whilst unfed -1 -1 infected fish had an average increase of 2.16 µM O2 g h (± 0.72, mean ± SEM). For a 100 g fish this translates into an extra 1.94 kJday-1 for a fed infected fish, and an extra 1.29 kJ day-1 for the unfed infected fish. It is thought that the increase in routine metabolic rate was a result of a significant increase in blood plasma osmolality seen in infected animals. This represents a significant increase in the energy expenditure required by the animal for routine functions. Maximum metabolic rate increased -1 -1 significantly for unfed uninfected fish with an average increase of 2.51 µM O2 g h

(± 0.61, mean ± SEM), whilst all other treatment maintained Mo2 max from pre- to post-infection levels. Maximum metabolic rate for infected Atlantic salmon did not change significantly from pre- to post-infection levels, suggesting that the increased costs associated with infection with T. maritimum were not compensated by an increase in maximum metabolic rate.

2 ) -1

.h 1 -1 .g 2

-1

-2 Metabolicscope (µM O

-3 * -4 * -5 Fed infected Fed uninfected Unfed infected Unfed uninfected

Figure 28. Mean (± SEM) change in metabolic scope from pre to post-infection of Atlantic salmon (S. salar) infected with T. maritimum. The asterix indicates significant change (P < 0.05) in metabolic scope from pre- to post-infection. Fed infected n = 19, unfed infected n = 8, fed uninfected n = 11, unfed uninfected n = 16. (From Jones et al submitted).

Amoebic Gill Disease Routine metabolic rate didn’t change for any of the groups from day 0 to day 10, however from day 10 to day 20 post-infection there was a significant increase in routine metabolic rate for both infected groups (fed and unfed). This corresponded to a significant increase in infection rates from day 10 to day 20. The change in routine metabolic rate effectively increased the energy requirements of the fish from a routine level of 4.07 kJ day-1 and 4.5 kJ day-1 for the fed and unfed treatments respectively, to 7.32 kJ day-1 and 7.49 kJ day-1. The increase in routine metabolic rate in fed infected fish effectively reduced metabolic scope by 40%, whilst unfed infected fish maintained metabolic scope (Fig. 29), despite the increase in routine metabolic rate, by increasing maximum metabolic rate. This increase in energy requirements will have a profound impact on the food conversion ratios of fish affected by AGD, as any increase in energy going towards maintaining the disease effectively means that it is being diverted away from processes such as growth. How pronounced this effect is

depends on a number of factors, predominantly the level of background infection maintained throughout a growing period, as the results suggest that an increase in routine metabolic rate only occurs above a certain threshold of infection. Infection levels on day 10 were around 10% infected lesions whilst on day 20 they were around 58% infected lesions, suggesting that the threshold lies between these two levels of infection. Maximum metabolic rate increased for all treatments except the fed infected treatment. This result suggests two things, firstly, the reduction in gill surface area commonly thought to occur due to AGD does not seem to impede the ability of Atlantic salmon to take in oxygen even when demand is high. Secondly, fish that have been starved over a long period of time may have the ability to increase maximum metabolic rate; this is thought to occur due a decrease in body mass whilst maintaining gill surface area, which effectively increases the gill surface area proportionally to body mass. However further investigation will be needed to fully understand the mechanisms behind the phenomena.

3 Fed infected Fed uninfected

) Unfed infected c -1 2 * Unfed uninfected .h bc -1

.g ns 2

0 ab

-1

-2 Change in metabolic scope (µM O

* a -3

-4 01020 time from infection (days) Figure 29. Mean (± SEM) change in metabolic scope from day 0 (time from infection) to day 10 and from day 0 to day 20 of Atlantic salmon exposed to AGD. Letters indicate significant differences between treatments on day 20, asterix indicates significant difference between day 0 and day 20. (From Jones et al submitted)

An adverse reaction to chloramine-T bathing

A commercial trial of chloramine-T in seawater was undertaken, with the support of the project team.

The treatments did not show any significant reduction in AGD and severe adverse reactions were seen in one of the three pens treated, leading to heavy losses of fish.

Clearly, on current knowledge, this treatment is neither sufficiently safe nor efficacious in standard commercial practice.

Benefits and Adoption This project has completed its objectives to investigate the feasibility and efficacy of alternative treatments for amoebic gill disease in salmon. It has used a tiered screening process to identify potential candidate treatments and screen the potential efficacy of a number of disinfectant or potentially in-feed amoebicidal compounds. From these studies several potentially useful in-feed amoebicides have been identified, as well as compounds to promote feeding and improved performance in AGD affected salmon. The primary focus of the project was for industry to have a direct input into the project with regard to how treatments were developed and commercialised. This meant that some avenues of research were terminated early with industry believing no further development was necessary (for example research into the use of AquaciteTM and BetabecTM). It should be noted that industry has not (at the time of compiling this report) adopted the results from this project or further commercialised its outcomes.

A commercial trial of Chloramine-T baths was unfortunately halted by the adverse reaction occurring in one of the trial pens. The lack of efficacy seen in the commercial scale trial contradicts the results of previous studies, although the conditions of its use in the commercial trial differed significantly from those studies (Harris et al 2004; 2005; Powell et al. 2005). These two issues suggest that seawater chloramine-T baths, at least under the conditions identified in the commercial trial, are unlikely to be a practicable treatment for AGD affected Atlantic salmon in Tasmania.

Further development Emerging from this project have been several potential in-feed amoebicidal treatments that appear to show some possibilities for further development. In particular, bithionol shows considerable promise with an approximately 50% reduction in AGD severity when fed to fish under laboratory challenge. This does, however, require further validation and small scale field testing would be a logical next step. In addition, the pharmacokinetic parameters for this drug also need to be determined for bithionol and withdrawal times established for Atlantic salmon under Tasmanian culture conditions. This latter step was beyond the remit of this project.

Although results with regard to the efficacy of ionophores were disappointing, there was some suggestion that marginal reductions in the severity of AGD was achieved with salinomycin. With this in mind it is reasonable that further investigation of these compounds as potential amoebicides be pursued. Of particular significance is the synergistic potential of some of these drugs (and others tested in vitro by Powell et al. 2005, such as amprolium) whereby increased efficacy can be achieved by the inhibition of physiological systems designed to eliminate the treatment drug in the host, so improving potential efficacy against a target. Since little is known about the effects of combined treatments in fish, this is an area where a great deal of further investigation is required.

Planned outcomes

The relevant outcomes stated in the Aquafin CRC Commonwealth Agreement were: (a) An effective and economic industry treatment regime for the removal of Neoparamoeba from the gills of farmed salmon. This was progressed as described below. (b) Measures to mitigate potential side-effects or residual toxicity of treatment(s) for AGD. As no commercial-ready treatment has emerged, this outcome could not be addressed.

The specific project outcomes were as follows: 1. Provide a cost-effective bath treatment for the control of AGD. This project has identified a number of treatments that have been developed to the point where industrial adoption could be considered. Of particular interest was chloramine-T in seawater, but it is clear that it is unlikely to be commercially practicable if used under standard farm conditions. However, AquaciteTM and BetabecTM treatments show some potential for improving salmon health and reducing mortality in general with some suggestions that growth may be marginally enhanced although the clinical signs of AGD were not reduced. The reduction in mortality was equivalent to or better than that demonstrated so far by current investigations into vaccination against AGD. The cost of Aquacite/Betabec treatment is likely to be in the range of a premium of $120 per tonne feed, equivalent to the immunostimulant diets currently being provided to the aquaculture industry by Skretting and Ridley’s feed.

2. Identify potential in-feed treatments for the control of AGD. This project has tested a number of candidate in-feed amoebicidal treatments and identified a number of successful candidates. The most promising results to date have been with the anti-protozoal drug bithionol. As a drug registered for use in humans and in other mammals it has potential to be extended to fish. This treatment when fed to fish at 25 mg kg-1 feed showed significant reductions in both the gross gill score as well as the severity of AGD as determined by histological lesions. At a cost of approximately$120 AUD per tonne of feed it is within an acceptable cost for commercial use. The purchase of significant quantities of bithionol may also reduce

its cost based on an economy of scale. It was unfortunate that the cheaper metabolite of bithionol (bithionol sulphoxide) was ineffective at reducing gill lesion numbers. Bithionol sulphoxide would add a premium cost of approximately $20 per tonne to feed. However, in laboratory studies, we have demonstrated that bithionol sulphoxide may have reduced mortality rates during the Neoparamoeba spp. Challenge, suggesting potential benefits in commercial scale production. This however, needs to be investigated further under commercial conditions.

Other in-feed potential amoebicides tested, such as ionophore treatments, do not appear to be as effective as bithionol. Similarly, unlike L-cysteine ethyl ester (LCEE), the cheaper mucolytic analogue N-acetyl cysteine (NAC) was ineffective at reducing AGD-lesion severity. Mucolytic treatments may have some commercial application for the control of AGD although these would have to be based upon LCEE rather than NAC.

Conclusion In conclusion, this project has undertaken a wide range of testing of potential treatments for the control of amoebic gill disease and maintenance of salmon health. Of these treatments, in vitro studies identified potential amoebicides that were then tested further for efficacy in vivo. In addition treatments identified in other studies (Powell et al. 2005) were tested further to commercial scale. It is possible that some of the candidate treatments, both amoebicidal (such as bithionol) and immunostimulatory (such as Aquacite/Betabec) may have potential for further commercial development.

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Appendix 1: Intellectual Property The intellectual property and valuable information arising form this report are: 1. Copyright of this report

Appendix 2: Staff

Dr. Mark D. Powell Project Leader Dr. Joy A. Becker Post-Doctoral Research Fellow Ms Renee L. Florent PhD student Mr Matthew Jones PhD student Ms Julie Ransome Research technician Ms Megan Barney Research technician Ms Christine Paetzold Casual research technician Assoc Prof Rahim Peyghan Visiting research associate Mr Raymond Duijf Visiting research associate

Appendix 3: Publications and presentations from this project (at time of going to press)

Peer-reviewed publications

1 Peyghan, R and Powell, M.D. (2006). Histopathological study of gills in experimentally amoebic gill disease (AGD) infected Atlantic salmon, Salmo salar L. Iranian Journal of Veterinary Research 7: (in press).

2 Florent, R.L., Becker, J.A. and Powell, M.D. (2006). Evaluation of bithionol used as a bath treatment for amoebic gill disease (AGD) treatment for amoebic gill disease caused by Neoparamoeba spp. Veterinary Parasitology (in press).

3 Powell, M.D., Ransome, J., Barney, M. Duijf, R. and Flik, G. Effect of dietary inclusion of N-acetyl cysteine on mucus viscosity and susceptibility of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) to amoebic gill disease. Journal of the World Aquaculture Society (in press).

4 Mlynarski, H., Becker, J.A., Mackie, J.A. and Powell, M.D. Effects of oral administration of beta-glucan-based nutritional supplements in Atlantic salmon (Salmo salar) challenged with the protozoan Neoparamoeba spp. (under revision).

5 Peyghan, R. and Powell, M.D. In vitro toxicity testing of amoebocidal effect of garlic extract and metronidazole against Neoparamoeba pemaquidensis, Page 1987 and isolated ameobae from the AGD affected Atlantic salmon. Aquaculture International (submitted).

6 Florent, R.L., Becker, J.A. and Powell, M.D. Efficacy of bithionol as an oral treatment for amoebic gill disease in Atlantic salmon Salmo salar (L.). Aquaculture ( in press).

7 Jones, M., Becker, J.A., Carter, C.G. and Powell, M.D. Effect of nutritional state and infection with Tenacibaculum maritimum on the metabolic rate of Atlantic salmon (Salmo salar). Diseases of Aquatic Organisms (submitted).

Conference presentations 1 Powell, M.D. (2004). Development of seawater treatments for amoebic gill disease. Australian Aquaculture 2004: profiting from sustainability. Sydney Convention Centre, Sydney NSW, Australia Sep 26-29.

2 Powell, M.D. (2005). Treatments for parasitic diseases. Aquafin CRC Conference. Corus Hotel, Hobart TAS, Australia Jul 5-7.

3 Mlynarski, H., Powell, M.D., Burgess, J., Dix, T., Taylor, R and Mackie, J. (2005). Efficacy on Aquacite on the Growth of Atlantic salmon, Salmo salar L. Aquafin CRC Conference. Corus Hotel, Hobart TAS, Australia Jul 5-7.

4 Jones, M.A., Powell, M.D. and Carter C.G. (2005). Metabolic scope of Atlantic salmon is reduced by disease. Aquafin CRC Conference. Corus Hotel, Hobart TAS, Australia Jul 5-7.

5 Burgess, J., Duijf, R., Barney, M. and Powell, M.D. (2005). The use of N-acetyl- L-cysteine as an in-feed retardant of AGD infection in salmonids. Aquafin CRC Conference. Corus Hotel, Hobart TAS, Australia Jul 5-7.

6 Villavedra, M., To, J., Lemke, S., Powell, M.D., Butler, R., Crosbie, P., Broady, K., Wallach, M. and Raison, R.L. (2005). AGD vaccine: an antibody approach. Aquafin CRC Conference. Corus Hotel, Hobart TAS, Australia Jul 5-7.

7 Powell, M.D. (2005). Pathophysiology of gill diseases. FRDC Aquatic Animal Health Subprogram Scientific conference Rydges Hotel Resort, Cairns QLD, Australia Jul 26-28.

8 Powell, M.D., Roberts, S.D., Duijf, R., Burgess, J. and Barney, M (2005). Oral mucolytic drugs and their protective effects against amoebic gill disease in

salmonids. FRDC Aquatic Animal Health Subprogram Scientific conference Rydges Hotel Resort, Cairns QLD, Australia Jul 26-28.

9 Jones, M.A., Powell, M.D. and Carter C.G. (2005). Metabolic scope of Atlantic salmon is reduced by disease. FRDC Aquatic Animal Health Subprogram Scientific conference Rydges Hotel Resort, Cairns QLD, Australia Jul 26-28.

10 Florent, R.L. and Powell, M.D. (2005). Examination of the efficacy and toxicity of bithionol as a bath treatment for rainbow trout Oncorhynchus mykiss affected by amoebic gill disease. FRDC Aquatic Animal Health Subprogram Scientific conference Rydges Hotel Resort, Cairns QLD, Australia Jul 26-28.

11 Florent, R.L. and Powell, M.D. (2005). Examination of the efficacy and toxicity of bithionol as a bath treatment for rainbow trout Oncorhynchus mykiss affected by amoebic gill disease. 12 th Meeting of the European Association of Fish Pathologists, Copenhagen, Denmark, Sept 12-15

12 Powell, M.D., Green, T., Florent, R., Jones, M.,Attard, M., Burgess, J., Mlynarski, H. and Mackie, J.A. (2005). Aquacite as a feed supplement maintains growth of Atlantic salmon smolts with amoebic gill disease under laboratory conditions. 12 th Meeting of the European Association of Fish Pathologists, Copenhagen, Denmark, Sept 12-15

13 Jones, M.A., Powell, M.D., Becker, J. A. and Carter, C.G. (2006). The effects of gill diseases on the metabolic rate of Atlantic salmon. 12th International Symposium on Fish Nutrition and Feeding, Biarritz France May 28-31.

14 Powell, M.D., Leef, M.J. and Jones, M. (2006). Respiratory, metabolic and cardiovascular responses of salmonids to infection with Neoparamoeba spp. 7th International Congress on the Biology of Fish, St. John’s Newfoundland, Canada. July 18-22.

15 Becker, J.A., Florent R.L. and Powell, M.D. (2006). Developing drug treatment models for aquaculture. 5th International Symposium on Aquatic Animal Health, San Francisco Marriott Hotel, San Francisco, California USA. Sept 2-6.