Improving the treatment of amoebic gill disease in salmonids with soft freshwater and the mucolytic drug L-cysteine ethyl ester

Amoebic gill disease (AGD) is the single most significant disease affecting Tasmanian marine salmonid farming. The current treatment of AGD is freshwater bathing affected sea-caged fish. However, this current method of treatment is becoming less effective with an apparent increase in bathing frequency over the years fuelling a rise in production costs to the Tasmanian Atlantic salmon industry. The research reported in this thesis primarily aimed to identify an improved method of treatment that not only has an enhanced efficacy compared to the current treatment, but also exhibits minimal physiological consequences to treated fish. Initial experiments aimed at assessing the pathophysiological effects of AGD and freshwater bathing, with the intention for later comparison to improved and novel treatments. Freshwater· bathing elicited a minor ionoregulatory, and possibly acid base, dysfunction that was enhanced in AGD-affected Atlantic salmon (Salmo salar L.). Significant histochemical changes to gill mucous cell populations were also documented in response to salinity and AGD. These results highlighted the potential importance of the mucous layer in differing saline conditions and disease events. Freshwater bathing was subsequently refined and its effectiveness enhanced with the use of soft freshwater as opposed to hard freshwater. Commercial salmonid farms have access to a number of freshwater sources that vary considerably in their total hardness (mg L-1 CaC03). We found that soft freshwater bathing delayed re-infection and subsequent pathology in treated Atlantic salmon by 2 weeks, representing a significant financial saving to the industry. From blood plasma analyses, no significant effect of soft freshwater bathing was found. It was documented that one of the therapeutic effects that :freshwater bathing offers is its ability to reduce mucus viscosity, aiding flushing of gill-associated amoebae and hyperplastic tissue. Soft freshwater was found to significantly reduce mucus viscosity by 1 h into bathing (P<0.05) whereas hard freshwater significantly reduced the viscosity by 3 h (P<0.05). Due to the effective action of freshwater bathing on fish mucus, our research progressed to the trial of the mucolytic agent L-cysteine ethyl ester (LCEE) as an in feed additive. Cysteine based mucolytics are widely used in human and domestic ' animal medicines, and LCEE has previously been tested in aquaculture to protect coho salmon (Oncorhynchus kisutch Walbaum) against a harmful diatom. We tested LCEE both in vitro and in vivo for its potential benefit at alleviating AGD. As an in feed additive, LCEE significantly delayed the progression of AGD pathology by 50 57 %, compared to controls, in Atlantic salmon. Prophylactic administration for 2 weeks at 45-50 mg LCEE kg-1 fish dai1 prior to, or at the lowest point of infection, was found to be the most successful oral delivery method. No detrimental physiological consequences were found in LCEE-treated fish, even when freshwater bathing followed prolonged medication. The mechanism of action of LCEE was in the alteration of the hosts mucous layer, with no effect on isolated gill amoebae. An underlying theme that required an intimate understanding for the assessment of improved and novel treatments was that of fish mucus. The limited knowledge in the literature on fish mucus also paved the way for some interesting and much needed research. Gill mucous cell histochemistry and cutaneous mucus viscosity and biochemistry showed significant differences between three salmonid species; Atlantic salmon (S. salar), brown trout (S. trutta) and rainbow trout (Oncorhynchus mykiss). Significant differences due to salinity and AGD were also documented. Some important findings were that mucus viscosity was significantly greater in seawater compared to freshwater fish for all three species, and significantly reduced due to AGD in Atlantic salmon and brown trout. Trends for all three salmonids were shifts in mucous cell glycoproteins from neutral mucins in freshwater to acidic mucins in seawater, and shifts towards neutral mucins, with an increase in mucous cell numbers, due to AGD. Such findings not only hold implications for disease management, but also aid in a better understanding of differing fish physiologies and disease susceptibilities. The research conducted for this thesis has identified improvements for the treatment of AGD and represents new pathophysiological information on a gill disease in three euryhaline salmonid species. In addition, significant contributions have been made to the infant field of fish mucology. The documented findings will hopefully pave the way for future research into fish disease treatments and pathophysiology.