Treatment of amoebic gill disease (AGD) caused by Neoparamoeba perurans in Atlantic salmon (Salmo Salar)

Neoparamoebae perurans is a marine protozoan parasite that infects gills of cultured Atlantic salmon, Salmo salar, causing amoebic gill disease (AGD) which is currently reported in most salmon producing countries. In Tasmania, the management of AGD in Atlantic salmon accounts for 20% of production costs. As a result, several studies have been done to explore alternative treatment methods. However, previous studies other than freshwater bathing focused solely on testing chemotherapeutants against AGD. With the exception of garlic Allium sativum and AQUI-S\\(^¬¨vÜ\\) (product based on clove oil), no other plant products have been tested against AGD. In the first study, the effects of clove oil and its derivative AQUI-S\\(^¬¨vÜ\\) against N. perurans were studied. N. perurans trophozoites were exposed to clove oil at 10, 20, 40 and 80 ˜í¬¿L L\\(^{-1}\\) for 10 min and 5, 10, 20 and 40 ˜í¬¿L L\\(^{-1}\\) for 120 min; AQUI-S\\(^¬¨vÜ\\) at 5, 10, 20 and 40 ˜í¬¿L L\\(^{-1}\\) for 10 min and 2.5, 5, 10 and 20 ˜í¬¿L L\\(^{-1}\\) for 120 min. There were no significant differences in viability and survival of trophozoites after exposure to the anaesthetics. When trophozoites were exposed to clove oil at 80 ˜í¬¿L L\\(^{-1}\\) for 10 min there was significantly (P < 0.05) more detachment from a plastic surface compared to the control. Continued use of clove oil and AQUI-S\\(^¬¨vÜ\\) at 40 ˜í¬¿L L\\(^{-1}\\) or at lower concentrations for up to 120 min is unlikely to have a detrimental impact on amoebae that are isolated and collected from salmon with AGD to be used for AGD research. In the second study, viability estimations using viability assays were compared to manual counting using trypan blue and neutral red vital dyes. The four viability assays examined were CellTiter-Blue\\(^¬¨vÜ\\), CellTiter-Glo\\(^¬¨vÜ\\), CellTiter-Fluor‚Äöv묢 and CytoTox-ONE‚Äöv묢. A stock solution comprising of 50% live cells and 50% dead cells (25% heat inactivated, 25% formaldehyde inactivated) was used. All viability assays and manual counting using neutral red estimated viability of cells in stock solution to be approximately 50%, except trypan blue which overestimated cell viability. A positive linear relationship between the signal generated and the number of live cells was observed when the maximum number of viable cells did not exceed 1500. However, positive linear relationship between signal generated and the number of live cells was only observed in CellTiter-Glo\\(^¬¨vÜ\\) and CellTiter-Fluor‚Äöv묢 when the maximum number of viable cells did not exceed 10000. CellTiter-Glo\\(^¬¨vÜ\\) was determined to be the most suitable viability assay due to the ease of use and the ability to maintain linearity over a wide range of viable cells. In the third study, efficacy of GTE (green tea extract) and OLE (olive leaf extract) against N. perurans was examined in vitro using gill isolated amoebae and exposing them to GTE at 9, 19, 38, 75, 150 and 300 mg L\\(^{-1}\\) and OLE at 12500, 6250, 1562, 780 and 390 mg L\\(^{-1}\\) . OLE and GTE were found to be effective against N. perurans at 780 mg L\\(^{-1}\\) and 150 mg L\\(^{-1}\\) respectively. Safety of GTE and OLE to Atlantic salmon were examined using bath treatment of either 1500 mg L\\(^{-1}\\) OLE or 75, 150 & 300 mg L\\(^{-1}\\) GTE in seawater; only OLE was considered safe. Subsequently in the fourth study, efficacy of OLE as bath treatment for AGD was studied by bathing AGD affected salmon at 780 mg L\\(^{-1}\\) for 1 h. OLE significantly reduced the prevalence of fish affected by AGD both immediately after and seven days after bath treatment. This thesis identified OLE as a potential therapeutic compound against AGD caused by N. perurans. This finding serves as a proof of concept that plant products such as OLE could have therapeutic effects against AGD. This indicates that OLE could be worth examining against other ectoparasitic diseases and OLE warrants further investigations as a potential in-feed treatment for AGD in Atlantic salmon.