Role of bacteria in amoebic gill disease

Neoparamoeba spp., are the causative agent of amoebic gill disease (AGD) in marine farmed Atlantic salmon, Salmo salar and AGD is the major problem faced by the salmonid industry in Tasmania. The only effective treatment to control AGD is freshwater bathing; however, complete removal of the parasite is not achieved and under favourable conditions AGD can reoccur within 10 days. Previous research on AGD suggests that gill bacteria might be one of the factors influencing colonisation of Neoparamoeba spp. onto Atlantic salmon gills. Therefore, the aim of this project was to investigate the role of salmonid gill bacteria in AGD. To obtain good understanding of the bacterial populations present on Atlantic salmon gills, bacteria samples were collected twice during this study. Initially, bacteria were cultured from AGD-affected and unaffected fish both from laboratory and farm. These bacteria were characterised based on the colony morphology and biochemical profiles and identified using 16S rRNA gene-based approach. The bacteria from the genera Winogradskyella and Staphylococcus predominantly colonised the gills of AGD-positive Atlantic salmon; these bacteria were absent on AGD-negative samples. AGD-negative fish had a varied distribution of Gammaproteobacteria (Shewanella baltica, Idiomarina spp., Pseudomonas spp. and Halomonas sp.) and Cellulophaga spp., Arthrobacter rhombi, Arenibacter troitsensis and Flavobacterium sp.. Further, a series of in vivo experiments were conducted to study the influence of bacteria in AGD. The result showed an apparent involvement of Winogradskyella sp. in AGD. Fish experimentally infected with this bacterium developed significantly more filaments (51% in chapter 5; 17% and 21% in chapter 6) with AGD lesions following challenge with Neoparamoeba spp. compared to the group that was exposed to Neoparamoeba spp. alone (16% in chapter 5 and 8% in chapter 6). Furthermore, this study also confirmed that Neoparamoeba spp. are able to infect salmonid gills and cause AGD with very low levels of culturable bacteria on the gills. Based on the above results, it was important to verify whether bacteria such as Winogradskyella spp. that could exacerbate AGD conditions frequently colonise the gills of AGD-affected fish. Hence, a field-based study was conducted and bacteria samples were collected from marine farmed Atlantic salmon gills on five different occasions over a period of 102 days. The results showed that Winogradskyella species were not consistently present on AGD-affected fish gills. However, it was not possible to determine which bacteria will colonise the gills of AGD affected fish in a given period of time because the changes occurring within the gill bacterial population appeared to be complex and unpredictable and were influenced by several factors. In conclusion, this study verified that the presence of bacteria on salmonid gills is not necessary to induce Neoparamoeba spp. infection; which further suggests that Neoparamoeba spp. is the primary causative agent of AGD. However, this study also demonstrated that the presence of Winogradskyella sp. on the gills during the process of AGD infection significantly increased AGD severity. This work forms the basis for future studies assessing the effect of bacteria and/or amoeba on fish and in investigating the role of other microorganisms that are recognized as potential factors influencing AGD.