Effects of catecholamines on Yersinia ruckeri and yersiniosis in Atlantic salmon (Salmo salar)

Since its inception in the 1980s Atlantic salmon (Salmo salar) aquaculture in Tasmania, Australia has expanded at an ever-increasing rate and is the highest value commercial fishery related industry in Australia. The growth of the Tasmanian industry is expected to continue as are concerns regarding the environmental impacts and potential diseases associated with the rapidly expanding industry. These concerns are common to all intensive culture environments where stress and its impacts on animal health cannot be avoided. The response of fish to stress is not unlike that of farmed terrestrial animals where stress induces the release of stress hormones including catecholamines that can negatively impact the animal’s immune response. Stress hormones may also affect potential bacterial pathogens as bacteria are able to sense catecholamines resulting in enhanced growth and/or virulence. Yersinia ruckeri is the aetiological agent of enteric redmouth disease or yersiniosis in Atlantic salmon. Despite the success of yersiniosis vaccination disease outbreaks are still reported implying a greater understanding of this bacterium and its relationship to the host and the aquaculture environment is needed to improve fish health management in response to this disease. This research aimed to explore the effects of catecholamines on virulence factors of Y. ruckeri in vitro and the host immune response and gut microbiome changes of Atlantic salmon exposed to catecholamine treated Y. ruckeri. Exposure of Y. ruckeri in vitro to catecholamines was found to enhance the growth and increase activity of known virulence factors of Y. ruckeri including lipase, haemolysin, siderophore and biofilm production while not altering the inhibitory effects of selected antibiotics on Y. ruckeri. More conclusively, epinephrine was found to increase the virulence of Y. ruckeri in vivo as demonstrated from an experimental infection challenge that found Atlantic salmon exposed to catecholamine-treated Y. ruckeri had a higher mortality compared to salmon challenged with untreated Y. ruckeri. To investigate the effects of stress on the host immune response and Y. ruckeri infection Atlantic salmon were subject to an acute stress by reducing the tank water levels to 50% and chasing the fish for 10 minutes. This treatment did not induce any significant changes in fish plasma cortisol when samples were taken from 12 hours onwards post-acute stressor. The survival rate of fish subjected to the acute stressor post challenge with Y. ruckeri was not significantly different to that of unstressed fish. Differential gene expression of select host immune-related genes was found to be related to both stress and/or disease challenge. Fish subjected to the acute stressor had a significant upregulation of whole blood interleukin 12β (IL12β) and downregulation of cluster of differentiation 8 alpha (CD8α) at day 2 after the stress event. When assessing the host response to disease challenge a 25-fold increase in the mRNA expression of the interferon IFNγ was found compared to unchallenged fish at day 1 post infection. The relative expression of interleukin 12 beta (IL 12β) in challenged fish was also significantly expressed at day 10 in comparison to day 1 while a significant reduction was observed in tumor necrosis factor alpha (TNFα), IFNγ and interleukin 6 (IL 6) at day 30 compared to day 1. Prior exposure Y. ruckeri to epinephrine significantly increased the expression of immunoglobin M (IgM) (day1) (2.5-fold), TNFα (3.8-fold) and interleukin 22 (IL 22) (8.4-fold) (day 30) compared to fish challenged with control Y. ruckeri. In contrast, the expressions of IFNγ and IL 6 were 39.5 and 17.2-fold lower in Y. ruckeri exposed to epinephrine at day 1 suggesting that a reduction in these two important immune genes is associated with increased virulence. Microbiomes were assessed using 16S rRNA amplicon sequencing where the most abundant phylum in the gut microbiome of Atlantic salmon was Proteobacteria (42%), Actinobacteria (19%) and Firmicutes (19%). At genus level, the major genera were Propionibacterium (19%), Sphingomonas (14%), Aeromonas (11%) and Staphylococcus (8%). No significant difference in alpha diversity indices was found. Analysis of beta diversity showed no difference in the community structure between the midgut and hindgut. Importantly, significant differences were identified the between stressed and unstressed fish, fish challenged (with and without epinephrine treated Y. ruckeri) and unchallenged as well as between different sampling times. Abundance of families Aeromonadaceae and Staphylococcaceae was more dependent on different challenge treatments while abundance of Sphingomonadaceae and Burkholderiaceae was related to sampling time. The findings of this thesis confirm that Y. ruckeri is able to sense stress-related hormones in the form of catecholamines and provides potential mechanisms at the transcriptional and functional level of how stress to the host translates to increased susceptibility to disease. These findings improve our understanding of the interaction between stress, virulence factors of Y. ruckeri and the Atlantic salmon immune response. Furthermore, apart from consolidating the accepted view that stress negatively impacts fish health and should be minimised it introduces the possibility of manipulating the host microbiome to potentially overcome or minimise the consequences of stressors on fish health that are common to aquaculture