LAUSR

Flavobacterium psychrophilum is one of the most important pathogens affecting rainbow trout (Oncorhynchus mykiss) worldwide at the fry stage of their life cycle. Studies have shown disruption of the fish's skin mucus to be a critical element in the establishment of F. psychrophilum infection in rainbow trout (Henriksen et al., 2013; Madetoja et al., 2000; Martínez et al., 2004). Mucus is the first barrier (biochemical and physical) of the fish, playing a vital role in the protection against pathogens, and contains various antibacterial and inflammatory factors such as lysozyme, immunoglobulin, complement, lectins, acutephase proteins and proteases (Shunsuke, 2016; Subramanian et al., 2007). Furthermore, the study of external fish mucus provides nonlethal alternatives for the early detection of infections. In the current study, we examined the proteins of skin mucus of rainbow trout following immersion vaccination with F. psychrophilum. The influence of the route of infection on mucus proteins was also investigated by comparing skin mucus collected from fish challenged with F. psychrophilum by intramuscular injection to fish challenged with immersion. Skin mucus was subjected to 2D sodium dodecyl sulphate– polyacrylamide gel electrophoresis (2D SDSPAGE) and spots differentially expressed between vaccinated and control fish were analysed by matrixassisted laser desorption/ionization timeofflight mass spectrometry (MALDITOF MS). Proteomic studies to elucidate host responses in fish vaccinated and/or infected with F. psychrophilum are limited, and to our knowledge, this is the first report on proteomics of skin mucus of trout in response to mucosal vaccination and mucosal challenge with this pathogen. Rainbow trout fry (3– 4 g) and 15g rainbow trout were maintained in flowthrough (5 L/min) dechlorinated tap water at 15°C at the Institute of Aquaculture, University of Stirling, UK. The fish were fed at 2% body weight/day (Inicio feed, BioMar). The F. psychrophilumfree status of the fish was determined by streaking samples of head kidney and spleen onto the modified Veggietone medium followed by a nested PCR (Toyama et al., 1994). Fry were immersionvaccinated with a polyvalent F. psychrophilum vaccine (Hoare et al., 2017); controls were immersed in tank water using the same procedure. At 630 degreedays postvaccination (dd pv), the fish were starved for 24 hr prior to immersion challenge according to Ref. (Hoare et al., 2017). The mucus of fish (vaccinated and unvaccinated) was sampled postvaccination prior to challenge, and again 32 days after the challenge as described by Ref. (Hoare et al., 2017). Briefly, skin mucus was sampled by placing three fish (from each duplicate tank giving 2 pools of mucus/group) into a plastic bag containing 5 ml of Trisbuffered saline (TBS: 10 mM Tris base and 0.5 M NaCl, pH 7.5) and gently massaging for 2 min. Fish were removed, and the mucus was collected into a centrifuge tube and placed on ice. Any mucus samples contaminated with blood were discarded. The mucus was vortexed vigorously (10 s) and centrifuged at 4000 x g for 15 min, and the supernatant was filtered through