LAUSR

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is present in all Atlantic salmon (Salmo salar L.) farming countries with significant economic and welfare impacts (Oldham, Rodger, & Nowak, 2016). Infected fish develop proliferation of the lamellar epithelium and increased production of gill mucus (Adams & Nowak, 2003). Oxidative stress has been suggested as one of the mechanisms involved in AGD pathogenesis (Loo, Sutton, & Schuller, 2012; Marcos-L opez et al., 2017; Wynne et al., 2008), but to the best of our knowledge, no enzymatic studies have been undertaken to date. Oxidative stress occurs when the balance between antioxidant enzymes and reactive oxygen species (ROS) is disrupted either because of depletion of antioxidants or accumulation of ROS. Oxidative stress in finfish gills has been widely studied in response to abiotic stressors, but less is known regarding the possible effects caused by pathogens. This study aimed to assess the antioxidant status in gills of farmed Atlantic salmon naturally infected with N. perurans through the analysis of the hydrophilic antioxidant activity (HAA) and the activity of major antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), as well as gene expression of SOD and CAT. Gill samples were obtained from a commercial farm in Ireland, where na€ıve S1 smolts were monitored from sea transfer to after first freshwater (FW) bath treatment against AGD (<3 practical salinity units (PSU) for 3 hr). Gill scoring for gross AGD lesions [as described by Taylor, Muller, Cook, Kube, & Elliott, 2009;] was undertaken weekly, and samples were taken at gill score (GS) 0, GS 2 and four days after FW treatment. At each sampling point, five fish from the same pen showing equal GS were euthanised with an overdose of anaesthetic [400 mg/L of tricaine methanesulfonate (tricaine 1000 mg/L, Pharmaq)] and gill samples excised for enzymatic activity and gene expression analysis. Separate samples comprising gill areas with and without lesions were taken. Samples were snap-frozen in dry ice and stored at 80°C. In addition, histology samples and gill swabs for N. perurans real-time PCR (Downes et al., 2015) were taken to confirm AGD and the presence of N. perurans. Average weight and water temperature for the three sampling points were 85.2g, 312.1g and 375.3g and 9°C, 15.0°C and 15.2°C, respectively. For the enzymatic assays, gill samples were homogenized in a potassium phosphate buffer (50 mM, pH 7.0), and total protein concentration was determined by the Bradford method. HAA was measured based on the ability of the antioxidants in the sample to reduce the radical cation of 2,20-azino-bis-3-(ethylbenzothiazoline-6sulphonic acid) (ABTS), determined by the decolouration of ABTS and measuring the quenching of the absorbance at 730 nm (Arnao, Cano, & Acosta, 1999). Sample values were compared with a standard curve of ascorbic acid and expressed as ascorbic acid equivalents (mM) per mg of protein. SOD activity was calculated by the inhibition of the reduction in cytochrome C. The superoxide radical Received: 23 April 2017 | Revised: 10 July 2017 | Accepted: 12 July 2017 DOI: 10.1111/jfd.12699