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Atlanctic salmon bacterial challenge

In order to investigate the immunomodulatory properties of the phytogenic compounds against bacterial infection, an experimental bacterial challenge with the strain IRTA-17-44 of Asalmonicida subsp. salmonicida was performed at the end of the nutritional trial. Bacterial suspensions of the selected strain were prepared from a stock stored in glycerol at -80°C. The inoculum was grown in TSA at 23.0 ± 1.0°C for 48 h. The bacterial inoculum was prepared to an OD of λ = 550 nm of 1.2, corresponding to a density of 108 CFU/ml previously established by serial dilutions and plate counting. The bacterial suspension was 10-fold serially diluted in sterile PBS, to prepare the desired inoculum, which was confirmed by CFU’s plate counting. Prior to the challenge trial, an Asalmonicida (IRTA-17-44) lethal dose of 50% (LD50) was determined for the experimental conditions to be assayed. For this purpose, 30 control Atlantic salmon were injected intraperitoneally (IP) with 0.2 ml of three concentrations of Asalmonicida inoculum, 106, 107, and 108 CFU/mL (10 fish injected with each inoculum concentration). Ten additional fish were injected with PBS as methodological control. The concentration of 107 CFU/mL was established as the nearest LD50 (data not shown).

For the challenge trial, 32 Atlantic salmon smolts (BW = 194.0 ± 29.1 g) per each dietary treatment were randomly distributed (https://www.randomizer.org) into quadruplicate tanks (four tanks per dietary treatment), with eight fish per tank (stocking density = 14–16 kg m-3). During the acclimation period (5 days), fish were fed ad libitum with the same experimental diets used in the nutritional assay. After acclimation, fish were anaesthetized and IP injected with 0.2 ml of 107 CFU/ml of Asalmonicida (IRTA-17-44).

Both the establishment of the Asalmonicida LD50 and the challenge trial were performed at IRTA’s biosafety challenge room, in 32 cylindrical tanks (100 l) connected to a RAS unit (IRTAmar®) equipped with real-time control of oxygen and temperature, mechanical filtration, biofiltration, and ultraviolet disinfection of the water. The outflow water was chlorinated, followed by ozone treatment before being discharged. The water quality conditions in terms of temperature and salinity were 13.1 ± 1.1°C and 32.3 ± 0.4 ppt, respectively.

Firmino, J., M. D. Furones, K. B. Andree, C. Sarasquete, J. B. Ortiz-Delgado, G. Asencio-Alcudia and E. Gisbert (2019). “Contrasting outcomes of Vibrio harveyi pathogenicity in gilthead seabream, Sparus aurata and European seabass, Dicentrachus labrax.” Aquaculture 511. Elsevier BV. doi:10.1016/j.aquaculture.2019.734210.

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