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Rainbow trout pathogenic bacterial challenge

To investigate whether the replacement of FM by increased levels of BSCP compromised the immune response of rainbow trout juveniles fed experimental diets, an in vivo bacterial challenge with the strain IRTA-17-44 of A. salmonicida subsp. salmonicida was performed at the end of the nutritional period. For this purpose, bacterial suspensions were prepared from a stock stored in glycerol at −80 °C, defrosted and grown in TSA (23.0 ± 1.0 °C for 48 h) and prepared to an optical density (OD550 nm) of 1.23, corresponding to a density of 108 CFU/ml that was 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, the lethal dose of 50% (LD50) for A. salmonicida subsp. salmonicida was determined using fish fed the control diet (D1). For this purpose, 30 rainbow trouts were injected intraperitoneally (IP) with 0.2 ml of three concentrations of the pathogenic bacterial 106, 107, and 108 CFU/ml (10 fish injected with each dose). Additionally, ten 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, twenty-four trout per dietary group weighting between 185 and 205 g were selected and transferred to forty 100 l tanks connected to an IRTAmar™ unit (8 fish per tank; stocking density = 20 kg/m3; three tank replicates per diet) at IRTA’s biosafety challenge room (Salomón et al., 2021). During the acclimation period (7 days) and during the bacterial challenge (15 days), fish were fed ad libitum with the same experimental diets used in the nutritional assay. After acclimation, fish were anesthetized, and IP injected with 0.2 ml of 2.3 × 107 CFU/ml of A. salmonicida subsp. salmonicida. Fish mortality occurring 12 h post-injection (hpi) was considered to be induced by the pathogen infection rather than handling stress, since no casualties were found in the control group injected with PBS. During the bacterial challenge, fish were supervised every 2 h, six times per day, including weekends. For ethical issues and to avoid unnecessary suffering, when the animals became moribund (i.e., loss of equilibrium, swollen abdomens, haemorrhaging in the anal area, and erratic swimming), they were euthanized with an overdose of MS-222. At the end of the trial, alive fish were sacrificed following the same procedure. Cause of death confirmation was determined by the recovery of the bacteria from all moribund animals (sampled head kidney platted on TSA), followed by specific PCR using A. salmonicida molecular tools as described in Salomón et al. (2021).

Salomón, Ricardo, M. Dolors Furones, Felipe E. Reyes-López, Lluis Tort, Joana P. Firmino, M. Angeles Esteban, and Cristóbal Espinosa Ruíz et al. 2021. “A Bioactive Extract Rich In Triterpenic Acid And Polyphenols From Olea Europaea Promotes Systemic Immunity And Protects Atlantic Salmon Smolts Against Furunculosis”. Frontiers In Immunology 12. doi: 10.3389/fimmu.2021.737601.

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