2.7. Serum Bactericidal Activity (SBA) Assay

Individual mouse sera collected from groups sacrificed at week 12 were tested for SBA assay, as previously described [47]. S. Typhimurium D23580 strain was used as target strain against STm, to perform SBA reactions [34]. Bacteria was grown in Luria Bertani (LB) medium to log-phase (OD: 0.2), diluted 1:30,000 in PBS to approximately 3 × 10³ colony forming units (CFU)/mL and distributed into sterile polystyrene U bottom 96-well microtiter plates (12.5 µL/well). Sera samples serially diluted 2 or 3-folds (starting from 1:100 dilution), were added to each well (final volume 50 µL, ~620 CFU/mL). Sera were heat-inactivated and Baby Rabbit Complement (Cederlane, Burlington, Canada) was used at 50% of the final volume. Each sample was tested in triplicate on three different days. One hundred microliters reaction mixtures from each well were spotted on LB-agar plates at time zero (T0) to assess initial CFU, and at 3 h (T180) after incubation at 37 °C. LB-agar plates were incubated overnight at 37 °C and the resulting CFU were counted the following day. Bactericidal activity was determined as serum dilutions necessary to obtain 50% CFU reduction at T180 compared with T0. Serum titres equal to 1 were designated when no bactericidal activity was detected.