Skimmed milk

The bacterial strains and plasmids used are listed in Additional file 1. We used B. suis bv2 CITA 198 (herein Bs2WT) because, although Bs2WT and the B. suis bv2 reference strain (B. suis bv2 Thomsen) have the same PCR-RFLP pattern [3 (link)], the former shows a virulence pattern in mice typical of B. suis bv2 field strains and the latter is attenuated (Additional file 2).
Bacteria were routinely grown either in standard tryptic soy broth (TSB; Scharlau, Barcelona, Spain) or TSA (TSB supplemented with agar [Pronadisa, Laboratorios Conda, Spain]) at 37 °C. For the studies in mice, vaccines and challenge strains were grown on Blood Agar Base (BAB; Oxoid, UK). When needed, media were supplemented with 5% sucrose, diaminopimelic acid (DAP [Sigma]; 1 mM), 0.2% activated charcoal, kanamycin (Km) at 50 µg/mL or at 35 µg/mL, ampicillin (Amp) at 100 µg/mL and/or chloramphenicol (Cm) at 20 µg/mL (all from Sigma). The lactate-glutamate-glycerol-vitamins synthetic medium of Gerhardt’s [35 (link)] was supplemented with 1 mM methionine (mGSM) (this amino acid is required for growth of some Brucella strains in synthetic media [32 (link)] including Bs2WT (Zúñiga-Ripa, unpublished observations). All strains were stored at − 80 °C in skimmed milk (Scharlau, Barcelona, Spain) or in TSB supplemented with 0.5% yeast extract (Pronadisa, Laboratorios Conda, Spain) (TYSB) and 7% dimethylsulfoxide.

The characteristics of the bacterial strains and plasmids are shown in Supplementary Table 1. The virulent B. suis biovar 2 (bv2) strain CITA 198 (henceforth Bs2WT) was selected for this study because the reference bv2 strain B. suis Thomsen is attenuated in mice (Aragón-Aranda et al., 2020 (link)). The choline-containing media used were tryptic soy broth (TSB, Scharlau, Barcelona, Spain), the same medium with 15% agar (TSA) or Blood Agar Base (BAB; Oxoid, United Kingdom), supplemented when necessary with 50 μg/ml kanamycin (Km), or/and 5% sucrose or/and 0.2% activated charcoal (Sigma) (see below). The choline-free medium was the lactate-glutamate-glycerol-vitamins medium of Gerhardt (1958) (link) supplemented with 1 mM methionine (a Bs2WT requirement; Zúñiga-Ripa, unpublished results) (henceforth mGSM). E. coli strains were cultured on TSA-Km (50 μg/ml), supplemented with 1 mM diaminopimelic acid (DAP [Sigma]) in the case of E. coli β2150. All strains were stored at −80°C either in skimmed milk (Scharlau, Barcelona, Spain) or in TSB containing 0.5% yeast extract and 7% dimethylsulfoxide. Work was performed under Biosafety Level 3 (BSL-3) conditions.

The bacterial strains and plasmids used are listed in Additional file 1. For construction of mutants, B. melitensis 16 M and Rev1 strains were grown at 37 °C in tryptic soy broth (TSB, Biomérieux, Marcy l’Etoile, France) or in this medium supplemented with agar (TSA, Pronadisa, Conda, Spain). B. ovis strains were grown at 37 °C in TSB supplemented with 0.5% yeast extract (Pronadisa, Conda, Spain) and 5% fetal bovine serum (TYSB-S) or this medium supplemented with agar (TYSA-S). For the studies in mice, vaccines and challenge strain were grown in Blood Agar Base (BAB, Oxoid) or BAB-S (supplemented with 5% fetal bovine serum). Where needed, media were supplemented with 5% sucrose (Sigma), diaminopimelic acid (DAP; 1 mM), 0.2% activated charcoal (Sigma), kanamycin (Km) at 50 µg/mL, chloramphenicol (Cm) at 20 µg/mL, ampicillin (Amp) at 100 µg/mL, polymyxin (Pmx) at 1.5 µg/mL or streptomycin (Strp) at 2.5 µg/mL. All strains were stored at −80 °C in skimmed milk (Scharlau, Barcelona, Spain) or TYSB-7% dimethylsulfoxide (DMSO).