Ft LVS (American Type Culture Collection 29,684) and Ft ΔclpB [obtained from Conlan et al. (2010) (link)] were grown to mid-log phase in modified Mueller-Hinton (MH) broth (Difco Laboratories, Detroit, MI), harvested, and frozen in aliquots in broth alone at−80°C (Baker et al., 1985 (link); Fortier et al., 1991 (link)). Ft strain SchuS4 (BEI Resources, Manassas, VA, NR-10492) was sub-cultured in modified cysteine partial hydrolysate (MCPH) broth to produce a sub-master stock and a working stock, which were frozen in aliquots with 20% glycerol at −80°C. Of note, Ft SchuS4 NR-10492 has been demonstrated to be identical and with similar level of virulence compared to Ft SchuS4 NR-28534 (Lovchik et al., 2021 (link)), which was used previously to evaluate vaccine efficacy in NHsd rats (De Pascalis et al., 2022 (link)). Bacteria were periodically thawed for quality control by quantification of viability on MH agar plates or chocolate agar plates (Hardy Diagnostics, Santa Maria, CA); in addition, quantification was also evaluated at the time of vaccination or challenge procedures. Although different media were used at each site, within each laboratory’s work with the three rat strains, bacteria were cultured using the same media.
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