Production of vaccine

Vaccine was produced according to published protocol ¹⁸ bypassing the lyophilization step ²⁵. Briefly, pET9c-ospA-transformed and untransformed E. coli BL21(DE3)pLysS were cultured in TBY media supplemented with 50μg/ml Kan, and grown at 37°C, 220 rpm, for about 3h to reach an OD₆₀₀=0.8. Protein expression was induced with 1mM IPTG/ml during 3h to reach OD₆₀₀=1 (~10⁹ E. coli cells/ml). The cells were harvested by centrifugation at 4000×g for 10 min at 4°C and the biomass was resuspended in 20% glycerol/phosphate buffered salt solution (Gibco, Grand Island, NY) in 1% of the initial volume. Cell suspensions in aliquots of 1 ml were snap frozen in a dry ice bath and stored at −80°C. Aliquots of 300μl (~3×10⁸ cells) were thawed and placed in a ball tipped disposable feeding needle (Fisher Scientific, Pittsburgh, PA) for oral gavage inoculation.