Mouse immunization and challenge

In each group, mice were intramuscularly immunized with 500 µl of vaccine on days 0, 7, and 14. Each 500 µl of vaccine contained 25 µg of Vo or Vo with 500 µg of aluminum adjuvant. Immunizations with blank nanoparticles, aluminum adjuvant and PBS alone were performed as negative controls. The mice were sacrificed on day 21, and their sera were collected to determine the titer of antigen-specific antibodies.

Mice were intraperitoneally injected with a lethal dose (1 × 10⁸ CFU per mouse) of E. coli K1 RS218 in 100 µl of sterilized PBS. The number of deaths was recorded daily for 5 days. For further analysis of the protective mechanism, another five mice were intraperitoneally challenged with a sublethal dose (1 × 10⁷ CFU per mouse) of E. coli K1. Their body weights were monitored daily for 5 days, and the percentage of the initial weight was calculated. In addition, five immunized mice in each group were sacrificed at 24 h after the challenge, and the bacterial loads in their blood and spleens were determined. The spleens of the sacrificed mice were collected, weighed, and homogenized in 1 ml of sterilized PBS buffer. The spleen homogenates and blood were plated onto LB plates at a tenfold serial dilution and cultured at 37 °C for 20 h. The number of colonies on the plates was counted and used to calculate the bacterial load. The CFU per gram of tissue was calculated for the comparison of bacterial load.