FIG. 4.

Virulence attenuation in independent infection experiments. (A) Five BALB/c mice were orally administered 10⁷ CFU of a single KLA-attenuated mutant strain. The survival rate of these mice was monitored daily for 2 weeks. MDD are shown in parentheses, and both mortality rate and MDD were determined by Kaplan-Meier analysis. Strains VA05, -07, -12, -14, -15, -18, -21, and -28, which were avirulent in mice during the observation period, were regarded as class I mutants. Strains VA01, -02, -04, -08, -10, -19, -20, -22, -24, -25, -32, and -33, which resulted in mortality rates of 20% to 40% in mice, had their virulence attenuated to a statistically significant level (P < 0.05) and were therefore categorized as class II mutants. The remaining VA strains, with no significantly attenuated virulence, were grouped as class III mutants. (B) Bacterial growth in different mouse tissues. Three BALB/c mice were orally administered 10⁷ CFU of a single KLA-attenuated mutant strain. Small intestine, colon, spleen, liver, and blood samples were harvested at 48 hpi to enumerate bacterial concentrations, which were expressed as numbers of CFU g⁻¹ tissue or CFU ml⁻¹ blood. The limit of detection was approximately 50 CFU. Samples which yielded no colonies were plotted as having values of 50 CFU g⁻¹ tissue or CFU ml⁻¹ blood.