At an optimal concentration of magnesium, highly virulent wild strains of serotype 0:8, with an LD50, for mice, of <10 cells intravenously, had an in-vitro requirement for calcium at 37°C but not at 26°C. Avirulent wild strains of (LD50 > 10 cells intravenously) did not have this calcium dependence. When grown on calcium-depleted media at 37°C, eight highly virulent strains yielded 0.5-6% large calcium non-requiring, avirulent colonies; the remaining colonies were slow growing, calcium dependent and highly virulent. Like wild avirulent strains, these calcium non-requiring mutants were quickly destroyed in organs within 48 h, even after large intravenous challenge. In contrast the slow-growing calcium-dependent colonies were highly virulent on intravenous inoculation, growing rapidly in the liver, spleen and lungs to produce multiple abscesses. Homogenates of heavily infected organs produced the original proportion of calcium non-requiring colonies when plated on media without calcium. Results of a fluctuation test suggested that the emergence of calcium non-requiring mutants is the result of induction rather than spontaneous mutation.


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