Improvement of the Brucella abortus B19 vaccine by its preparation in a glycerol based medium

The Brucella abortus B19 vaccine strain differs from other Brucella strains in its sensitivity to erythritol. However, erythritol tolerant (Eri(t)) mutants arise from sensitive cultures of B19 at high rate, and may cause persistence and/or abortion when the vaccine is inoculated on adult cattle. Twelve different batches of B19 have been examined for the presence of Eri(t) mutants. All contained Eri(t) variants at a proportion ranging from 10(-4) to 10(-6). In order to eliminate these mutants from the vaccine cultures, we have developed a minimal medium with glycerol as the sole carbon source, named MMG30. Growth of the parental strain B19 (erythritol sensitive) in this medium was fairly good compared with the growth of its Eri(t) derivatives. Culture of the 12 different batches of B19 in liquid MMG30 produced up to a thousandfold decrease in the proportion of Eri(t) mutants present in the vaccine cultures. Use of this medium to grow B19 could represent an easy and considerable improvement of the vaccine, by the reduction of the presence of potentially dangerous Eri(t) mutants.