Identification of Brucella melitensis vaccine strain Rev.1 by PCR-RFLP based on a mutation in the rpsL gene.

The live attenuated strain B. melitensis Rev.1 is considered the best vaccine available for the prophylaxis of brucellosis in sheep and goats. The Rev.1 vaccine was obtained in the 1950s by a two-step selection involving firstly streptomycin resistance and dependence and secondly reversion of dependence but keeping streptomycin resistance. Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. Nucleotide sequencing revealed one mutation in the rpsL gene of vaccine strain Rev.1 compared to that of reference strain 16M leading to an amino acid Pro-to-Leu change at codon position 91 (Pro91Leu). This mutation resulted also in the lack of a NciI restriction site in the gene. PCR-restriction fragment length polymorphism (PCR-RFLP) using NciI applied to a large number of Brucella reference and field strains showed that the mutation detected was specific of vaccine strain Rev.1.