Identification of the Brucella melitensis Vaccine Strain Rev.1 in Animals and Humans in Israel by PCR Analysis of the Pst I Site Polymorphism of Its omp2 Gene

Abstract

ABSTRACT Adverse effects of strain persistence and secretion in milk have been encountered with the Brucella melitensis vaccine strain Rev.1. Field isolates obtained from vaccinated animals and from a human resembled the vaccine strain Rev.1 by conventional bacteriological tests. The lack of a specific molecular marker that could specifically characterize the commercial vaccine strain prevented confirmation of the homology of the Rev.1-like field isolates to the vaccine strain. The composition of the omp2 locus from two gene copies with differences in their Pst I restriction endonuclease sites was used to establish an epidemiologic fingerprint for the omp2 gene in the Rev.1 vaccine strain. Primers designed to amplify DNA sequences that overlap the Pst I site revealed a single 282-bp DNA band common to all Brucella spp. Agarose gel electrophoresis of the Pst I digests of the PCR products from strains 16M and the vaccine strain Rev.1 revealed a distinctive profile that included three bands: one band for the intact 282-bp fragment amplified from omp2a and two bands resulting from the digestion of the amplified omp2b gene fragment, 238- and 44-bp DNA fragments, respectively. Amplified fragments of 37 Rev.1-like isolates, including 2 human isolates, also exhibited this pattern. In contrast, DNA digests of all other Israeli field isolates, including atypical B. melitensis biotype 1 and representatives of the biotype 2 and 3 isolates, produced two bands of 238 and 44 bp, respectively, corresponding with the digestion of both omp2a and omp2b genes. This method facilitates identification of the Rev.1 vaccine strain in both animals and humans in Israel.