Use of polymerase chain reaction to identify Brucella abortus strain RB51 among Brucella field isolates from cattle in Italy

Brucella abortus strain RB51, a tough mutant of the B. abortus 2308 virulen t strain, was recently approved in the United States as the official vaccin es for brucellosis in cattle. Following recent evidence of unauthorized use of RB51 vaccine in Italy. where the use of vaccines For brucellosis is no longer allowed, the suitability of an RB51-specific polymerase chain reacti on assay for identifying the RB51 strain among Brucella field isolates from cattle in Italy was investigated. The oligonucleotide primers used in this study, belonging to a six-primer cocktail for Brucella species previously described by other authors, allowed the amplification of a 364-base pair (b p) fragment specific for RB51 and its parent strain 2308, and a 498-bp prod uct specific for B. abortus In addition, unresolved bands ranging from 600 to 700 bp were observed from RB51 strain. Brucella abortus biovars 1, 2 and 4 have only one specific sensitive 498-bp band. The B. abortus biovars 3, 5 and 6 did not give any signal. The 498-by product from a reference Brucel la strain aas sequenced and submitted to EMBI. with the accession number AJ 271968. while the 364-bp fragment from RB51 strain was submitted to EMBL da tabase with accession number AJ271968. The sequence studies confirmed the s pecificity of the detected fragments. No amplification was obtained by test ing DNA from strains antigenically related to Brucella, such as Yersinia en terocolitica coli O:157, Salmonella urbana and Pasteurella multocida. The r esults of this study indicate that this technique, in combination with spec ific serological tests, could be a useful diagnostic method to verify the u se of RB51 vaccine and can contribute to the creation of a databank of circ ulating strains.