The relatedness of spp. and was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis. Whole-cell and soluble proteins of brucellae and showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of spp. Numerical analysis of Western blot profiles of whole-cell extracts showed that LMG 3301 was closer to spp. than to LMG 3331a result not obtained by protein profiling. These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile. Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331was in a separate cluster. The LMG 3301 and the LMG 3331clusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA primers, and both methods showed strains of both clusters among clinical isolates classified as by conventional tests. These results and those of previous DNA-DNA hybridization studies [Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Int J Syst Bacteriol 38, 406–416] show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name sp. nov. is proposed (type strain is LMG 3301= NCTC 12171= CNS 2-75).


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