The gene and surrounding sequences from reference strains of the six species were amplified by the polymerase chain reaction (PCR) with primers chosen according to the published sequence of the gene and studied for polymorphism with nine restriction endonucleases. The restriction patterns were identical for all species with all restriction endonucleases tested except for strain 16M that showed a different pattern with RV, consistent with the presence of a single site instead of two for the other species. The absence of the second RV site for 16M was confirmed by DNA sequence analysis. The second RV site in other species was located in a 12-bp segment, which was missing from the published sequence of , between the stop codon of the gene and its putative transcription terminator sequence. The difference between strain 63/290 and 16M was due to an additional base-pair in 16M. Subsequently, 71 other field, vaccinal and reference strains of the six species and their different biovars were studied for restriction fragment length polymorphism (RFLP) of the locus with RV. The presence of a unique RV site was specific to strains. Southern blot analysis of whole genomic DNA digested with RV and with the gene used as probe also detected a distinct pattern for . These results indicate that both PCR-RFLP and Southern blot analysis of the locus can be used to distinguish strains from the other species and may be useful for typing and diagnostic purposes as well.


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