Optimized ribotyping protocol applied to Hungarian Bordetella bronchiseptica isolates: identification of two novel ribotypes.

We reported previously that ribotype patterns generated with PvuII and a probe derived from the Escherichia coli rrnB gene could be used to differentiate isolates of Bordetella bronchiseptica. In the present study we report modifications made to the original ribotyping procedure that permit detection in the formerly characterized isolates of an additional 8 fragments with homology to rrnB. Ribotypes were redefined to include these fragments. Although this modification did not permit the detection of novel ribotypes from the previously characterized isolates, it did result in a more accurate reclassification of five of these isolates to other existing ribotypes. It was hypothesized that the additional fragments could form the basis for novel ribotypes in future analyses, and this was supported by the subsequent evaluation of 101 previously uncharacterized pig, rabbit, and dog B. bronchiseptica isolates from Hungary. A total of six different patterns were detected from this group, including two previously not identified that were designated ribotypes 17 and 18. The profile of ribotype 17 includes a novel fragment not associated with any other ribotype. A subset of the fragments constituting ribotype 18, essential for its differentiation from other ribotypes, is only detectable under the modified conditions reported here. Hungarian swine isolates are highly clonal, since 98.2% were identified as ribotype 3. Similarly, 83.7% of rabbit isolates from Hungary are also ribotype 3. Cluster analysis revealed that despite the existence of numerous ribotypes, B. bronchiseptica isolates display limited heterogeneity. The ability to detect additional ribotypes under the modified conditions described in this study strengthens the usefulness of ribotyping as an epidemiologic tool.