USE OF AN ENRICHMENT BROTH CULTIVATION PCR COMBINATION ASSAY FOR RAPID DIAGNOSIS OF SWINE ERYSIPELAS

We have previously described the creation by Tn916 mutagenesis of avir ulent transposition mutants from a highly virulent strain of Erysipelo thrix rhusiopathiae, the causative agent of swine erysipelas, In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 inserti on in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection off. rhusiop athiae. According to the sequences of this region, oligonucleotide pri mers were designed to amplify a 937-bp fragment of the E. rhusiopathia e chromosome by PCR. The specificity of the PCR was investigated by an alyzing 64 strains of Erysipelothrix species and 27 strains of other g enera different from Erysipelothrix. A 937-bp DNA fragment could be am plified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a r apid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, whic h used a commercially available DNA extraction kit, to identify E. rhu siopathiae in the specimens from swine with arthritis, After samples w ere enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR w as 10(3) CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.