RAPID DETECTION OF SERPULINA-HYODYSENTERIAE IN DIAGNOSTIC SPECIMENS BY PCR

A PCR assay for the detection of Serpulina hyodysenteriae in diagnosti c specimens was developed on the basis of sequence analysis of a recom binant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3- kb DNA fragment unique to S. hyodysenteriae, aas identified by screeni ng a plasmid library of S. hyodysenferiae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibo dy 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested gen omic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 23-kb DNA fragment of pRED3C6 indicated that the cloned seque nce was present exclusively in the seven serotypes of S. hyodysenteria e. An oligonucleotide primer pair for PCR amplification of a 1.55-kb f ragment and an internal oligonucleotide probe were designed and synthe sized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenf eriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intest inal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the o rder Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amp lified with the oligonucleotide primer pair in a hot-start PCR. The 1. 55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of th e 1.55-kb, products for S. hyodysenteriae was confirmed on the basis o f production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and po sitive hybridization signal with the S. hyodysenteriae-specific intern al oligonucleotide probe. By using total DNA obtained from normal swin e feces inoculated with decreasing concentrations of S. hyodysenteriae cells, the sensitivity of the PCR assay was calculated to be between 1 and 10 organisms per 0.1 g of feces. The PCR assay was 1,000 times m ore sensitive than conventional culture of dysenteric feces on selecti ve medium. There was complete agreement between the results of PCR ass ays and anaerobic culture on selective agar medium with diagnostic spe cimens (n = 9) obtained from six farms on which there were cases with clinical signs suggestive of swine dysentery. Detection of S. hyodysen teriae by PCR amplification of DNA has great potential for rapid ident ification of S. hyodysenteriae in diagnostic specimens.