Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs

Abstract Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10 2 –10 3 cells/g of faeces for 16S rRNA gene PCR and 10 4 cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.