Identification of surface protective antigen (spa) types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real-time and conventional PCR assays

Abstract

To develop spa multiplex real-time and conventional PCR assays to detect and differentiate between spaA, spaB and spaC genes within Erysipelothrix spp. For evaluation of the assays, 28 Erysipelothrix spp. reference strains, 25 tissues from pigs inoculated with reference strains of serotypes 1, 2, 5, 10 or 18, and 15 diagnostic samples were used. SpaA was found to be present in Erysipelothrix rhusiopathiae serotypes 1a, 1b, 2, 5, 9, 12, 15, 16, 17, 23 and N; spaB was detected in E. rhusiopathiae serotypes 4, 6, 8, 11, 19 and 21 and spaC was detected in E. sp. strain 2 serotype 18. Spa-related genes were not detected in E. tonsillarum strains (serotypes 3, 7, 10, 14, 20, 22, 24, 25, 26) or E. sp. strain 1 (serotype 13). With the spa multiplex real-time PCR assay, it was also possible to further differentiate spaB into spaB1 (serotypes 4, 6, 8, 19 and 21) and spaB2 (serotype 11). Overall, spaA was detected in seven experimental tissue samples and six diagnostic tissue samples, and spaC in two experimental tissue samples. The detection limits were determined to be five colony-forming units (CFU) per reaction for the spa multiplex real-time PCR assay and 4000 CFU per reaction for the conventional PCR assay. Both spa PCR assays were specific and reproducible in the identification of spa types in Erysipelothrix spp. The described spa PCR assays may be useful tools for investigating spa prevalence among strains isolated from field tissues and to determine the role of the Spa proteins in vaccine protection and pathogenesis.