Serological assay for swine erysipelas using nitrocellulose particles impregnated with an immunodominant 65 kDa antigen from Erysipelothrix rhusiopathiae.

ABSTRACT

Pigs (n = 10) that were experimentally challenged with an arthritogenic isolate of Erysipelothrix rhusiopathiae (strain VRS 229; serotype 1a) developed arthritis in at least one of twelve major limb joints. Immunoblots using sera obtained from these pigs at necropsy revealed a major band of immunoreactivity against a subunit polypeptide of apparent molecular mass 65 kDa. The usefulness of the 65 kDa immunodominant subunit as an assay reagent in an ELISA test was examined by presentation of antigen impregnated onto nitrocellulose particles (AINP). This was prepared by electro-transfer of bacterial polypeptides from SDS-PAGE gels to nitrocellulose. Protein bands were visualized by staining with amido black and a strip of nitrocellulose bearing the 65 kDa band was excised and extracted with formic acid. Nitrocellulose particles impregnated with the 65 kDa antigen (65-AINP) were precipitated from solution by neutralization with ammonium hydroxide. 65-AINP was suspended in water and the optimum dilution for ELISA assay was determined by titration to be 0.1 A650 units. Sera from all pigs challenged with VRS 229 reacted against the 65-AINP antigen in the ELISA assay while sera from control, and experimental pigs prior to challenge, failed to do so. The 65-AINP antigen could also be used efficaciously to quantify serological reactivity of pigs experimentally infected with other strains of E. rhusiopathiae representing the three major serotypes (1a, 1b and 2) that are most commonly associated with swine erysipelas infections. Mouse immunizations with 65-AINP also confirmed that nitrocellulose particles bearing the immunodominant subunit antigen will elicit murine antibodies that are monospecific against this determinant.