Analyses of the immunodominant antigens of Corynebacterium pseudotuberculosis

Antibodies to 7 antigens in a whole cell lysate C. pseudotuberculosis ranging in molecular mass from 22 to 120 kDa were present in sera of 40 sheep and goats infected with C. pseudotuberculosis. Three antigens of about 120, 68 and 31.5 kDa in size were consistently detected with sera from all animals and 22 sera had antibodies to 64, 43, 40 and 22 kDa antigens. None of these antigens was detected by sera from 160 sheep in a C. pseudotuberculosis-free research flock. An NaCl extract of C. pseudotuberculosis cells contained one major protein of about 31.5 kDa and 4 minor proteins 68, 64, 43 and 22 kDa in molecular mass as shown by Coomassie Blue staining. Immunoblot analysis demonstrated that the 3 immunodominant antigens identified in the whole cell extract by fast-protein liquid chromatography gel filtration to near homogeneity. The purified 31.5-kDa protein showed phospholipase D activity as indicated by synergistic haemolysis with Rhodococcus equi factors and sphingomyelinase activity. The 31.5-kDa protein reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis. This serum also had phospholipase D neutralizing activity. On the basis of its molecular mass, biological activity, N-terminal amino acid sequence analysis, and immunoreactivity, the 31.5-kDa protein was identified as the phospholipase D exotoxin of C. pseudotuberculosis.