Immunoblot and ultrastructural analysis of antigens extracted from Dichelobacter nodosus with potassium thiocyanate

NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Menangle, Australia.

The antigens extracted from Dichelobacter nodosus with potassium thiocyanate were analyzed by western blotting with sera from footrot-free sheep and from sheep infected with D. nodosus to identify components specific for infection. Several components with molecular mass < 33 kD were associated with infection, particularly bands of 32.5 kD, 30.5 kD, and 28 kD. Components with molecular mass > 56 kD may be responsible for false-positive reactions observed when sera of footrot-free sheep react with the potassium thiocyanate extract in solid phase enzyme-linked immunosorbent assays (ELISAs). Bands of 62.5 kD, 56 kD, 40 kD, and 19 kD were recognized for most sheep regardless of their disease status or ELISA reactivity and therefore do not appear to be detected by the ELISA. No components of the potassium thiocyanate extract were completely specific for infection. Antigens in the extract were identified primarily on the cell envelope by immunogold electron microscopy. Labeling was concentrated in the periplasm, with minor labeling of the cell surface. The extract consisted of tangled strands of particles with electron-dense cores, and few vesicular structures were observed.