Myriam De Kesel

Composition and immunological properties of the protein fraction of A36, a major antigen complex of Mycobacterium paratuberculosis.

TMA (thermostable macromolecular antigens) are major mycobactcrial complexes present in all mycobticteria. We have purified A36, the TMA complex of M. paratuberculosis, the etiological agent of paratuberculosis (Johnes disease), and shown by the immune electron microscopy approach its presentation at the cell surface. The immunodominance of the A36 complex in Johnes disease was suggested by comparative ELISA analysis of infected bovine sera, using either A36 or M. paratuberculosis total soluble sonicate as antigens. The cross‐reactivity of TMA complexes from diifcrcnt tnycobacteria was evaluated by immunocn/ymometri c measurements. Percentage of shared epifopes was high tor the couple M. paratuberculosis‐ M. avium. and somewhat lower for the couple M. paratuberculosis and M. bovis Immunological kinship between M. paratuberculosis and M. leprae was suggested by the finding that oul of eleven anti‐M. lepraemonoclonals, four cross‐reacted with A36 proteins. The specificiiy missing al the level of the whole A36 complex was sought at the level of its protein components. Comparative immunoblot analysis of electrophoresed A36 proteins indicated three of them to contain epitopes not shared by M. bovis proteins, and one of them to contain epitopes specific wilh respect lo M. avium, M. bovis and M. phlei. The latter component, a 34‐kDa protein, could be an ideal reagent for a serological tesi for, Johnes disease, being immunodominant in infected cattle and endowed with species‐specific epitopes.

Induction of cellular immune reactions by A36, an antigen complex of Mycobacterium paratuberculosis : comparison of A36 and Johnin components

Paratuberculosis (Johnes disease) is a chronic enteritis syndrome of ruminants, which is due to infection by Mycobacterium paratuberculosis. Cutaneous testing with proteins extracted from a mycobacterial culture fluid (johnin‐PPD) is currently used to evaluate the cellular immune status. We have compared the components of johnin‐PPD with those of the A36 complex, a thermostable macromolecular antigen (TMA) present in the cytoplasm and associated with the cell wall of M. paratuberculosis. The presence in the johnin‐PPD of fifteen A36 components has been shown by Western blotting. Moreover, monoclonal antibodies, which bind respectively to the 65‐kDa M. leprae heat shock protein, the 28‐kDa M. leprae superoxide dismutase, and M. tuberculosis lipoarabinomannan, recognized components of the johnin‐PPD. The ability of A36 to trigger delayed hypersensitivity reactions in sensitized rabbits, and to induce the proliferation of T lymphocytes from the lymph nodes of A36‐sensitized mice, matched that of johnin‐PPD. The homology levels of T epitopes between A36 and the TMA complexes of M. phlei, M. bovis, M. tuberculosis and M. avium were estimated, in a lymphoproliferation assay, to be 51, 52, 59 and 94% respectively. A strong cross‐reactivity of A36 with an M. leprae sonicate was also observed by cutaneous testing. The A36 components within the 45.2–26.8‐kDa and the 21.6–19.8‐kDa ranges were proved to induce the proliferation of T lymphocytes from sensitized mice. This work supports the possible use of the A36 complex, and of some of its components, for cutaneous tests and lymphocyte proliferation assays, in order to monitor cellular immunity in Johnes disease.