Detection of Mycobacterium avium subsp. paratuberculosis in infected tissues by new species-specific immunohistological procedures

Abstract

We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930-4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947-954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne's disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity.

FIG. 1

Evaluation of antibody specificity by Western blotting. Soluble sonic extracts (50 μg of protein/sample) of M. paratuberculosis (lanes A and B) and M. bovis (lanes A′ and B′) were fractionated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and reacted with either anti-a362 polyclonal serum (lanes A and A′) or anti-a362 monoclonal antibodies (lanes B and B′). Antigen-antibody complexes were revealed by peroxidase-labelled secondary antibodies.

FIG. 2

(a) Ziehl-Neelsen staining and immunohistological detection of M. paratuberculosis in infected tissues. Sections of the ileocecal mucosa from a pluribacillary form of paratuberculosis (A, B, and C) and sections of a mesenteric lymph node from a paucibacillary form of paratuberculosis (A′, B′, and C′) were subjected to the following histological procedures: Ziehl-Neelsen staining (A and A′) and immunohistochemical detection with anti-a362 polyclonal serum (B and B′) and anti-a362 monoclonal Ig (C and C′). Note that the number of positive cells is higher in panel B′ than in panel A′. The chromogen used in panels B and B′ was DAB (yielding brown staining in numerous macrophages), whereas the chromogen in panels C and C′ was AEC (yielding red staining). Note the presence in panel C′ of two multinucleated giant cells, one positively stained and the other negative. (b) Specificity testing of immunohistological procedures. Sections of the ileocecal mucosa from a paratuberculous cow were incubated with either anti-a362 polyclonal serum (A and A′) or anti-a362 monoclonal Ig (B and B′), either before (A and B) or after (A′ and B′) immunoneutralization of anti-a362 antibodies with a preparation of the a362 recombinant polypeptide. Note the negative reaction occurring after immunoneutralization.