Cloning and expression of portions of the 34-kilodalton-protein gene of Mycobacterium paratuberculosis: its application to serological analysis of Johne's disease

Abstract

Paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle that is caused by Mycobacterium paratuberculosis, is characterized by incoercible diarrhea and fecal shedding of bacteria. The present work aimed at developing a specific serological test for this disease. We have recently shown that a 34-kDa protein belonging to the major antigen complex A36 of M. paratuberculosis is immunodominant and contains epitopes specific with respect to all mycobacteria tested, including Mycobacterium bovis and the closely related species Mycobacterium avium. From a Agtll genomic library of M. paratuberculosis, three portions of the gene coding for this 34-kDa protein have been isolated. Two of them expressed cross-reacting mycobacterial epitopes. One portion (in clone a362) expressed a polypeptide which cross-reacted with all tested M. paratuberculosis strains but not with 20 other bacteria tested, including many strains of the M. avium-Mycobacterium intracelulare-Mycobacterium scrofulaceum group. The occurrence at the M. paratuberculosis surface of epitopes corresponding to the a362 polypeptide was shown by immune electron microscopy. The recombinant a362 polypeptide was used as reagent for an enzyme-linked immunoassay for paratuberculosis. This assay correctly diagnosed all the tested blood samples from infected cattle at all stages of the disease.

Comparative analysis of three sensitins used in cutaneous testing for tuberculosis and paratuberculosis in cattle

“…1, tracks 8,9,10) or anti-bovine tuberculin-PPD ( Fig. 1, tracks 11,12,13). Most of these bands present in the patterns of the three organisms are presumably re- paratuberculosis 2E (lanes 2, 5, 8, 11), M. avium D4 (lanes 3, 6, 9, 12) and M. boris BCG (lanes 4, 7, 10, 13) were fractionated by polyacrylamide gel electrophoresis (SDS-12% PAGE) and transblotted to a nitrocellulose membrane.…”

“…To the sec- ond group were assigned the 49.8-and 38.3-kDa proteins of M. avium and the 58.7-and 54.0-kDa proteins of M. bovis, which are endowed with at least one species-specific epitope with respect to the other two mycobacteria. Screening procedures based on antisera pre-adsorbed on heterologous bacterial sonicates were previously found to be very useful to identify proteins endowed with species-specific epitopes in complex protein mixtures [11,12]. By the combined approach used in this work, several cross-reactive proteins (presumably responsible for the non-specificity of tuberculin-like assays) and a few proteins endowed with at least one species-specific epitope (putative reagents for specific cutaneous tests) were identified.…”

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family

“…Genomic DNA was extracted as previously described [9]. All other DNA manipulations were carried out by standard methods [10].…”

B‐Cell Epitopes in the Immunodominant p34 Antigen of Mycobacterium avium ssp. paratuberculosis Recognized by Antibodies from Infected Cattle

“…The aminoterminal hydrophobic region is buried within the bacterial envelope, whereas the hydrophilic carboxyterminal is exposed at the cell surface [14]. In addition, the B-cell epitopes at the C-terminal region were localized by immune electron microscopy to the cell surface [15]. Using a panel of sera from cattle, naturally infected with tuberculosis or paratuberculosis, De Kesel et al [15] concluded that the 98-amino acid carboxyterminal region of p34 is specific for M. paratuberculosis.…”

“…In addition, the B-cell epitopes at the C-terminal region were localized by immune electron microscopy to the cell surface [15]. Using a panel of sera from cattle, naturally infected with tuberculosis or paratuberculosis, De Kesel et al [15] concluded that the 98-amino acid carboxyterminal region of p34 is specific for M. paratuberculosis. This observation was consistent with the reactivity of serum from rabbits immunized with this polypepetide.…”