Identification of a 25-kilodalton protein of Mycobacterium bovis BCG to distinguish BCG strains from Mycobacterium tuberculosis
Journal of Clinical Microbiology
Mycobacterium bovis BCG vaccine strains were compared with Mycobacterium tuberculosis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 25-kDa protein observed in the BCG strains was absent in M. tuberculosis. Rabbit antibodies specific to the 25-kDa protein uniquely identified this protein in BCG strains but not in M. tuberculosis. It is suggested that the 25-kDa protein and polyclonal antibodies directed against this antigen can be exploited to distinguish BCG strains from M.
... strains from M. tuberculosis. The Mycobacterium bovis BCG vaccination has been used extensively to prevent tuberculosis and for immunostimulation in neoplasia and is a preferred treatment for superficial stages of bladder cancer (11). Nevertheless, BCG vaccination may cause disseminated BCGitis, an illness caused by the vaccine itself in patients suffering from severe immune deficiency. Recently, such a case was confirmed in a human immunodeficiency virus-infected individual who displayed reactivation of M. bovis BCG 30 years after vaccination (1). Therefore, it is very important to distinguish M. bovis BCG strains reliably from Mycobacterium tuberculosis to differentiate between BCG reactivation and reinfection with M. tuberculosis. From time to time, a battery of tests to distinguish BCG strains from M. tuberculosis has been suggested (3-9, 12, 13, 15). We have compared four vaccine strains of M. bovis BCG with virulent strains of M. tuberculosis and found that a 25-kDa protein of BCG could be used as a marker to distinguish BCG strains from M. tuberculosis. The following mycobacterial strains were used: four M. bovis BCG strains, namely, the Indian vaccine strain (Tuberculosis Research Centre, Madras, India), the Bulgarian strain (Tuberculosis Hospital, Lucknow, India), the Russian strain (World Health Organization, Geneva, Switzerland), and the BCG-Tokyo strain (S. Nagai); M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. smegmatis (our collection); M. simiae, M. szulgai, M. triviale, M. kansasii, and a clinical isolate from the M. avium-M. intracellulare complex (R. Schwalbe); and clinical isolates of M. tuberculosis (A. K. Singh, A. Rattan, and R. Schwalbe). The clinical isolates were obtained from different patients at different times. All mycobacterial cultures were routinely grown on Lowenstein-Jensen slants, unless otherwise specified, at 37ЊC. Bacterial growth from slants was scraped and suspended into 500 l of 1ϫ sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10% glycerol, 5% ␤-mercaptoethanol, 0.025% bromophenol blue). After incubation for 15 min in a boiling-water bath, the sample was centrifuged and the supernatant containing the soluble proteins was separated by SDSpolyacrylamide gel electrophoresis (PAGE) with 4% stacking and 12% resolving gels (10). After electrophoresis, the gel was