Detection of Mycobacterium bovis lymphocyte stimulating antigens in culture filtrates of a recombinant Mycobacterium smegmatis cosmid library

Culture filtrates derived from a Mycobacterium bovis cosmid library in Myco bacterium smegmatis were screened for bovine lymphocyte stimulatory antigen s using peripheral blood mononuclear cells (PBMC) from cattle vaccinated wi th a low dose of Mycobacterium bovis BCG. Lymphocyte proliferation and inte rferon-gamma (IFN-gamma) production were used as cellular response markers for antigen recognition. In the primary screen, approximately 28% of all cu lture filtrates (CF) stimulated responses by PBMC from at least two out of four vaccinated cattle. In one of these CF, the M. bovis Ag85-B antigen was detected by Western-blot analysis. Despite heterogeneous lymphocyte respon ses of the animals, twenty-four of the culture filtrates stimulated lymphoc yte proliferation and IFN-gamma production from at least six out of eight v accinated animals in a secondary screen. Analysis of the cosmid DNA associa ted with these positive CF demonstrated that several contained homologous D NA sequences. It appears that the lymphocyte screening has detected M. bovi s antigens that are immuno-dominant in cattle vaccinated with,M. bovis BCG. (C) 1999 Elsevier Science Ltd. All rights reserved.