Setting: Molecular techniques are now available to develop new live tuberculosis vaccines by producing avirulent strains of the Mycobacterium tuberculosis complex with known genes deleted.

Objectives: Determine if removal of esat-6 from new live tuberculosis vaccines with known attenuating mutations affects their vaccine efficacy and if it could enable the development of discriminating diagnostic tests.

Design: Remove the esat-6 gene by allelic exchange from two illegitimate mutants of Mycobacterium bovis that had previously been shown to have similar vaccine efficacy to BCG in a guinea pig vaccination model. Determine the effect this removal has on virulence, vaccine efficacy and skin test reactivity in guinea pigs.

Results: Two double knockout strains of M. bovis were produced and their virulence and vaccine efficacy were compared to their parent strains. Removal of the esat-6 gene had no significant effect on vaccine efficacy. In skin tests, animals inoculated with the double knockout strains reacted to PPD but not ESAT-6, whereas those inoculated with the parent strains had similar skin test reactivity to both PPD and esat-6.

Conclusion: Removal of esat-6 from new live tuberculosis vaccine candidates has no significant effect on vaccine properties but does enable the use of skin tests to distinguish between vaccination and infection.