An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine.
METHODS
Strains of the Mycobacterium tuberculosis complex are being rationally attenuated in order to develop better tuberculosis vaccines than BCG, and it would be helpful if new vaccines lacked an immunogenic protein which could be used as a skin test reagent for determining infection status.


OBJECTIVE
To delete the esat6 gene from a virulent Mycobacterium bovis strain and determine (i) whether this mutant sensitizes guinea pigs to a skin test based on ESAT6 and (ii) what effect this has on the virulence of M. bovis.


METHODS
An homologous recombination technique was used to produce an esat6 knockout mutant of a virulent strain of M. bovis. Guinea pigs were inoculated with either the mutant or parent strain and their reactivity in intradermal skin tests was determined to bovine purified protein derivative (PPD) and recombinant ESAT6 protein.


RESULTS
Production of an esat6 knockout strain was demonstrated by Southern blot hybridization and the polymerase chain reaction. Guinea pigs inoculated with either the esat6 knockout strain or its virulent parent had positive skin test reactions to PPD but only animal inoculated with the parent strain had positive skin test reactions to ESAT6. Gross pathology, histopathology and mycobacterial culture of tissues indicated that the knockout strain was less virulent than its parent.


CONCLUSIONS
If an effective live tuberculosis vaccine can be produced by inactivation of virulence genes in M. bovis, then prior or subsequent knockout of the esat6 gene could contribute to the loss of virulence and enable the development of a test to distinguish between vaccinated and infected animals.
Wards
B J
BJ
Wallaceville Animal Research Centre, AgResearch, Upper Hutt, New Zealand. wardsb@agresearch.cri.nz  
de Lisle
G W
GW
Collins
D M
DM
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001500:BCG Vaccine / Q000276:immunology*; D001426:Bacterial Proteins; D015139:Blotting, Southern; D002417:Cattle; D005260:Female; D017353:Gene Deletion; D006168:Guinea Pigs; D009163:Mycobacterium bovis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D012882:Skin Tests; D014376:Tuberculosis / Q000235:genetics / Q000517:prevention & control*; D014613:Vaccines, Attenuated; D019444:Vaccines, DNA; D014774:Virulence
D000942:Antigens, Bacterial; D001500:BCG Vaccine; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D014613:Vaccines, Attenuated; D019444:Vaccines, DNA