The effect of passaging in liquid media and storage on Mycobacterium bovis - BCG growth capacity and infectivity

The effect of successive cultures - undergoing or not cycles of freezing, storage and thawing - on the growth curves of the Mycobacterium bovis Bacille Calmette-Guerin (BCG) Moreau strain and a recombinant-BCG (rBCG) vaccine preparation were evaluated. The results showed that both strains going through three rounds of freezing and thawing were not able to grow efficiently in the third stage of liquid culture. This effect and also long-term frozen storage appeared to be more preeminent in cultures that had been harvested at 0.8 optical density (OD at 600 nm) prior to freezing and storage, as in comparison to their 0.4 OD counterparts. Altogether, the data suggest that cultures inoculated with samples harvested at lower OD are less sensitive to the limiting effects of serial cultivation, regardless of being BCG or rBCG. Successive cultivations without freezing and thawing also affect growth of BCG culture inoculated with cells at later exponential phase (0.8 OD). Finally, macrophage infectivity with BCG cells from the third growth passage was significantly lower than from the first passage. These results draw attention to the importance of using fresh, low-passage and/or growth and infection capacity-controlled vaccine stocks for the evaluation of strains of BCG or rBCG.