Growth of Mycobacterium lepraemurium in cultures of mouse peritoneal macrophages

Abstract

Successful growth of Mycobacterium lepraemurium was observed in cultures of mouse peritoneal macrophages. The optimal host cell maintenance medium was composed of 40% horse serum, 50% of the chemically defined medium NCTC 109, and 10% of a 1:5 dilution of beef embryo extract, supplemented with both liver extract and ferric nitrate. Multiplication of the bacilli was observed in 1 week and maximal growth in 6 to 7 weeks. All macrophages were filled with tens to hundreds of the organisms in cultures showing maximal growth. Glycerol caused an increase in the normal length of M. lepraemurium, without a corresponding increase in the number of the bacilli. Elongation of M. lepraemurium was observed 3 or 4 days after infection. Rapid and uniform growth of M. lepraemurium was achieved in serially transferred cultures (subcultures). The cumulative increase of the number of intracellular bacilli was 1.4 x 10(20)-fold in 14 transfers over a period of 68 weeks in one series, and 10(17)-fold in 12 transfers over a period of 56 weeks in another series. The generation time of M. lepraemurium was 7 days, a growth rate which approximates the fastest growth of the organisms in vivo. Organisms harvested from cultures at various stages of growth produced murine leprosy in mice, but showed no growth in bacteriological media. The present model offers an opportunity for studies on the host-parasite relationship without the complication of extracellular growth of the parasites.