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 1020-fold in 14 transfers over a period of 68 weeks in one series, and 1017-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.

Multiplication of Mycobacterium lepraemurium in Mouse Foot‐Pad Cell Culture

“…However, some bacteria at-clusters in different numbers were observed days in the Leighton tube culture. These results are not as rapid as those reported by Rees and Garbutt [8] who noted an average generation time of 15 days, and Clung, Anderson, and Vaituzis [2] with their results of 7 days.…”

“…Chang and his associates [2] studied on the effect of mycobactin, glycerol, liver extract, liver homogenate, ferric nitrate, and various combinations of these substances, and found that liver homogenate, liver extract, and ferric nitrate, and combination of L-fraction of liver or iron and glycerol had definite stimulatory effects on the growth of M. lepraernuriurn. Addition of other substances which might be found useful for promoting the growth of this organism will also be examined in future experiments.…”

“…Furthermore, they transferred heavily infected cells to fresh cells and also obtained satisfactory results. The procedures employed by Chang and his associates [2] were always to transfer infected cells to newly isolated mouse peritoneal macrophages at appropriate intervals. In the case of the present study, infected cells in the culture flask were suspended in fresh culture medium two times as large as the preceding culture on each occasion of subculture.…”

“…lepraemurium occurred in subcultures of the 14pf strain of rat fibroblasts with an average generation time of about 15 days. Chang, Anderson, and Vaituzis [2] reported more rapid and uniform growth of the organisms in subcultures of mouse peri-…”

“…In the past decade, reports [1-3, 5, 7-9] were presented in which multiplication of Mycobacterium lepraemurium had been observed to some extent in cultures of mouse and rat spleen explants and of peritoneal macrophages of the mouse and rat as well as in established cell line cultures. However, successful multiplication of the organisms in subcultures has only been reported by Rees and his coworkers [3,8] and Chang and his associates [1,2]. According to Rees and Garbutt [8], multiplication of Al.…”