To grow Mycobacterium leprae in cultured mouse peritoneal macrophages, studies were made on 1) the purification of M. leprae from lepromatous nodules by trypsinization, 2) growth experiment of purified M. leprae in cu1tured macrophages by in vivo infection-in vitro cultivation technique and 3) the observation of pathological changes in sp1eens of mice induced by intraperitoneal inoculation of purified M. leprae. Results are summarized as follows. 1. A simple and effective procedure is described for purification of M. leprae from biopsied nodules of lepromatous leprosy patients by trypsinization and high speed centrifugation. The procedure resulted in a good yie1d of homogeneous preparation of M. leprae with a negligible contamination of tissue debris. 2. Significant decreases were observed in the numbers of acid-fast bacilli in cultured macrophages and of macrophages harboring acid-fast bacilli by the length of intervals between the time of intraperitoneal inoculation of purified M. leprae and the time of initiation of macrophage cultures. 3. Microscopic examination of stained preparations of macrophages cultured by in vivo infection-in vitro cultivation technique indicated that an apparent increase in the number of acid-fast bacilli in the macrophages occurred when the cultures made at 24 hours and 1 week after inoculation were maintained in vitro up to 2 months or more. 4. Pathological changes in the spleens of mice inoculated with purified M. leprae were of mainly degenerative nature in the red pulp. No multiplication of M. leprae was observed in the spleens of mice up to 5 months after inoculation.
Adolescent ; Adult ; Animal ; Cells, Cultured ; Female ; Human ; Leprosy/microbiology* ; Macrophages/microbiology* ; Male ; Mice ; Mycobacterium leprae/growth & development* ; Mycobacterium leprae/isolation & purification ; Peritoneum/microbiology*

OBJECTIVETo understand the genotypic mapping of Mycobacterium leprae identified in China and to compare with those from other countries to select suitable alleles for epidemiological investigation in the transmission chain of leprosy.

METHODSVarious number of tandem repeat(VNTR) in genomic DNA of Mycobacterium leprae was used in the present genotyping study. 33 skin biopsies from Wenshan prefecture,Yunnan province and 17 from other parts of China were studied. DNA extracted from skin biopsies of leprosy patients was subjected to PCR followed by agarose gel analysis and DNA sequencing to determine the number of repeats.

RESULTSLoci GGT-5,12-5,21-3 and 23-3 were as highly homogenous as 100%; The homogeneity of loci AC-8, 18-8, 27-5 and rpoT were 97%, 94%, 97% and 85% respectively. Loci GTA-9, AC-9 and 6-7 showed significant allelic diversity in isolates and the diversity of GTA-9 in Mycobacterium leprae isolated from China was also different from those identified other countries. We had subjected loci GTA-9 and the ten loci to phylogenetic tree analysis respectively.

CONCLUSIONThe present study revealed that the genotype of Mycobacterium leprae identified from China was close to the strains from the Philippines and India although a few loci were somehow differentiate. Locus 12-5 manifested as only 3 copies in China whereas 4-5 copies predominating in other countries. 12-5 locus might serve as a useful marker to diffrentiate Chinese strains from those in other countries. However, further study on the diversity of GTA-9 was needed in China. The molecular typing of Mycobacterium leprae from different geographic areas might be useful in studying the transmission of leprosy.

Alleles ; China ; epidemiology ; DNA, Bacterial ; Genotype ; Humans ; Leprosy ; epidemiology ; transmission ; Molecular Epidemiology ; Mycobacterium leprae ; genetics ; isolation & purification ; Polymerase Chain Reaction ; Skin ; microbiology