我々はヌードマウスを用いてらい菌の継代をしてきている。継代株の内の一つタイ53株かららい菌DNAを分離し, lambda-gtllファージを用いてゲノムDNAライブラリーを作製した。これは平均3kbのインサートを持つリコンビナントDNAが106個近くから成るライブラリーであり,らい菌の全DNAを充分にカバーできるものであると考えられる。事実,PCRによる検索でらい菌特異遺伝子の存在が確認された。

Although the viability of Mycobacterium leprae suspended in distilled water with or without 10% fetal calf serum was reduced approximately 10(-2) to 10(-4) from that of the starting material during the process of lyophilization, bacilli capable of multiplication in nude mouse foot pads were found in the lyophilized samples stored for 4 years at 4 degrees C. The multiplication rate of the lyophilized bacilli which were suspended in 10% serum-water was much higher than that of the bacilli suspended in water only. On the other hand, no reduction of the viability of M. leprae suspended in 10% skim milk-water was demonstrated during the process of lyophilization as well as storage for 2 years at 4 degrees C. From the results obtained here, it could be suggested that M. leprae might be preserved in vitro by means of lyophilized M. leprae was extremely stable during cryopreservation when the bacilli were suspended in 10% skim milk-water. Therefore, the composition of the solution for suspending the bacilli is definitely critical for the maintenance of M. leprae viability by means of lyophilization.

When BALB/c mice were infected with Mycobacterium leprae and orally treated 6 times weekly with a dose of 8 mg/kg cyclosporin A (CsA) for 19 months, the number of organisms was slightly higher at 19 months as compared with mice in which the dose of CsA was gradually decreased after 6 months and discontinued at the 8th month (p less than 0.01 for the 15th and 19th months). Lymphocyte blast transformation (LBT) showed that spleen cells from CsA-treated mice 4 weeks after infection with M. leprae and 3 weeks after CsA treatment was stopped responded to the sonicated supernatant of M. leprae suspension (SS), M. leprae (Ml), and concanavalin A (ConA) less than those cells from mice not treated with CsA. This response was dose-dependent. At week 15, 14 weeks after CsA administration was stopped, the LBT response to SS and Ml by cells from M. leprae-infected mice exceeded that of mice without CsA treatment, and the response to ConA in M. leprae-infected mice was less than that in uninfected mice without CsA-treatment. Thus, if CsA was administered, the T-cell functions were suppressed. However, when CsA treatment was discontinued for longer periods, the T-cell function was activated. From these results, we speculate that M. leprae would have the capability of growing more abundantly in mice treated with CsA 100 mg/kg for 1 week every month.

When leprosy bacilli grown in nude mouse foot pad were used for culture experiments, cultivable acid-fast bacillus was sometimes isolated as a contaminant. Whenever bacilli were inoculated to nude mice, the same leprosy bacilli were killed by autoclaving and were inoculated in to foot pads of 5 nude mice for examination of this cause of the contamination. Acid-fast bacillus was cultivated on 3% Ogawa egg medium at 33 degrees C from homogenates of foot pads of nude mice infected with M. leprae after one year and a while of infection. Foot pad of nude mouse injected with leprosy bacilli was cut off, ground in mortar and passed through sterile absorbent cotton and the filtrate was centrifuged at 10,000 rpm for 30 minutes. The sediment was inoculated on 3% Ogawa egg medium after treating with a small amount of sterile 1 N sodium hydroxide. Acid-fast bacilli were isolated from 3 out of 41 mice inoculoted with heat killed bacilli. The isolated acid-fast bacillus did not be observed in the same experimental group inocudated with live bacilli, positive cases were scattered in another groups. Four out of 16 tubes were positive for acid-fast bacilli in mice infected with Kurume-naha and 5 out of 7 tubes in the Amami-KM infected mouse group. The two negative tubes were discarded due to contamination. Kurume-Oki strain which has yellow colonial morphology was isolated from one out of 6 culture tubes. Strains Kurume-naha and Amami-KM have the same characteristics as follows: slow grower with pale yellow smooth colonial morphology, strongly positive for niacin production and ureas; positive for nicotinamidase, pyradinamidase and 68 degrees C catalase; no growth at 45 degrees C, negative for nitrate reduction, hydrolysis of Tween 80, diamine oxidase, heat stable acid-phosphatase and arylsulphatase; resistant to streptomycin, isoniazid, rifampicin and B 663. Two isolates were identified as Mycobacterium simiae from these characteristics. Characteristics of a Kurume-Oki isolate was as follows: slow grower with yellow smooth colonial morphology, positive for urease, 68 degrees C catalase, hydrolysis of Tween 80 and arylsulfatase; no growth at 45 degrees C, negative for niacin production, nicotinamidase, pyradinamidase, nitrate reduction, daimine oxidase and heat stable acid-phosphatase; resistant to streptomycin, isoniazid, rifampicin and B. 663. This bacillus was identified as Mycobacterium gordonae from these characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

In an effort to preserve Mycobacterium leprae in vitro, the effect of freezing and drying, i.e., lyophilization, on viability of M. leprae was studied. The viability of the bacilli was quantitatively measured with foot-pad inoculation method using nude mouse. The results obtained demonstrate that the viability of M. leprae was reduced approximately 10(-2) to 10(-3) from that of the starting material, during the process of lyophilization; no viable bacilli were detected in the lyophilized sample containing less than 1.8 X 10(3) bacilli. On the other hand, the bacilli capable of multiplication in nude mouse foot-pads were found in the lyophilized sample with more than 10(5) bacilli. From the results obtained here, it could be suggested that there might be a possibility to preserve M. leprae in vitro by means of lyophilization.

The 65-kDa protein of Mycobacterium leprae was produced in an Escherichia coli strain carrying a plasmid harboring the recloned gene coding for the protein. The protein was purified through affinity chromatography prepared with the IgG fraction of a monoclonal antibody which was prepared against the 65-kDa protein. The purified 65-kDa protein also reacted immunologically with the monoclonal antibody IIIE9, which recognizes the epitope for M. leprae, prepared by Buchanan, et al. BALB/c mice were inoculated with M. leprae and 4 months later were skin tested with the purified 65-kDa protein. Gross changes were observed at the skin-test site. The role of the protein in protective immunity against M. leprae foot pad infection in mice was also studied.

Quatre anticorps monoclonaux diriges contre la proteine de 65KD de M. leprae sont obtenus par la technique des hybridomes. Un des AcM reconnait des epitopes presents sur 3 especes mycobacteriennes seulement parmi les 21 especes testees. Ces anticorps peuvent etre des reactifs utiles pour l'analyse des epitopes qui stimulent les cellules T, pour la purification de la proteine 65KD et pour l'identification des mycobacteries

Convit used the mixture of heat-killed leprosy bacilli and live BCG as a leprosy vaccine. It seems that some devices could be applied to inactivate the bacilli instead of heat-killed bacilli of Convit's vaccine. When heat-killed bacilli were inoculated to mouse, many stainable acid-fast bacilli were detected in the injected site for a long time. However, in the case of live leprosy bacilli, stainable acid-fast bacilli disappeared comparatively ear-lier in the injected area. Leprosy bacilli of live but defected growth ability may be more efficient for vaccine than heat-killed bacilli.Purified leprosy bacilli were prepared from leproma produced in nude mouse foot pad infected with Mycobacterium leprae, Thai-53 strain, by ficoll gradient centrifugation me-thod and treatment of 1 N sodiumhydroxide. The bacillary counts were 1.6×1010 in 16.3ml of 0.1M P. B. (pH 6.4). The first group of nude mice were inoculated with live bacilli into their right hind foot pads, the second group were injected with heat killed bacilli, the third group were injected with formalin treated bacilli, the fourth group were inoculated with rifampicin treated bacilli, the fifth group were injected with Co60 irradiated bacilli and the sixth group were injected with bacilli cultivated on Ogawa egg yolk medium for 4 months. The first to the third groups consisted of 5 mice and the fourth to sixth groups consisted of 8 mice. Bacterial suspension of 0.05ml were injected in right hind foot pad. For the first to the third groups, observation was carried out at 14 months after inocula-tion and for the fourth to the sixth groups, one mouse was killed every 2 months. Bacterial numbers in the foot pad were counted. In the first group injected with live bacilli, all 5 mice produced lepromas in the injected sites of right foot pads. In the second group injected with heat-killed bacilli, bacterial count was not done because of some trouble. In the third and fifth groups injected with the formalin treated and Co60 irradiated bacilli respectively, bacterial counts were almost the same in the 0 time and after 14 months as seen in Table 1. In the sixth group injected with the cultivated bacilli, the bacillary counts after 14 months were less than that of 0 time and their acid-fastness were weak. In the fourth group, the bacilli treated with rifampicin grew gradually in the foot pads at 8 months after injection and reached to 10 times growth after 14 months.Leprosy bacilli kept in the ordinary medium at 33°C for 4 months may be regarded as dead bacilli. Moreover, if they have been treated with rifampicin, they are probably inactive in growth. If nude mice could not produce leproma in foot pad after injection of leprosy bacilli kept in culture media for a long time, the cultivation method would be hopeless.

Two aspects of the immune deficiency of nude mice make these animals particularly useful tools for leprosy research. Nude mice are capable of supporting multiplication of M. leprae to levels approaching 10(10) per g in peripheral body tissues. In addition, nude mice may be inoculated with greater than 10(4) (in fact, with as many as 10(8) organisms per foot pad, without provoking an immune response that prevents multiplication of the organisms. Thus, the nude mouse should be particularly suitable for detecting persisting M. leprae in treated patients, and as a model of the patient for evaluating chemotherapeutic regimens.

The ability of Mycobacterium lepraemurium (Mlm) to adhere to A31 cells in culture decreased with the number of passages of the bacilli on Ogawa egg-yolk medium. Pathogenic Mlm consistently grew in tissue culture cells but growth was not seen with attenuated Mlm isolated from a smooth colony. After prolonged incubation, attenuated Mlm became adapted to tissue culture growth. The pathogenicity of the attenuated bacilli was restored partially by the adaptation to tissue culture cells and restored almost completely by passage in mice. After restoration of pathogenicity by these methods, the Mlm formed rough-type colonies on Ogawa egg-yolk medium although the colonies were not completely of the rough type. Attenuated Mlm did not interfere with the growth of in vivo-derived Mlm in tissue culture or in mice.

Cat leprosy bacilli passaged in mice could be isolated on 1% Ogawa yolk medium. The isolated cat leprosy bacilli which were cultivated successively four times on 1% Ogawa yolk medium produced a leproma in mice. All characteristics of the isolated cat leprosy bacillus were the same as isolated murine leprosy bacillus, as follows: slow grower, light yellowish-white rough colony, production of much coproporphyrin on the medium, heat-resistant catalase negative, heat-resistant phosphatase negative, arylsulfatase negative, niacin negative, hydrolysis of Tween 80 negative, urease negative, nicotinamidase positive, pyrazinamidase positive, cytochrome b1 at 560 nm positive, cytochrome a2 at 630 nm positive, and cytochrome c at 550 nm negative. Cats are susceptible to both cat and murine leprosy bacilli; the bacilli produced a leproma in a newborn cat at 3 to 4 months and in an adult cat at 2 months after inoculation. Many globi of acid-fast bacilli (AFB) were observed in the histopathological sections and the smear preparations of the newborn cat's lepromas, especially in the necrotic areas of the lepromas. Many AFB and polymorphonuclear leukocytes were seen in the histopathological sections and the smear preparations of the adult cat's lepromas. These lepromas formed ulcers by autolysis and healed or absorbed without ulcer formation over the course of months. Large lepromas remained for a long time without ulcer formation and caseation in some cats. Secondary infections with cat and murine leprosy bacilli were done respectively to the right and left femoral subcutaneous regions of newborn cats carrying primary lepromas. After one month, granulomas in which many AFB were observed were produced in both infection sites. Cats are susceptible to infection with cat and murine leprosy bacilli; however, the bacilli did not invade progressively to internal organs or other subcutaneous areas. Cat leprosy bacilli which were passaged in the mouse are identical to murine leprosy bacilli.

The study results indicated that thymus transplantation was effective in suppressing the growth of Mycobacterium leprae in the nude mouse, and also suggested that thymus transplantation was effective as immunotherapy of experimental leprosy in nude mice. The histopathological findings revealed the induction of reversal reactions in those animals receiving thymus transplants.

Beige mouse which has partially defective natural killer (NK) function, is known as a model of Chediak-Higashi syndrome in man.Six-week-old beige mice, C57BL/6J-bg/bg and W/Wνbg/bg, were provided from Okamura and used in the experiment. CBA and C3H mice or nude mice (BALB/c-nu/nu) were added as controls.Bacillary suspensions of M. leprae were prepared from 3 materials. Kurume-naha strain, 6th and 7th generation, was obtained from nude mouse developed experimental leprosy (obtained from a previously untreated patient and was passaged in nude mice). Suruga-YM and Amami-KM strains were prepared from a leproma taken from a relapse case of a lepromatous patient and from a newly untreated patient, respectively. The mice were inoculated with 5.0×103, 1.1×105, 5.0×105, 5.0×106, 2.0×107 and 7.0×107 M. leprae per 0.05 ml into their right hind foot pads.No significant differences were observed in the growth of M. leprae between the beige and normal mice even though the proliferation was remarkable in nude mouse. When a small number of M. leprae, 5.0×103, 1.1×105 and 5.0×105 was inoculated in beige mice, Bacillary proliferation reached up to 106 in the foot pad the same as normal control group. On the other hand, no any proliferation found in the mice inoculated with a large number of the bacilli.The results indicate that the susceptibility of beige mouse to infection of M, leprae was not over than that of normal mice.

The in vivo effects of rifampin and dapsone on immunological responses were investigated using mice immunized with sheep erythrocytes. The number of cells producing antibody was not affected by a clinical (1 CD) or a threefold excess dose (3 CD) of the drugs administered for ten days. A similar result was obtained in an experiment using a mouse strain known to be low responders to the antigen. Induction of suppressor cells acting on antibody production was not influenced by 3 CD or 6 CD of the drugs. Neither delayed-type hypersensitivity nor induction of the suppressor cell population acting on delayed hypersensitivity was affected by 3 CD of the drugs. Phagocytosis of sheep erythrocytes by peritoneal cells and the growth of a tumor were not altered by 6 CD of the drugs.

Fairly pure leprosy bacilli were easily collected from nude mouse foot pad lepromas by the Ficoll density gradient centrifugation and alkali treatment methods. The yield of bacilli available for biochemical study was 42.6%. The density of Mycobacterium leprae was very heterogeneous. The percent of solid bacilli in the light bacilli fraction was 23%; that in the heavy bacilli fraction was 40%. The endogenous respiration activity in the heavy bacilli was greater than that in light bacilli. The average coefficient of respiration in M. leprae was 1 microliter O2/mg X hr. In the whole cells of M. leprae, a cytochrome b1 absorption peak and its Soret peak were detected at wavelengths of 560 nm and 426 nm, respectively. However, a cytochrome a2-like peak (which was observed in M. lepraemurium), and a cyt c and cyt a were not detected. Catalase activity was not found in whole cells, the cell-free extract, or particle fractions of M. leprae. Any catalase activity associated with M. leprae suspensions is a tissue contaminant. NAD-peroxidase activity was also not detected in the cell-free extract of the leprosy bacillus. These results would indicate that leprosy bacilli cannot degrade hydrogen peroxide.