In the present paper, it was described that newly improved cell-free culture me-dium, referred to as NC-5, permitted more abundant multiplication of M. lepraemurium than that of NC-3 medium which was previously reported. The NG-5 medium was established by the method in which 1-cysteine HC1 was added to NC-3 medium. Opti-mal concentration of 1-cysteine HC1 to be added was 0.03%. When three strains of M. lepraemurium, Hawaii, Kurume No. 42, and Kumamoto, were inoculated to and cultivat-ed in NC-5 medium at 30°C, these strains easily multiplied, whereas there were some differences of growth abilities among the three strains in the case of NC medium.

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It is important to clarify the source of natural infection of murine leprosy since we have found in previous experiment that murine leprosy bacillus is present in apparently healthy mice, an attempt was therefore made to isolate acid-fast organisms from the or-gans of fresh water fish bred and reared in pond closely related to earth and then in-oculate the organisms isolated to SPF (specific pathogen free) mice rearing under aseptic conditions in Vinyl-Isolator system.
For examination of bacilli in fish, stamp smears were prepared from liver, spleen, . kidney and gill. The slide was stained by Ziehl-Neelsen method, examined, closely under the microscope and organ showing bacilli was emulsified in Hanks solution, centrifuged lightly, and a small quantity of india ink added to supernatant, and a part of material prepared was inoculated subcutaneously into the back of SPF mice and the other of its was cultivated. The inoculated mouse carefully examined by method of spread prepara-tion from connective tissue surrounding the site of inoculation, marked by the india ink.
The organs of 3 carp and 280 goldfish were examined for the presence of acid-fast bacillus, and the bacilli were found in the organs from 3 goldfish. In 2 out of 6 primary inoculated mice (origin ; No. 243), though a leproma was not formed, there was pronounc-ed bacterial proliferation at site of injection, and then the material from subcutaneous tissue showing the proliferation of the bacilli was inoculated into next generation of SPF mice. Four of 6 animals in second generation showed bacterial proliferation and a mild leproma, and moreover 3 of 4 animals showed pulmonary lesion with leproma. All animals inoculated with material from second passaged mouse developed subcutaneous lepromata of murine leprosy in third generation (C3H mouse reared in conventional).
In the control, proliferation of acid-fast bacillus was not found in animals both groups in inoculated with heat-killed material and untreated, and there was no growth on the usual media for acid-fast bacteria at 33°C or 37°C.
As a result of the finding in this study, it was shown that acid-fast bacilli were found in organs of fish grown in fresh water pond and the typical leproma of murine leprosy was produced at site of inoculation in mice inoculated with living material. It seems that these findings are important in considering the origin of natural infection of the murine leprosy, but it is difficult to obtain the conclusion from only the present data because there are several questions at present time. It will be able to clarify the difficult problem according to development of culture technique of M. lepraemurium in the future.

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The bacillus employed in the test was the 6th subculture, 2-month-old on the 1% egg yolk medium, of the supposed Hawaiian strain. Male mice of ddN strain were divided into 6 groups of 10 each and the groups were inoculated either subcutaneously or intravenously with each 3 different doses (2×10-1, 2×10-2, 2×10-3mg and 10-1, 10-2, 10-3mg). The mice inoculated were sacrificed at the end 4, 6, 9 and 11 months (Table 1).
Observation of gross diseases were made at necropsy. Spleens were weighed and their weight per gram of body weight were recorded. The internal organs, superfi-cial lymph nodes and local lesions were removed, homogenized, and then submitted to microscopic examination and cultural recovery (Tables 2 and 5). The Ziehl-Neelsen stained smears were rated not only for the number of acid-fast bacilli under oil-imme- rsion but also for relative numbers and maximum and minimum sizes of globi under low magnification (Tables 2, 5, 4 and 6). In some instances histopathologic examinationswere also performed (Table 3). The results obtained indicated that in the mice inoculated with each of the doses subcutaneously or intravenously there was gross and bacteriologic evidence of infection, supported in some by histopathologic evidence. The findings were more pronounced in the animals intravenously inoculated than in those subcutaneously inoculated. In either case, however, they showed a tendency to become more marked in the course of infection. It seemsed reasonable to conclude that the disease in mice was fairly consistently reproduced with the test organisms, even when small inoculum doses, such as 10-1, 10-2, and 10-3 mg were used.

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The bacillus employed for the test was the 2 nd subculture, 3-month-old on the 1% egg yolk medium, of the supposed4) Keishicho strain. Male mice of ddN strain were divided into 6 groups of 12 each and the groups were inoculated either subcutaneously or intravenously with 3 different doses (10-1, 10-2 and 10-3mg) of the bacillus. The mice subcutaneously inoculated were sacrificed at the end of 3, 6, 8, 10 and 13 months and the mice intravenously inoculated at the end of 3, 6, and 8 months (Table 1).
Observations of gross disease were made at necropsy. Spleens were weighed and their weight per gram of body weight were recorded. The internal organs, super-ficial lymph nodes and local lesions were removed, homogenized, and then submitted to microscopic examination and cultural recovery (Tables 2, 3, 4 & 7). The Ziehl-Neelsen stained smears were rated not only for the number of acid-fast bacilli under oil-immersion but also for the relative numbers and maximum sizes of globi under low magnification (Tables 6&8). In some instances histopathologic examinations were also performed (Table 5).
The results obtained indicated that in the mice inoculated with each of the doses subcutaneously or intravenously there was gross and bacteriologic evidence of infection, supported in some by histopathologic evidence. The findings were more pronounced in the animals intravenously inoculated than in those subcutaneously inoculated. In either case, however, they showed a tendency to become more marked in the course of infec-tion. It seemed reasonable to conclude that the disease in mice was fairly consistently reproduced with the test organisms.

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As stated in our previous reports, this author considered from comparative observa- tions on the development of experimental murine leprosy, after subcutaneous infection, in various inbred strains of mice that mouse leprosy could be classified into two polar types, benign and malignant. The former type is represented by the case of C57BL/6 strain and the latter type is represented by that of C3H strain.
However, no significant differences were observed concerning the development of visceral lesions between these two strains, C57BL/6 and C3H, after the intraperitoneal infection.
On the other hand, long-term observations, up to 80 weeks, were made in various inbred strains of mice with the subcutaneous infection by the same manners previously examined. From the results of these observations, mouse leprosy should be classified into three types, benign, intermediate and malignant. In the case of intermediate type, newly classified, the leproma at the inoculation site shows the benign feature but the visceral lesion develops severe with time as observed in the malignant case.
Therefore, further long-term observations were made in 7 inbred strains (C3H, CFW, CF#1, KK, BALB/C, C57BL/6 and DDD) of mice, after the intraperitoneal infection, on the development of visceral lesions and survival time of the experimental animals.
Except the cases of DDD strain, the development of visceral lesions showed similar tendency to that observed in the previous experiment, in the cases of these 6 strains. There were no significant differences among these 6 strains, concerning the survival time of mice inoculated with 0.5ml of a 1:100 suspension. In the cases inculated with 0.5ml of a 1:10000 suspension, significant but slight prolongation of the survival time was observed in the cases of C57BL/6 strain which was representative of the benign type after the subcutaneous infection. But no marked difference was observed in survi-val time among the cases of the remaining 5 strains.
In contrast, mice of the DDD strain showed remarkable prolongation of the survival time compared with those of the other strains. Especially, all the DDD mice which were inoculated with 0.5ml of a 1:10000 suspension were survived throughout the ex-perimental period, although almost all the mice of the other strains died at the earlier stage of infection.
It is emphasized from the findings of this experiment that remarkable delay in evolu-tion of the visceral lesions followed murine leprosy infection is a distinctive characteristic of the DDD strain.

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