Biological properties of Myc. lepraemurium have not been completely understood. The presenting paper is to report the fundamental data concerning the effects of chemical treatments on the infectivity of Myc. lepraemurium in order to contribute toward the experimental attempts of cultivation of Myc, leprae which is, at present, an obligate intracellular parasite,
1) Phenol: The infectivity of the bacilli was completely destroyed within 30 min. by treatment of more than 5 per cent phenol.
2) NaCl: Hypertonic salt solution, especially 7% NaCl, destroyed the infectivity of the baci.lli for 3 days at 37°C.
3) SDOC and urea: Protein denaturing agents, such as sodium desoxycholate (SDOC) and urea, had no inactivating effect on the infectivity of Myc. lepraemurium, under the conditions of treatment in the concentration of 0.01% to 1.0% for 60 to 120 min. at 37°C.
4) Organic solvents: Among the organic solvents used, chloroform, ether, toluene and butanol had inactivating effect, but petroleum ether had no destroying effect on the infectivity of the bacilli.
5) Dye: Crystal violet solution in the concentration of 0.1% inactivated the infectivity for 24 hours at 37°C, however, it would be seemed that the solution of 0.00001% crystal violet enhanced the pathogenecity of the bacilli to mice.

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The fate of Myc. leprae and lepraemurium followed by the mouse-joint inoculation which had not been attempted before was observed and the results obtained demonstrated that Myc. lepraemurium developed well in the foot-joint cavity. The part of the bacillus multipliaction was the joint-sack, not joint fluid. No additional effects of mucine, crystal violet, and trypan blue to the bacillary suspension for stimulating the development of the bacilli were observed.
In the case of Myc. leprae experiments, there were no increases of the bacilli in this part, when observed direct smear preparations. However, the further experiments shoud be needed for the determination of the increases of the bacilli, because, in this experiment, the number of bacilli in the materials was not mathe- matically calculated.

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Myc, lepraemurium was inoculated into different parts of a mouse by subcutaneous and intrajoint inoculation with respect to looking for the most susceptible part to development of the bacillus.
The results obtained indicate that rapid and regular increases of the bacilli were observed in the parts of neck, foot-pad, and foot-joint. The mechanism of these findings is not yet dissolved, but it might be thought that the bacilli inoculated did not spread but localize in these parts.

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Attempts have been made to inoculate M. leprae intracerebrally in the adult mouse but there are no reports of intracerebral inoculation in the suckling mouse. The intracerebral inoculation method in suckling mice is recently being used in the isolation of some viruses and a study was carried out to see if M. leprae could be grown by this method. It was also believed that this would be a more appropriate method since it is known that the skin of the healthy mouse is contaminated by murine leprosy-like acid-fast bacilli. Suckling mice, 1-4 days after birth, were inoculated intracerebrally with bacterial suspension prepared from untreated leproma. The control group was inoculated with heat-killed bacterial suspension or left untreated.
The animals were sacrificed and examined 6 months-1 year after inoculation. Of 78 animals inoculated with the live bacillus, 14 showed an increase over the original count (2×10⁵) and of these, over one-haf showed a 100 to 300-fold increase in the number of organisms. Numerous bacilli were found in site other than the brain such as the subcutaneous tissue, lymphglands and other organs. In the group inoculated with the heat-killed bacterial suspension, proliferation was found in 2 nd in the untreated group, acid-fast bacilli were found in brain of 8 of 15 animals. It would be difficult to believe that the isolated organism was the inoculated bacillus and it would be more rational to consider that contamination by murine leprosy-like acid-fast bacilli had taken place. The results suggest that M. leprae cannot be grown in the brain of suckling mice and by rearing the animals for a long time, contamination by murine leprosy-like acid-fast bacilli takes place.

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Relationship between the tuberculin reaction and the reaction to Dharmendra's antigen have been investigated in peoples contained all population, and it was recognized that these relation of both reactions were different each other in the examined towns or villages. Therefore, the relationship between M. leprae and M. tuberculosis or BCG related to tuberculin reaction was studied on the skin test in experimental animals.
The reaction to Dharmendra's antigen was compared with the reaction to old tuberculin or PPD-s prepared from M. tuberculosis hominis and the reaction to Mitsuda's antigen was compared with the reaction to the heat-killed tubercle bacilli suspension with tissue prepared from healthy persons in guinea pigs sensitized by the heat-killed tubercle bacilli or BCG with adjuvant.
The averaged sizes of reactions to old tuberculin, PPD-s or the heat-killed tubercle bacilli suspension with tissue were about equal each other in both sensitized guinea pigs, but the averaged sizes of reactions to Dharmendra's or Mitsuda's antigens in BCG-sensitized animals were larger than the sizes of both reactions in animals sensitized by tubercle bacilli. From these results, it seems that the common antigen contained in M. leprae and BCG is more than that contained in M. leprae and M. tuberculosis.
The size of reaction to Mitsuda's antigen and the heat-killed mycobacteria suspension with tissue in healthy or sensitized guinea pigs were investigated for four weeks after the injection of antigen. The largest size of reaction was seen at 24 hrs, after the injection of antigen and the size of reaction at 5_??_28 days was smaller than that at 1_??_3 days after the injection of antigen. Therefore, the phenomenon of "pre existence hypersensitivity" explained by Wade was not observed in these animal experiments.

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It is assumed that similar to the human leprosy bacillus, the source of infection of murine leprosy is the infected mouse. The infection may be readily passed from the lesion of the infected animal to the next generation of animal, the infective bacillus, like the leprosy bacillus, is difficult to cultivate outside the living organism and the survival outside of the body of the host was believed impossible.
We have found^{(1, 2)}, however, that murine leprosy-like acid-fast bacilli are present in the subcutaneous tissue and lymphglands of symptom-free, apparently healthy mice which have not come in contact with infected animals and the source of natural infection of murine leprosy has become a problem.
The first source which must be considered is, of course, earth which contains numerous acid-fast organisms. The report by Kubica et al⁽³⁾. that an atypical acid- fast bacillus was isolated from earth by cultivation suggests that this is not without basis but in view of the difficulty in cultivating the murine leprosy bacillus, the attempt was made to isolate acid-fast organisms from the organs of fresh water fish bred and reared in ponds, which are, of course, closely related to earth and then transmit the organisms so isolated, to animals.
Several strains of acid-fast bacillus were isolated from the crucian carp and gold- fish and the findings are presented.
The organs of crucian carp, 3 carp and 79 goldfish, grown in fresh water ponds, were examined for the presence of acid-fast bacillus and acid-fast organism was isolated from 2 crucian carp and 10 goldfish. The bacillus was most prevalent in the gill, and a little bacilli were found in the kidney, liver and spleen of the crucian carp. The attempt was made to cultivate and to infect mice using material from 6 cases show- ing relatively large numbers of bacilli. Acid-fast bacillus, not pathogenic for the guinea pig and mouse was isolated by cultivation from material of a crucian carp and 2 gold-fish and acid-fast bacillus capable of proliferating vigorously in the mouse, especially the lung, and producing a leproma was isolated by inoculation in mice from material of 1 crucian carp and 3 goldfish.
It was thus shown that acid-fast bacilli are present in fish grown in fresh waterponds and some are similar to the murine leprosy bacillus and proliferate and produce lepromata in the mouse but cannot be cultivated. It is believed that these findings are important in considering the origin of the murine leprosy bacillus.

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There has been a marked decrease in new cases of leprosy from 1903 to today. If the ratio of new cases of leprosy to the total number of patients visiting the Dermatology Department in 1938 taken as 100, the ratio in 1963 is 14. This sharp decrease may be attributed to improvement in hygienic environment, isolation of patients and the widespread practice of giving BCG vaccinations. It is not clear, however, which of these factors has played the major role.
Of the 183 patients treated in the out-patients clinic of the Institute in the past one year, 129 (70%) are home therapy cases and 53 (30%) have been treated in a leprosarium at one time or another. Though 70% of the out-patients are being treated at home without hospitalization, there has been no special increase in new cases of leprosy in the Osaka- Kobe area because of this.
In the Meiji (1903-1911), Taisho (1912-1925) and early Showa (1926-1938) eras, the peak in the age range of new cases of leprosy was at 20-30 years but recently (1950-1963), two peaks, one at 20-30 years (mostly the L-form) and another at 50-60 years (almost all T-form) have become apparent. New cases in the 50-60 year range have, of course, occur- red previously but the number of cases was small compared to the cases in the 20-30 year range and did not form a peak. The extension of the life span with an increase in the number of old aged has also contributed to this. The reason for infection in the aged is not clear but it can be assumed that, similar to childhood, there is a decline in resistance to the leprosy bacillus.
The ratio of new cases of T-form to L-form, which was 7.1: 1 in the Meiji era (1903- 1908) has gradually declined and is 1: 0.9 today. In Japan, the T-form was previously predominant similar to India and Southeast Asia but today, there has been a marked decrease in the T-form but the decrease in the L-form has not been so pronounced.
This increase in new cases in the aged and the reversal in the ratio of T-form to L-form are extremely interesting from the epidemiological viewpoint.
This report was prepared in commemoration of the 35th anniversary of the founding of the Osaka Dermatological Institute.

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