The utilization of the lepromin reaction in the L-type patient is useful in differentiating leprosy bacillus from other acid-fast bacilli. A problem encountered here, is the influence of tissue components on the skin reaction other than the bacterial cell.
To clarify this, antigens were prepared by the Mitsuda method from the skin of healthy mouse, skin of healthy man, leproma of marine leprosy and leproma of leprosy patient, and prepared from M. ulcerans suspension. Fifty-six cases of L-type and 15 cases of T-type leprosy patients were injected with the various antigens and the reddening which had developed, was comparatively examined. Similar experiments were twice carried out.
The development of the reddening as an early stage reaction (average 3.6-5.3mm) and the late stage reaction (average 2.3-3.2mm) was observed, when the mouse skin antigen was tried, indicating then higher values of 1.2-2mm for the early reaction and 0.4-2.1mm for the late reaction in comparison with the human skin antigen.
In conclusion, a lepromin reaction tests, when determinating it with the antigen made from the acid-fast bacilli multiplied in the inoculated mouse, should be carried out with the antigen prepared by Dharmendra's method, rather than Mitsuda's antigen. Otherwise, the antigen should be diluted as much as possible and when determinating it, it is important to consider the reddening difference.

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In order to examine the influence of tissue components contained in the Mitsuda antigen upon the lepromin reaction, the skin reaction caused by tissue suspension was compared with the Mitsuda's reaction. The tissue suspensions were prepared from the skin of a leprosy patient free from leprosy bacilli or the skin of a healthy person by means of the preparation procedure of the Mitsuda antigen. The results obtained were as follows:
1) The size of reaction to the tissue suspension was compared between the two different sources of antigen material; namely the skin of a leprosy patient free from leprosy bacilli and the skin of a healthy person. No significant difference was observed between them, and this observation denied the possibility that some tissue components might be involved in the Mitsuda's reaction. Therefore, the reaction caused by tissue components can be cosidered as non-specific one unrelated to the essential lepromin reaction provoked by the Mitsuda antigen.
2) With the increment of tissue components in the antigen, the size of skin reaction became larger, particularly in the reaction read at 15 days after the injection of antigen. This observation indicates the need of eliminating the contamination with the tissue components in the Mitsuda antigen to a possible extent.
3) The size of skin reactions caused by the tissue suspension was compared with that of reactions provoked by the Mitsuda antigen through the frequency distribution curves of reaction size. This comparison indicated that the reactions of 9 to 13mm in the size read at 48hrs and the reactions of 3 to 10mm in size at 15 days after the injection of antigen were a mixture of the specific reaction to leprosy bacilli and the non-specific one due to the tissue components. This observation will be the key point for the settlement of the criteria for reading the reaction.
4) Concerning the mode of appearance of the skin reaction caused by the tissue suspensions, there was no difference between the two clinical types of leprosy at 48hrs reading. At 15 days reading, however, the tissue suspension of higher concentration than the Mitsuda antigen induced the skin reactions of larger size in the non-lepromatous type than in the lepromatous type.
From the above results, the criteria for reading the Mitsuda reaction at 15 days after the injection of antigen, particularly, the positivity in the Mitsuda reaction was discussed on the difference between the size of reaction caused by the tissue suspension and that provoked by the Mitsuda antigen.

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The studies were carried out in regard to the phosphorous metabolism of leproma.
After intravenous injection of ³²P-orthophosphate, rats were killed at serial intervals and the subcutaneous leproma were resected. The rate of incorporation of ³²P into leproma was considerably rapid and increased consistently for the first 60 minutes, and relatively high levels of ³²P were maintained following 24 hours. The murine leprosy bacilli were collected. The ³²P was taken up by the bacilli somewhat delayed compared with leproma, but a parallel relationship was found between them.
The phosphrous fractions of leproma resected at serial intervals one hour after injection of ³²P were obtained by the Schneider's method. The ³²P was rapidly taken up by the acid-soluble fraction and the specific activity increased consistently for one hour. The incorporation of ³²P into the acid-insoluble fraction was significantly high in the phospholipid fraction in contrast to the low levels in the nucleic acid and phosphoprotein ones. The turn over rate of the phosphorous, however, was more active in the nucleic acid fraction than in others.

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For the purpose of biological potency test on lepromin, particularly on the Dharmendra antigen, guinea pigs were sensitized with defatted leprosy bacilli suspended in liquid paraffine, and various conditions necessary for the optimal sensitization as well as the specificity of cutaneous reactions were examined, comparing with those in animals sensitized with heat-killed tubercle bacilli.
It was found that the sensitization of guinea pigs to defatted leprosy bacilli is achieved by the injection of about 10 times larger amount of the bacilli and after longer period of incubation than the sensitization with dead tubercle bacilli. However, the cutaneous reaction in guinea pigs sensitized with the former was relatively specific to the Dharmendra antigen.
Adjuvant effect of lipid fractions extracted from leprosy nodules on the sensitizing as well as cutaneous reactivity of defatted leprosy bacilli was also examined, and it was found that the alcohol-insoluble fraction has an intensifying effect on the protency of the Dharmendra antigen, in contrast to the inhibitory action of the alcohol-soluble one. However, skin-sensitizing activity of defatted leprosy bacilli was not influenced by the addition of acetone-insoluble lipid which composed of the above two fractions, because of contradictory effect of each other.

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The aim of the present study was to examine the relation between the grade of difficulty in cultivation of mycobateria and their development of cytochrome system as well as the difference of cytochrome system between the same strain of mycobacteria grown in vitro and in vivo. For this purpose, spectrophotometric investigations were made on cell suspensions or particulate fractions of four species of mycobacteria grown in vitro; M, smegmatis, BCG, M, tuberculosis avium, Flamingo strain and M, paratuberculosis and two species grown in vivo; BCG in vivo and M, lepraemurium.
BCG cells grown in vivo were isolated from the hard nodule at the great omentum region of guinea pig, thumb size, formed due to the growth of the organism which had been injected intraperitoneally. The method of isolation consists of protease digestion of tissue fragments and centrifugal fractionation of bacterial cells. The viable cell number per unit weight of the cells grown in vivo was found to be approximately the same as that of BCG cells grown in vitro. The cells of M, lepraemurium, Hawaiian strain, were obtained from subcutaneous leprous nodules formed in mice, the isolation of the cells were carried out as described previously elsewhere.
The results obtained indicated the presence in M, smegmatis of cytochromes of the a, b₁ and c types, as well as a CO-binding pigment similar to cytochrome o, in BCG and M, tuberculosis avium, Flamingo strain, of cytochromes of the a, b and c types and in M, paratuberculosis of cytochromes of the a, b₁ and c types. While no clear interrelation were evident between the cytochrome patterns or their cytochrome contents of these organisms and the ease with which they can be cultivated in vitro, the cells of BCG grown in vivo and these of M, lepraemurium were found to be completely deficient in cytochrome pigments.
From the point of this similarity in the respiratory system of BCG grown in vivo and murine leprosy bacillus, that is the defect of cytochrome, it was presumed that further similarities in their respiratory mechanism might exist among these mycobacteria grown in vivo. Accordingly, it seems to be quite useful to study the respiratory system of BCG grown in vivo, which could be independent on cytochrome system, in order to contribute to the study on the metabolism of murine leprosy bacillus.

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