The subcutaneous connective tissue, lymphnodes and organs of healthy mice were examined in detail by the spread tissue preparation and stamp smear methods and staining with the Ziehl-Neelsen stain. Material from bacillus positive animals was cultivated onto artificial media or inoculated into other mice and numerous acidfast bacilli were isolated. Leproma was produced in mice inoculated with the isolated organisms and identification tests showed many of the organism to have properties similar to the murine leprosy bacillus.
The organism has been called the murine leprosy-like acid-fast bacillus since there are several points of difference such as the absence of active infection in the original animal, the lack of leproma production but proliferation in the lungs in the next generation of mice and the simple conglomeration of the organism rather than the presence in the cells. The source of infection is discussed.
As a result of the findings in this study, it is suggested that caution must be exercised in experiments in which mice are inoculated with the leprosy bacillus, and that acid-fast bacillus present in natural circumstances must be noted as an infectious agent of natural murine leprosy.
This study was supported in part by a grant from WHO.

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Une émulsion dense des tissue broyés de lésions sous-cutanées de rat blanc a été inoculée dans un milieu liquide et incubée à 30°C. (1) Aprés trois à quatre semaines quelques modifications ont été déjà remarquées dans les tubes incubés à 30°C en comparaison avec ceux mis à 5°C comme témoins. Ces modifications sont devenues les plus nettes aprés huit à dix semaines et étaient à supposer comme les conséquences du développement in vitro. (2) Les formes longues, de 10 à 15μ de long ou plus, qui se sont développées étaient à considerer comme les vrais filaments mycéliens. Elles se sont segmentées souvent et ont projetté nombre de ramifications. Ces éléments c'est-à-dire les filaments et leurs ramifications ont formé, en se groupant ou en s'enroulant les uns sur les autres, une construction assez compliquée comme les branches ou les racines d'un arbre c'est-à-dire un mycélium vraisemblable. (3) Du point de vue de la morphologie et de l'arrangement de ce germe développé in vitro, it est apparent qu'il n'appartient pas au Genre Mycobacterium, mais à un autre Genre qui doit etre etabli à nouveau dans le voisinage du Genre Nocardia dans la Famille Actinomycétacées.

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Un milieu solide a été inocule avec l'émulsion dense des tissues broyés de rats blancs atteints et incubé à 30°C: Quoique quelques modifications ont été déjà remarquées, aprés trois ou quatre semaines, dans le tube incubé en comparant avec le tube des témoins mis à 5°C, ce fut encore aprés six à huit semaines que la modification devint tellement nette qu'on a pu la juger facilement comme un développement in vitro. Nombre de filaments mycéliens de 10 à 15μ de long ou plus se sont évolués, en projettant un certain nombre de ramifications longues ou courtes et en se divisant eux-memes en plusieurs segments. Ces éléments, en se groupant ou en s'enroulant les uns les autres, ont formé souvent une construction compliquée comme un filet ou un disque composée de cordons larges ou fins. Elle était à considérer comme un mycélium vraisemblable. Du point de vue de la morphologie et de l'arrangement que ce microbe a formé en se développant sur ce milieu solide, il est déjà clair que ce microbe n'appartient pas au Genre Mycobacterium, mais à un autre Genre qui doit etre établi à nouveau dans le voisinage du Genre Nocardia dans la Famille Actinomycétacées. Ces observations nous ont indiqué d'un cote une certaine période plus favorable où la culture primaire, réussie comme ci-dessous, peut etre repiquée avec succés, mais elles semblaient nous suggérer d'un autre côté que le repiquage en série ne sera réalisé qu'apres l'invention d'une nouvelle technique.

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In the experimental murine leprosy the infection is usually produced by subcutaneous or intraperitoneal inoculation. Under these conditions the long period of observation, up to 2-3 months, is generally required. On the other hand, the observation period can be shortened by using bacillus counting as the criterion for evaluating the bacillary multiplication. However this method is very complicated technically. Therefore, it is desirable to find a simple and reliable method by which the experimental period should be shortened.
Since the good results were obtained on our works by using the spread tissue preparations to find acid fast bacilli in the subcutaneous tissues of healthy mice, this technique has been used in the early stage of subcutaneous infection to investigate the development of murine leprosy.
In this experiment, mice of the C57BL/6strain, being of benign type, were inoculated subcutaneously in their back with 0.25ml of a 1:1000 leproma suspension and sacrificed on 1, 5, 10, 15, 20 and 25 days to examine the spread preparations from the site of inoculation. One day after the inoculation, short acid-fast bacilli similar to the initial ones were found in cytoplasm of mononuclears. After 5 days, elongation of the bacilli was observed in mononuclears without increase in number. The maximum length of bacilli was about 2-3 times the initial size. On 10th day, fairly numerous bacilli of long forms were observed. This finding suggests that the bacillary multiplication has begun to occur. Marked increase in number of the bacilli was observed for 15 to 25 days after the inoculation. Therefore the enlargement of mononuclears, each of which was being crowded with longer bacilli, could easily be observed microscopically in a low magnification.
Elongation of the bacilli and increasing in number were examined in C3H mice, being of malignant type, in the same way. A pattern similar to those observed in C57BL/6 mice was also obtained. No significant difference was found between the mouse strains.
On the basis of these observations, this experimental method can be recommended for an early evaluation of development of murine leprosy.

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Having been found that the Dharmendra antigen combined with certain lipid fraction of leprous nodule (the "combined" antigen) gives stronger cutaneous reactions than the regular antigen in guinea pigs sensitized with leprosy bacilli, comparative tests between the above antigens as well as with a standard lepromin were done in leprosy patients, in order to find whether the effect of lipid fraction is seen in Fernandez reactions and/or Mitsuda reactions. The combined antigen was prepared by adding dried residue of the alcohol-insoluble lipid fraction to the Dharmendra antigen so that a weight-ratio of lipid to bacilli is 1: 4, considering the yields of them from the same pool of leproma.
The results of comparative tests between the combined antigen and the Dharmendra demonstrated that the former gives more strong Fernandez reactions than the latter in 85 cases of non-lepromatous type, since a mean of differences in reactionsizes to the both antigens is statistically significant; while that in 406 cases of lepromatous type is not. With respect to the Mitsuda reactions, however, these antigens showed no difference in reaction-size of lepromatous as well as non-lepromatous cases.
Comparing with the standard lepromin, the combined antigen gave somewhat strong Fernandez reactions in 21 cases of non-lepromatous type. On the contrary, the combined antigen gave weaker Mitsuda reactions than the standard lepromin in 98 cases of lepromatous type.
These results indicated that the alcohol-insoluble lipid of leproma has an intensifying effect only to the Fernandez reactions in non-lepromatous cases. In spite of this fact, usefulness of Mitsuda or late reactions to the Dharmendra or combined antigen was discussed, so far as observations in leprosy patients, according to a criterium of positive reactions different from that used for a lepromin.

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It has been reported by many workers that acid-fast bacilli produce large quantities of riboflavin and as it has also been stated that diaphorase, which is considered a flavin enzyme is shown by the murine leprosy bacillus, it was assume that riboflavin would be present in the murine leprosy bacillus but examination of the difference spectrum of cytochromes of murine leprosy bacillus revealed the absence of an absorption band equivalent to riboflavin. In order to clarify this discrepancy, quantitative determinations of riboflavin of murine leprosy bacillus was attempted and noteworthy results were obtained. The lumiflavin method of Yagi et al. was followed, and the riboflavin content of a heated extract of murine leprosy bacilli was measured. The riboflavin content of 1.0g of dried murine leprosy bacillus was 7.5μg and about 1/10 that of rapid grower acid-fast bacilli and about 12.5 that of BCG. The results show that the greater rate of proliferation of the bacillus, the higher was the riboflavin content and the speed of proliferation is in direct proportion to the quantity of riboflavin.
The presence of FAD in the heat extract of murine leprosy bacilli was proven with FAD-free D-amino acid oxidase but quantitative determination was not possible.
Quantitative determination of FAD arid FMN was attempted on an extract prepared with cold TCA according to Bessey's method but FAD could not be found in murine leprosy bacilli though a equal volum to that obtained with the lumiflavin method was verified with acid-fast bacilli grown in the test tube. In other words, the FAD of murine leprosy bacillus is not extractable with cold TCA.
Similar to succinic dehydrogenase, enzyme protein and FAD are firmly bound in this flavin enzyme and the two are not easily separated, and FAD moreover has lost the character of spontaneous oxidation. It is suggested that FAD is present in this form in the murine leprosy bacillus so that it is not extracted by cold TCA and as it is not converted to the oxidized form by aeration in air, it is not revealed in the difference spectrum between oxidation and reduction. It is believed that the electron-transport stops at the stage of flavin enzyme in the murine leprosy bacillus which is lacking in cytochromes. Since the flavin enzyme of murine leprosy bacillus have not the ability of spontaneous oxidation, energy production will not occur so long as there is no mechanism for oxidizing this reduced form of flavin. In order to grow murine leprosy bacillus in vitro, it is believed that a method which will oxidize this reduced form flavin enzyme is required.

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It has become clear that the reduced coenzyme I (NADH) produced by malic dehydrogenase is oxidized by Diaphorase I but the route by which NADH is oxidized in the murine leprosy bacillus is not know. Since there is no cytochrome system in the murine leprosy bacillus, catalyzation by oxygen in air will not take place. In cultivating the organism in vitro, a pathway for the hydrogen of NADH must be made inorder that energy may be produced, else cultivation is not possible. Elucidation of the conditions which will adequately fulfil the flow of electrons will become an important starting point for the in vitro cultivation of the murine leprosy bacillus.
As the initial step, it has been found that NADH is converted to NAD and water by NADH peroxydase in the presence of H₂O₂. This NADH peroxydase is present not only in murine leprosy bacillus but is also present in vitro cultivated M. Jucho and BCG. The enzyme is easily inactivated by heat but is stable to dialysis.
When the murine leprosy bacillus is present in the host cell, H₂O₂ is produced by the cell so the flow of electrons may proceed smoothly and energy produced through the action of NADH peroxydase but utilization of this pathway is difficult in vitro.

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When animals are inoculated with an antigen during foetal or early postnatal life, a phenomenon known as immunological tolerance takes place which makes it difficult for the animal to produce antibodies against the same antigen when challenged after maturity.
The attempt was made to infect mice with the leprosy bacillus after eliminating the resistance utilizing this phenomenon.
The experiment was carried out a total of 4 times starting in March 1962. The mice were of the ICF strain and a total of 605 animals were used.
Inoculant : Lepromata from 4 untreated and 10 relapsed leprosy cases were collected, stored under refrigeration for 1 day to 70 days, sliced and an emulsion prepared with YLH solution. A small quantity of india ink was added. The inoculum contained 3×10⁶-3.6×10⁷ organisms per milliliter. For the pre-treatment antigen, the live bacterial suspension was used since it was believed that a degenerative change may take place in the bacterial cell component with heating. It need not be emphasized that all the procedures were carried out under sterile conditions.
Experimental Method: The animals were divided into two groups A and B.
The first group was further divided into 3 subgroups, I, II and III, according to the number of pre-treatments given and the control group, was divided into 2 subgroups, IV and V. I consisted of newborn mice and 0.05ml of live leprosy bacillus suspension was injected intraperitoneally within 24 hours after birth. II was treated in the same way as I but an additional injection of 0.01ml of bacterial suspension was given 1 week after the first. III was given 2 additional injections of 0.01ml basides the first at intervals of one week.
One to 3 weeks after the pre-treatment, 0.1ml of bacterial suspension was injected subcutaneously in the back. The animals were sacrificed after 10 - 20 months and examined for bacterial proliferation.
In IV, live bacterial suspension was injected intraperitoneally and subcutaneously in the back at the same time in the newborn mouse while in V, the live bacillus was injected subcutaneously in the 3-week-old animal without pre-treatment.
A piece of subcutaneous connective tissue was excised from lesions which could be observed macroscopically while in the animals in which no change was apparent, a piece of tissue was removed from the site of injection marked by india ink. A spread tissue preparation was made with the connective tissue specimen. Smears of the lyrnphnodes and organs were also prepared. These were stained with Ziehl-Neelsen's stain and examined microscopically for bacteria. Material showing proliferation of acid-fast organism was inoculated onto culture media and into the next generation of mouse, including both tolerance treated and non-treated animals. Two types of culture media, neutral medium for alkalinetreated and then neutralized material and acid medium, for alkaline-treated material were used. Cultivation was carried out at 33°C and 37°C.
From the findings in the present study, it is clear that pre-treatment to give immunological tolerance had no effect on the proliferation of leprosy bacillus inoculated in the mouse. Acid-fast organism which could be considered a true leprosy bacillus furthermore could not be isolated from any of the leproma material from 14 cases of leprosy. It should be noted, however, that numerous murine leprosy-like acid-fast bacilli similar to those reported by Nishimura were isolated. In the previous study, murine leprosy-like acid-fast bacillus grew in 14 of 711 suckling mice (ca 2%) inoculated subcutaneously with the live leprosy bacillus while in the present study, proliferation of an acid-fast organism which could be passed to other mice was found in 32 of 576 newborn mice (5.6%) inoculated intraperitoneally.
The reason for this increase of about 3% is not clear and further studies must be carried.

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