The Sakurane Award for 1974 was made to a research concerning the above title; the details are to be found in seven articles of a series of our recent communications "Studies on Murine Leprosy Bacillus." In brief, the supposed Hawaiian and Keishicho strains of M. lepraemurinm have been cultured in the form of grossly visible colony on a cell-free solid medium.
The method used for primary isolation is as follows: Mice are injected with the murine leprosy bacillus either subcutaneously or intravenously and killed at a time when the bacillus multiples abundantly in their tissues; each of the specimens removed (spleen, liver, lung, lymph nodes and leproma) is homogenized, treated with one percent sodium hydroxide solution, and then inoculated onto the egg yolk medium; the tubes are incubated at 37°C for more than three months. The validity of this method has been confirmed by several workers (Sato 1972; Mori 1974; Koseki et al. 1972).
Our isolates have been kept in continuous passage using the egg yolk medium. At present, the supposed Hawaiian strain is now in the 21th transfer and the supposed Keishicho strain in the 16th transfer. The in vitro and in vivo characteristics of these two strains are different in many respects from those of other known cultivable acid-fast bacilli. Similar findings were reported by Koseki et al. (1972). Inoculation of as small as 10^-6mg of the bacillus will suffice to produce a progressive disease in mice; the lesions are indistinguishable from those of murine leprosy. The acid-fast bacilli recovered from these lesions were identified to be the same as inoculated originally. It seems, therefore, that our isolates fulfil Koch's four postulates. It may be concluded that the bacillus we isolated is in fact M. lepraemurium. Similar deduction for the supposed Hawaiian strain was made by different investigators on the basis of the results obtained by fluorescent antibody method (Abe 1971), diffusion chamber method (Mori 1972), and the subcutaneous injection into both C3H and C57BL/6 strains of mice (Kawaguchi 1972), respectively.
It must be pointed out, however, that our medium and method are not satisfactory because in primary isolation the culture results are decidedly inferior to those obtainable in the case of the tubercle bacillus and in subcultivation no visible growth is attained by the use of the bacterial suspension. Further studies into these problems are necessary.

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A follow experiment of the Ogawa's method was began at April 1971 and identification of cultivated M. lepraemurium passaged 11 generations was carried out by a method of injection to mouse, rat, hamster, rabbit, guinea pig and quail and by a diffusion chamber method.
Efficiency of isolation culture was better in cases of a subcutaneous leproma treated with alkali and using air loose stopper than in cases of no treatment and using air tight stopper. When the cultivation was carried out under 33°C for long time, several microcolonies of M. lepraemurium were seen after one year. As M. lepraemurium grows very slowly, it takes so long time that primary isolation should be kept low temperature to avoid a degradation of medium composition. The isolation of M. lepraemurium succeeded in 83% out of all test tube media on the case of air loose condition, while succeeded in only 50% on the case of air tight condition. Moreover, piled up and big colonies were seen in air loose condition. Some of these colonies were able to passagedon new 1% Ogawa yolk media but not on 1% Ogawa whole egg media.
A 3 months old culture of M. lepraemurium passaged 11 generations was donated from Dr. Ogawa and then its bacterial suspension was inoculated to back subcutaneous tissue of ddO white mouse. After 5 months a small leproma was palpable in the local place injected more than 1.8×10⁴ bacilli. At the same time, the bacterial suspension was mixed with mouse peritoneal cells and enclosed into the millipore diffusion chamber, which was inoculated in abdominal cavity of mouse. After 5 months many globi were seen in peritoneal cells mixed with 0.9×10³ bacilli. When the bacterial suspension grown in the diffusion chamber was inoculated to abdominal subcutaneous tissue of ddO white mouse, 5 bacilli was able to form a palpable subcutaneous leproma after 5 months. The second passage of the leproma which was grown on the back of ddO mouse by an injection with the cultivated M. lepraemurium, was carried out in C3H mice. After 8 to 13 months, big leproma was formed in the injected site of all mice.
At the present, for definition as a M. lepraemurium the organism must form a leproma contained globi of acid fast bacilli to mouse, rat and hamster and must not form a leproma to guinea pig, rabbit and bird, and the organism does not grow on the culture media for M. tuberculosis. Therefore the cultivated M. lepraemurium was injected into rat, hamster, guinea pig, rabbit and quail. After 6 to 10 months thinly developed leproma was seen in the injected local of rat and hamster, but was not seen in guinea pig, rabbit and quail.
From these results, the acid fast bacillus isolated on 1% Ogawa yolk medium by Dr. Ogawa can identify as M. lepraemurium.

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Six strains of M. leprae taken from lepromatous leprosy patients were inoculated into the testes of "¹³¹I-F₁" mice, which were divided into two groups, but the first group were born from females which had been subcutaneously injected with 131I-100μc during pregnancy ; and the second group were born from females which had been injected before pregnancy.
The results were as follows ;
The "¹³¹I-F₁" mice, born from females injected "during" pregnancy with ¹³¹I-100μc, and then inoculated with leprous bacilli described above, showed the presence of the so-called "globi" in the testes. When samples of leprous bacilli (LL28, LL32, LL33) taken from patients who had not been receiving anti-leprous drug treatments were used in the above ¹³¹I-F₁ mice, then globi were found in this investigation (Relationship, I, in Table 8). When the leprous bacilli were secured from leproma removed from patients being treated, and then injected into mice born from females which had been injected with ¹³¹I-100μc either during or before their pregnancy, no globi were found in the mice (Relationship, II-b). Even if the bacilli (LL32, LL33, LL34) taken from non-treated patients were injected into those mice which were born from those females injected with ¹³¹I-100μc "before" pregnancy, no globi were found (Relationship, II-a)

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As reported previously, an attemp to produce the disease in mice with the supposed Hawaiian strain was repeated four times and each of the tests gave positive results. In the present study, with an aim to know a minimum infecting dose, similar animal experiment was performed by the use of much smaller doses of the bacilli.
The animals, ddN strain, were injected with three different doses of the 13th subculture (10^-4, 10^-5 and 10^-6mg of bacilli) either subcutaneously or intravenously, and killed at intervals between 7 to 11.5 months after inoculation (Table 1). The macroscopic apperance of the organs was noted ; smears of spleen, liver, luns, kidneys, superficial lymph nodes and local lesion were stained by Ziehl-Neelsen's method and rated for the numbers of acid-fast bacilli as well as for the numbers of globi. In some instances histopathologic examination was also performed. All of the specimens removed were sumbitted to cultural recovery.
The experiment showed that the injection of the bacillus in each dose, whether subcutaneously or intravenously administered, produced the disease, which was characterized by such positive findings as macroscopic and microscopic involvement, increase in acidfast bacilli, appearence of globi, and recovery on culture of the tissues (Tables 2, 3, 4, 5 and 6). The characteristics of the bacilli reisolated were almost the same as those of the bacilli injected originally.
It must be conceded that reproduction of the disease was successful even with the amallest dose (10^-6mg) of the bacilli. The infection was slowly progressive and the extent of disease was more pronounced in animals inoculated intravenously than in those subcutaneously. Finally a word must be said about the decontaminating agent. In this study 4% sodium hydroxide was used, for the first time, for treating some of the specimens. The result in dicates that this stronger decontamination method is also available for the isolation of murine leprosy bacilli from pathologic materials (Table 7).

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The growth of M. lepraemurium was observed in various modified media which were composed of components eliminated from the NC-5 medium. The results obtained by morphological observation and bacillary counting method, indicate that the same growth to that obtained in NC-5 medium could not be observed when any component in the NC-5 medium was eliminated. It was noted that α-ketoglutaric acid was able to be replaced by oxaloacetate.
From the results mentioned above, it could be concluded that a component essential for the multiplication of M. lepraemurium might be α-ketoglutaric acid, and other components in NC-5 medium would be growth stimulators. However, it might be thought that α-ketoglutaric acid should not complete the growth, but act at the starting step of the growth of bacilli.

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