Factors involved in the inoculum of M. lepraemurium for the growth in NC-5 medium as well as in mice were studied and the results obtained are as follows: 1. Significant multiplications of M. lepraemurium obtained from infected subcutaneous tissue, liver, and spleen in NC-5 medium were observed. Therefore, it is obvious that the growth of bacilli in NC-5 medium is independent from the sourses of the materials used. The multiplication ability of the bacilli in NC-5 medium is kept for 2 months at-20°C (in a freezer). 2. No effects of treatments with 0.1% trypsin, 0.2% pronase, and 0.1% desoxycholate at 37° C for 60min on the potentiality of the growth in NC-5 medium were recognized. The treatment with petroleum ether somewhat destroyed the ability. 3. The growth rate of purified bacilli was superior than that of crude material. 4. The potentialities of the growth of M. lepraemurium in NC-5 medium as much as in mice were completely destroyed by the treatment with below pH 6 at 37°C for 60 min and by heating at 50°C for 30min. On the other hand, a complete destruction of the growth potentiality of bacilli in NC-5 medium was resulted by UV irradiation for 2.5min, whereas the leproma producing ability in mice was maintained even by irradiation for 60min.
In order to improve the NC-5 medium, the Dubos medium (pH 7.3) was used as a basal medium, instead of Kirchner medium. The complete medium thus prepared is referred to as ND-5 medium. In this medium, Mycobacterium lepraemurium quickly multiplies in the form of binary fission without an extraordinary elongation. Possible generation times could be calculated by repeated experiments as 1.4-2.6 days. A slightly degenerative changes in the cells during prolonged cultivation was observed by electron microscopy. This medium has some advantages for inhibiting other bacterial contaminations. Serial subcultivation is not tested yet.
A strain of acid fast bacillus was isolated from a leproma of armadillo infected with M. leprae during the cultivation trial. Colonies were easily formed on Ogawa egg medium one-two weeks after inoculation, and were yellow. This isolated mycobacterium was identified as a type of Scotochromogen, which is belonged to Group II atypical mycobacterium, by biological and biochemical characterizations.
The technical procedures of experiment were explained briefly in Table 1. Bacterial suspension prepared from lepromas was injected into mice once or several times at weekly intervals by subcutaneous or intravenous route, or by both routes. Animals. were killed at various intervals 2-16 months after injection. At necropsy, lesions were sought by gross inspection. Portions of various organs were removed and ground in mortar to make the homogenates. Smears made from the homogenate were stained by Ziehl-Neelsen's method and examined microscopically. The homogenate treated with one percent sodium hydroxide solution and then inoculated onto the egg yolk medium (for M. lepraemurium; also for M. leprae (?)) and Ogawa 1% egg medium (for cultivable mycobacreria). The tubes were incubated at 37°C for over 3 months. The details of single inoculation experiments and multiple inoculation experiments were shown in Table 2 and 3. Ten experiments containing four with single inoculation and the other six with multiple inoculation were carried out. But one experiment, Expt. (4), exhibited a probable contamination and its results will be described in the following paper separately. In nine experiments, gross findings were all negative. Cultivation trials showed a few, smooth, and buff colonies, supposedly atypical mycobacseria, from two specimens only, but no colonies of mycobacteria, especially suspected of M. leprae, have been isolated. On the other hand, microscopic examination revealed the presence of acid-fast bacilli in the tissues of various organs. Among the two experiments, Expts. (3)-1 & -2, showed remaykable microscopscal findings were summasized in Table 6. As shown, in the subcutaneous experiment Expt. (3)-1, acid-fast bacilli found in the injection site and superficial lymph nodes, and none of the tissue of viscera. In the intravenous experiment, Expt. (3)-2, acid-fast bacilli were detected in the spleen, liver and lungs, but smaller in number, comparing with those of the injection site just mentioned. No bacilli were found in the superficial lymph nodes. In both of the experiments the acidfast bacilli had a tendency to decrease in number steadily. In where numbers of bacilli were present, globi were often seen, but these were usually small in size and loose in arrangement. And, indeed, it was uncertain whether the bacilli had multiplied within the tissue or not. The microscopic findings obtained in all the experiments were summarized in Table 7. As the materials of leproma used differed from experiment to experiment, it was impossible to compare directly the values of percentage for smear-positive specimens. As a whole, however, it seemed fairly justified in concluding that the microscopic findings were superior in the multiple inoculation than in the single inoculation. This fact was accorded with the observations reported by previous workers. ACKNOWLEDGMENT This work was supported in part by grants from U. S.-Japan Cooperative Medical Science Program.
Thirty three cases with so-called phobic reaction to Hansen's disease were studied.They were divided into three groups by the following points ; what, how and way to be complained of, etc. (1) The cases of group I (12 cases) showed a transitory anxiety to become ill, but they well understand the results of medical examinations. In these cases may the items concerned with Hansen's disease in their life history be found. The appearance of anxiety in their mind may be related with three factors, such as patient's character, life history and something like inducement. They used to consult us only once or twice. (2) In the cases of group II (16 cases), their anxieties were stronger and more lasting than those in group I. Their chief complaints are within the main complaints of the patients with Hansen's disease although those are often changed during treatment. They may be often diagnosed and be treated as neurosis (true phobic reaction to Hansen's disease) or masked depression. (3) There is one group of cases (5 cases) who complain of peculiar and strange complaints such as delusion of ozochrotia, acoasma, dysmorphophobia etc., including usual appeals. One of these cases is under treatment in other university hospital as a schizophrenia. Their phobic state differs from that of true phobic reaction in the point of the severity of captive conditions ; the former is very mild in nosophobic point. These cases are in group III. (4) Many cases in group II and all cases in group III must consult psychiatrists and must be treated with help of them although all cases in group I and some cases in group II may be seen by us with medical examinations or supportive psychotherapy. (5) The thema of this phobic reaction, Hansen's disease was discussed especially on the socio-familiar problems in Japan.