The relationship between M. leprae and peripheral nerves has been studied since the beginning of the scientific research of leprosy by many workers, especially from histo-pathological point of view.
In this paper we present the data of our experimental studies concerning the affinity of M. leprae to peripheral nerves. We have studied this problem by the following experiments and observations, i. e. "Intraneural inoculation of acid-fast bacilli, " "Tissue culture of leprous peripheral nerves" and "Biomicroscopy of leprous corneal nerves" etc.
1) Intraneural inoculation of acid-fast bacilli.
We inoculated M. leprae, M. leprae murium, Tubercle bacillus (human, bovine and avian types), B. C. G., M. ulcerans, Smegma bacillus in sciatic nerves of rabbits, guinea-pigs, rats and monkey, and examined histo-pathologically the affinity of these acid-fast bacilli to the peripheral nerves of these mammalians.
We have found that acid-fast bacilli multiply in peripheral nerves of susceptible animals, especially in the endoneurial cells. For instance, M. leprae murium causes intraneural leprom in the sciatic nerves of rats, when inoculated directly in these nerves. Tubercle bacillus multiplies in the sciatic nerves of susceptible animals, and makes tuberculous lesions in these nerves. M. leprae does not multiply in. the peripheral nerves of monkey, rat, guinea-pig and rabbit. The most strong lipoid degeneration was observed in the lesions of intraneural inoculation of M. leprae into rabbits. Strong calcification occurs very often, when smegma bacillus is inoculated into the sciatic nerves of rabbits.
2) Tissue culture of the leprous peripheral nerves.
We have cultured 14 biopsy specimen of leprous peripheral nerves in vitro, and examined the possibility of multiplication of M. leprae in the growing cells.
Endoneural fibroblasts are most frequently seen in the tissue culture of lepromatous nerves, on the contrary tuberculoid nerves show scanty cell growth in vitro. Sometimes small number of M. leprae was found in the growing cells, but we could not conclude from such finding the multiplication of M. leprae in vitro. The growth of Schwann cells as observed frequently in the tissue culture of normal peripheral nerves was rarely encountered in the culture of leprous nerves. This fact is perhaps related to the deficiency of nerve regeneration in leprosy cases.
3) Biomicroscopy of leprous corneal nerves.
We have examined biomicroscopically the corneal nerves of leprosy patients and confirmed the "beaded corneal effect" in both lepromatous and tuberculoid cases. This finding indicated the affinity of M. leprae to the peripheral nerves of both polar types of leprosy.
We have also examined the corneal nerves of rabbits by polarizing microscope and found that there is a thin bi-refringent layer over the axon even in the so-called amyelinated portion of corneal nerves.
4) Histo-chemical study of leprous peripheral nerves.
In the tuberculoid nerves both acid and alkaline-phosphatase were found histochemically, and in case of lepromatous nerve lesions we have found acid phosphatase, but no alkaline phosphatase was confirmed. We think alkaline phosphatase in tuberculoid nerve lesions might play a role in the calcification of tuberculoid nerve lesions.
5) Study of the nerve regeneration in leprous nerves.
We examined the nerve stump of re-amputated legs of leprosy cases with polarizing microscope, and found that this microscope is very convenient to trace the remaining myelin sheath in the highly degenerated nerves.
6) Summary and conclusion.
M. leprae has an affinity to the axon in both lepromatous and tuberculoid leprosy, and after reaching peripheral nerves, it causes different lesions according to the tissue resistance of the individual toward this microbe.

View full abstract

Inoculating small amounts of the emulsions of leprous tissues into certain nutrient media, some workers in our country recently said that the leprosy bacilli, though in small amount, seemed to be able to develop in vitro. This may perhaps be the first step towards the solution of the problem, even though these workers did not, however, explain why the succesive cultures were not successful in any way.
Is it possible to verify the growth of leprosy bacilli in vitro, without using the method of transferring succesively in ordinary way? It may be verified, if one recognize 1) more or less considerable increase in number of bacilli, though within microscopic range, in comparison with the control tubes not incubated, 2) characteristic arrangement of the multiplied bacilli; formation of the cords; construction of the colonies, though within microscopic width, and 3) process of development; growth phase.
After being examined, from this point of view, many cultures in fluid and solid media, it became clear that the leprosy bacilli were able to develop in vitro in somewhat characteristic manner. The observation was made with-the cultures in Kirchner's fluid and solid media. The smears made, without stirring, with a drop of the sediments were examined weekly. One recognized that the leprosy bacilli developed with almost similar rapidity as the human or bovine tubercle bacillus showing their maximum growth after 3 to 5 weeks of incubation. They constructed fine acid-fast net-works visible with weak magnification covering many debris found in the smears (Fig. 1). And also in the smears made from the cultures on the slants, the bacilli often appeared forming peculiar round colonies though within microscopic width (Fig. 2).
It was above all striking that the bacilli could multiply only in small amounts; i. e. withh the limited growth ratio.
If one reminds the growth curves of other mycobacteria, it may be possible to suppose by analogy, that the number of viable leprosy bacilli in the cultures also begins to decrease as soon as they show the maximum development after 3 to 5 weeks of incubation. Thus if one try to transfer the cultures, it may be preferable to do at these periods of time. Actually the transfers were, however, not successful, although careful attention was taken to give more or less dense sediments of the primary cultures into new culture media. This might be due to the fact that the leprosy bacilli grow only with the minimum growth ratio as already written.
Some factors being supposed to influence the development of the bacilli were also able to be examined with success by means of the method described above. One inoculated a drop of more or less dense emulsion of tissues into Kirchner's fluid medium and examined, with weak magnification, the smears made with a drop of sediments of the cultures. For example, the bacilli developed better at 37°C than at 30°C, and also better in the medium containing glycerol of 2 to 4% than without it. As to the serum added to the medium, those of rabbit and rat promoted the development better than those of goats, men and guinea pigs.

View full abstract

Reported in the following are the findings of the joint studies of lepromin test carried out on a total of 3, 134 in-patients of the national leprosaria and on the 238 school children in non-leprous areas.
1. Comparison of Mitsuda's antigen and Dharmendra's antigen
Mitsuda's antigen is a preparation obtained by grinding and emulsifying the leproma with physiological saline to have a certain difinite concentration and it contains both leprosy bacilli and tissues. On the other hand, Dharmendra's antigen is prepared from the leproma in a certain special manner as would contain mostly leprosy bacilli.
Investigations were made on the active principles of those 2 antigens which participate in the lepromin test. For this purpose, Mitsuda's antigen was injected intracutaneously into the flexor surface of an forearm and Dharmendra's antigen into the other arm exactly in the same manner each in a dose of 0.1ml on 3, 137 leprosy patients. The sizes of erythema read after 48 hours (early reaction) and 15 days (late reaction) were compared for knowing the correlation between those antigens.
The findings were as follows:
a. Correlation of the 48 hous reading between Mitsuda's antigen and Dharmendra's antigen. Correlation coefficient: r=0.8466 n=3, 137
b. Correlation of the 15th day reading between Mitsuda's antigen and Dharmendra's antigen Correlation coefficient: r=0.8652 n=3, 137
c. Correlation between the reading of Mitsuda 15th day and Dharmendra 48 hours Correlation coefficient: r=0.7396 n=3, 137
d. Correlation between Mitsuda 48 hours and Mitsuda 15th day Correlation coefficient: r=0.7674 n=3, 137
e. Correlation between the reading of Dharmendra 48 hours and Dharmendra 15 th day Correlation coefficient: r=0.8056 n=3, 137
Thus, it appears that the same active principle participates in either case of the early reaction or the late reaction.
The findings of the comparisons based on the sizes of induration were of the same tendency, but as the measurement of erythema yielded better comparison than that of induration, in which errors involved were considerable, the intensity of the skin reactions was expressed in the term of so much mm in the diameter of the erythema rather than that of the induration.
2. Criteria for the reading of the reactions of lepromin test
Randomly selected 3, 137 leprosy patients were found to consist of 2, 321 L type patients and 816 of NM type and this ratio of L type and NM type is considered to represent the ratio of those types of leprosy patients in Japan. (Note : L type is the lepromatous type. N, M type means the N type including tuberculoid which was classified in Cairo Congress.) The criteria for the reading of the reactions of lepromin test was established by the analysis of frequency distribution curves of the sizes of erythema regardless to the type of the disease.
The frequency distribution curve of the erythema of 48 hours reading presented a bimodal curve which is the composition of 2 normal distribution curves. The first elevation of the curve was considered to be non-specific while the second elevation specific.
Through the statistical manipulation of those 2 curves, the following criteria have been attained for the reading of the 48 hours reactions of lepromin test.
a. Mitsuda's antigen
i. With the risk of 2% errors: The arithmetical mean of the two diameters of the size of erythema, when below 6mm, the reaction is negative; 7 to 10mm, doubtful positive; 11mm and over, positive.
ii. With the risk of 4% errors: erythema 9mm and below, negative; 10mm and over, positive, and in this case no reaction is taken as doubtful.
b. Dharmendra's antigen
i. With the risk of 1% errors: erythema 9mm and below, negative; 10 to 12mm, doubtful posstive ; 13mm and over, positive.
ii. With the risk of 2% errors, the erythema below 11mm, negative; 12mm and over, positive, and in this case no reaction is taken as doubtful.

View full abstract