[Where do we stand with BCG vaccination programme in Japan?].

The current BCG vaccination program of Japan is critically discussed based on recent knowledge, especially with regard to its epidemiological aspects, in order to put the problem into perspective for Japan's future tuberculosis control program. 1. EFFICACY AND OVERALL EFFECTIVENESS: Various indicators of BCG efficacy have been proposed, and the meticulous analysis on the variability and the quality of these indicators seems to have formed a consensus on the efficacy, as seen in the recent meta-analysis studies. However, much has been left unanswered concerning the conditions under which the efficacy is guaranteed. The impact of the vaccination program on the population should also be considered in order to make decisions on the program. Comparing the age-specific tuberculosis notification rate between Japan and the USA, where there has been no BCG vaccination program, shows that the rate for 0 to 4 year olds is clearly lower in Japan than in the USA, while it is more than five times higher for all ages in Japan than in the USA. The statistics for Japanese children are superior to those of US children with respect to the speed of decline in notification rate as well. These observations support the overall effectiveness of BCG vaccination in Japan. 2. MECHANISMS OF BCG VACCINATION EFFICACY AND ITS DURATION: Two possible mechanisms of how BCG works to prevent tuberculosis were proposed. Epidemiological models based on each mechanism were subsequently tested by simulating 20 years' development of cases in the BCG vaccination trial by BMRC. In mechanism 1, the BCG-induced immunity is assumed to boost TB immunity in inhibiting the clinical breakdown of tuberculosis during the 10 to 15 years after the vaccination. In mechanism 2, the immunity makes the infection process abort (presumably, at 90%, during the seven years after infection, for example), leading to a smaller risk of future clinical development. So far, most epidemiological models implicitly assume mechanism 1 above. In animal experimental models, however, it has been difficult to simulate the mechanisms differentially, which has been one of the drawbacks to this argument. 3. EFFECTIVENESS OF REVACCINATION: Revaccination with BCG vaccine aims to restore or to endow immunological resistance through repeating vaccination to those who have partially or totally lost the immunity acquired from the primary vaccination. Although some animal experiments support the efficacy of revaccination with BCG, studies in humans have been rare and the results are variable. The observation of Polish infants and schoolchildren is suggestive of the efficacy, but it is not randomized and of questionable value. The recent study of Malawi is a randomized trial. It demonstrated that BCG revaccination protects against leprosy, but does not protect significantly against tuberculosis. It is possible, however, that it does protect against tuberculous lymphadenitis. The two above-mentioned possible mechanisms of BCG immunity were applied to a model analysis of BCG revaccination. It was known that revaccination effectiveness is very limited under any assumption, given the current Japanese epidemiological situation of tuberculosis, so that the demerits due to revaccination, such as strong local reactions, must not be neglected but should be carefully considered. At the same time, we should remember that this model analysis assumes that the primary vaccination is given to new borns with advanced and uniform techniques, which is not always true, and revaccination may supplement the technical failure of the primary vaccination. 4. DECIDING ON THE TOTAL DISCONTINUATION OF BCG VACCINATION PROGRAMME, JAPAN: The recommendations of WHO or IUATLD on the discontinuation of the BCG vaccination program are just conventional ones and the theoretical reasonings is difficult to accept. After all, the decision making should depend on the lay decision makers' subjective judgment balancing benefit and loss in terms of costs and health incurred by the policy, as shown by Waaler and Rouillon. The current Japanese BCG vaccination program is very expensive, but brings about some, though very small, benefit. This balance was compared with that of Sweden around 1975, when the program was discontinued. The comparison clearly showed that the cost-effectiveness of the program in Japan today in superior to that of Sweden in 1975.

[Specific odor component produced by Mycobacterium lepraemurium on Ogawa yolk medium].

When Mycobacterium lepraemurium is grown on the 1% Ogawa yolk medium, it produces a specific odor. This odor was not observed in other easily cultivable acid-fast bacilli. Therefore, identification of the components responsible for the specific odor produced by M. lepraemurium was attempted. The odor components were extracted for overnight with sterilized and distilled water from the Ogawa yolk medium on which M. lepraemurium had been cultivated for two months. The odor components in the extract was adsorbed on refined charcoal. After washing with distilled water for three times, the charcoal was dried. Then the odor components were eluted from the charcoal with ethanol and the eluate was condensed under nitrogen gas flow at 40 degrees C. The condensate was analyzed by Gas-Chromatography-Mass-Spectrum (GC-MS). Phenylethanol and phenylacetic acid were identified as major odor components. A mixture of authentic phenylacetic acid, its methyl and ethyl esters, smelled similar to the odor of cultivated medium of M. lepraemurium. Thus, phenylacetic acid was identified as the key odor component produced by M. lepraemurium. When initial isolation culture of M. lepraemurium from murine leproma was cultivated on the Ogawa yolk medium by adding phenylacetic acid, growth inhibition was brought by the compound.

Genetic control of delayed type hypersensitivity to Mycobacterium leprae antigen.

The mechanism of genetic control of Mycobacterium leprae-specific delayed type hypersensitivity (DTH) in mice was investigated in terms of footpad swelling response. Studies with various congenic and recombinant inbred strains of mouse revealed that M. leprae-specific DTH was controlled by genes within the I-A subregion. The footpad swelling response showed an antigen-specific pattern, revealing that M. leprae antigen was not cross-reactive with M. tuberculosis antigen. Treatment of immune lymphoid cells with monoclonal antibodies plus complement revealed that the cells responsible for DTH transfer were Lyt-2-, L3T4+ T cells. Suppressor T cell activity was not detected in low-responder mice.

[Acid-fast bacilli isolated from foot pads of nude mice infected with leprosy bacilli].

When leprosy bacilli grown in nude mouse foot pad were used for culture experiments, cultivable acid-fast bacillus was sometimes isolated as a contaminant. Whenever bacilli were inoculated to nude mice, the same leprosy bacilli were killed by autoclaving and were inoculated in to foot pads of 5 nude mice for examination of this cause of the contamination. Acid-fast bacillus was cultivated on 3% Ogawa egg medium at 33 degrees C from homogenates of foot pads of nude mice infected with M. leprae after one year and a while of infection. Foot pad of nude mouse injected with leprosy bacilli was cut off, ground in mortar and passed through sterile absorbent cotton and the filtrate was centrifuged at 10,000 rpm for 30 minutes. The sediment was inoculated on 3% Ogawa egg medium after treating with a small amount of sterile 1 N sodium hydroxide. Acid-fast bacilli were isolated from 3 out of 41 mice inoculoted with heat killed bacilli. The isolated acid-fast bacillus did not be observed in the same experimental group inocudated with live bacilli, positive cases were scattered in another groups. Four out of 16 tubes were positive for acid-fast bacilli in mice infected with Kurume-naha and 5 out of 7 tubes in the Amami-KM infected mouse group. The two negative tubes were discarded due to contamination. Kurume-Oki strain which has yellow colonial morphology was isolated from one out of 6 culture tubes. Strains Kurume-naha and Amami-KM have the same characteristics as follows: slow grower with pale yellow smooth colonial morphology, strongly positive for niacin production and ureas; positive for nicotinamidase, pyradinamidase and 68 degrees C catalase; no growth at 45 degrees C, negative for nitrate reduction, hydrolysis of Tween 80, diamine oxidase, heat stable acid-phosphatase and arylsulphatase; resistant to streptomycin, isoniazid, rifampicin and B 663. Two isolates were identified as Mycobacterium simiae from these characteristics. Characteristics of a Kurume-Oki isolate was as follows: slow grower with yellow smooth colonial morphology, positive for urease, 68 degrees C catalase, hydrolysis of Tween 80 and arylsulfatase; no growth at 45 degrees C, negative for niacin production, nicotinamidase, pyradinamidase, nitrate reduction, daimine oxidase and heat stable acid-phosphatase; resistant to streptomycin, isoniazid, rifampicin and B. 663. This bacillus was identified as Mycobacterium gordonae from these characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid-fast bacilli detected in umbilical codes and skins of human at cases of surgical operation.

Acid-fast bacilli were detected in 13 (27%) of 49 skin samples in surgical operation under the procedures of collection of bacilli by centrifuging the filtrate of tissue homogenate through adsorbent cotton. Ten specimens (20%) contained cultivable organisms, including M. simiae (9 specimens) and M. gordonae (one specimen). The other 3 specimens did not contain any cultivable organism, although microscopic observation revealed the presence of acid-fast bacilli. Eight (17%) of 48 raw umbilical codes of babies received Cesarian operation were positive for acid-fast bacilli in the smear preparation. Six (13%) were positive in cultivation and the organisms were identified as M. simiae (4 cases), M. scrofulaceum (1 case) and M. avium-complex (1 case). The remaining two specimens were negative in cultivable bacteria in spite of obvious presence of acid-fast bacilli. In the case of frozen umbilical codes, 9 specimens (16%) were positive in acid-fast bacilli, only 3 cases of which were positive in cultivable organisms, including M. gordonae (2 cases) and M. scrofulaceum (one case). M. simiae was not detected in cultivation of frozen materials. The purpose of this experiment was to isolate the microscopically detectable but uncultivable acid-fast bacilli, using experimental infection system induced in nude mouse. However, two experiments separately performed failed to achieve this purpose, because of contamination of the cultivable acid-fast bacilli among mice or death of the organisms during storage.

Identification of cat leprosy bacillus grown in mice.

Cat leprosy bacilli passaged in mice could be isolated on 1% Ogawa yolk medium. The isolated cat leprosy bacilli which were cultivated successively four times on 1% Ogawa yolk medium produced a leproma in mice. All characteristics of the isolated cat leprosy bacillus were the same as isolated murine leprosy bacillus, as follows: slow grower, light yellowish-white rough colony, production of much coproporphyrin on the medium, heat-resistant catalase negative, heat-resistant phosphatase negative, arylsulfatase negative, niacin negative, hydrolysis of Tween 80 negative, urease negative, nicotinamidase positive, pyrazinamidase positive, cytochrome b1 at 560 nm positive, cytochrome a2 at 630 nm positive, and cytochrome c at 550 nm negative. Cats are susceptible to both cat and murine leprosy bacilli; the bacilli produced a leproma in a newborn cat at 3 to 4 months and in an adult cat at 2 months after inoculation. Many globi of acid-fast bacilli (AFB) were observed in the histopathological sections and the smear preparations of the newborn cat's lepromas, especially in the necrotic areas of the lepromas. Many AFB and polymorphonuclear leukocytes were seen in the histopathological sections and the smear preparations of the adult cat's lepromas. These lepromas formed ulcers by autolysis and healed or absorbed without ulcer formation over the course of months. Large lepromas remained for a long time without ulcer formation and caseation in some cats. Secondary infections with cat and murine leprosy bacilli were done respectively to the right and left femoral subcutaneous regions of newborn cats carrying primary lepromas. After one month, granulomas in which many AFB were observed were produced in both infection sites. Cats are susceptible to infection with cat and murine leprosy bacilli; however, the bacilli did not invade progressively to internal organs or other subcutaneous areas. Cat leprosy bacilli which were passaged in the mouse are identical to murine leprosy bacilli.

Pyrolysis gas chromatography-mass spectrometry of mycobacterial mycolic acid methyl esters and its application to the identification of Mycobacterium leprae.

Pyrolysis gas chromatography-mass spectrometry of methyl mycolates from 32 species of mycobacteria, including Mycobacterium leprae, was carried out. The mycobacteria could be classified into four groups in respect of the fatty acid ester patterns detected within the range C20 to C26. The applicability of this pyrolysis-gas chromatographic method for identifying M. leprae is discussed.

Restored pathogenicity of attenuated Mycobacterium lepraemurium in mice.

The ability of Mycobacterium lepraemurium (Mlm) to adhere to A31 cells in culture decreased with the number of passages of the bacilli on Ogawa egg-yolk medium. Pathogenic Mlm consistently grew in tissue culture cells but growth was not seen with attenuated Mlm isolated from a smooth colony. After prolonged incubation, attenuated Mlm became adapted to tissue culture growth. The pathogenicity of the attenuated bacilli was restored partially by the adaptation to tissue culture cells and restored almost completely by passage in mice. After restoration of pathogenicity by these methods, the Mlm formed rough-type colonies on Ogawa egg-yolk medium although the colonies were not completely of the rough type. Attenuated Mlm did not interfere with the growth of in vivo-derived Mlm in tissue culture or in mice.

Prophylactic/therapeutic effects and induction of reversal reactions with thymus transplantation in Mycobacterium leprae-infected nude mice.

The study results indicated that thymus transplantation was effective in suppressing the growth of Mycobacterium leprae in the nude mouse, and also suggested that thymus transplantation was effective as immunotherapy of experimental leprosy in nude mice. The histopathological findings revealed the induction of reversal reactions in those animals receiving thymus transplants.

Respiration in Mycobacterium leprae.

Fairly pure leprosy bacilli were easily collected from nude mouse foot pad lepromas by the Ficoll density gradient centrifugation and alkali treatment methods. The yield of bacilli available for biochemical study was 42.6%. The density of Mycobacterium leprae was very heterogeneous. The percent of solid bacilli in the light bacilli fraction was 23%; that in the heavy bacilli fraction was 40%. The endogenous respiration activity in the heavy bacilli was greater than that in light bacilli. The average coefficient of respiration in M. leprae was 1 microliter O2/mg X hr. In the whole cells of M. leprae, a cytochrome b1 absorption peak and its Soret peak were detected at wavelengths of 560 nm and 426 nm, respectively. However, a cytochrome a2-like peak (which was observed in M. lepraemurium), and a cyt c and cyt a were not detected. Catalase activity was not found in whole cells, the cell-free extract, or particle fractions of M. leprae. Any catalase activity associated with M. leprae suspensions is a tissue contaminant. NAD-peroxidase activity was also not detected in the cell-free extract of the leprosy bacillus. These results would indicate that leprosy bacilli cannot degrade hydrogen peroxide.

Collection method for Mycobacterium leprae from infected armadillo liver.

Leprosy bacilli were separated from infected armadillo liver almost free from tissue contaminants by a Percoll gradient centrifugation. The yield of bacilli was 46.7%. This is a very simple and effective method without enzyme treatment.

Failure to validate the growth of Mycobacterium leprae on M-Y 14b agar medium.

Confirmation experiments on colony formation of M. leprae on the M-Y 14b agar medium developed by Murohashi and Yoshida were carried out for two years independently by individual members of an organized research group, according to the method described by Murohashi and Yoshida. The results obtained can be summarized as follows: a) No colony production by M. leprae on M-Y 14b agar medium was seen. b) No increase in the number of cells of M. leprae on M-Y 14b agar medium during cultivation was seen. c) Light and electron microscopic observation indicated that there was an increase in the number of non-solid bacterial cells and ghost cells with time of cultivation. d) It was found by mouse foot pad inoculation that four of six samples of M. leprae used as inocula were definitely viable. e) By means of mouse foot pad inoculation, it was shown that viability of M. leprae inoculated onto M-Y 14b agar medium was lost within approximately seven weeks of cultivation. From these results, can be definitely concluded that there is no evidence indicating that multiplication of M. leprae took place on M-Y 14b agar medium.

Growth and drug sensitivity of M. lepraemurium by tissue culture applying monolayer and agar suspension technique.

M. lepraemurium grow well in a Balb/c 3T3 recloned cell line (A31). In monolayer culture, the average generation time of M. lepraemurium in A31 cells was 5.3 to 9.4 days at 37 degrees C. A31 cells are very sensitive to infection with M. lepraemurium. Bacterial increases were readily apparent 30 days after inoculating 2 X 10(5) A31 cells in monolayer culture with only six bacilli. The intracellular bacilli were well transferred without apparent losses by host cell transfer. The growth of intracellular bacilli was inhibited by streptomycin 100 micrograms/ml, clindamycin 25 micrograms/ml, INH 5 micrograms/ml, and rifampin 5 micrograms/ml. When streptomycin or clindamycin was removed from the culture medium after 41 days of treatment and the cultivation continued in drug-free medium, the intracellular bacilli began to multiply once more without a lag period. When the intracellular bacilli were treated with INH for 35 days or rifampin for ten days, growth resumed, but only after lag periods after removal of these drugs. We utilized agar suspension techniques for the cultivation of host cells M. lepraemurium because normal cells or transformed cells ceased undergoing cell division and remained healthy for long periods of time in agar medium. M. lepraemurium grew well in A31, A31 transformed by polyoma virus, nude mouse foot pad, chick embryo, and human neuroblastoma cells, utilizing the agar suspension technique. The agar suspension cell culture method should provide useful clues for the cultivation of M. leprae.