On April 26, 1951, a leproma developed in the subcutaneous tissue of the rat inoculated with the Fukuoka-strain of Myc. lepraemurium was homogenized in sterile physiological saline to make a 20 per cent solution. This suspension was divided into four aliquots and each aliquot was added respectively to an equal volume of physiological saline, 10% inactivated bovine serum water, 4% glycerine water, and Kirchner's medium containing 10% bovine serum. One ml of each mixed material was frozen, and then was dried by a rotary pump at room temperature. After drying these ampules were sealed and stored in a refrigerator at 4°C. On April 26, 1961, the infectious activities of the bacilli in the ampules stored in the cold for ten years were determined by subcutaneous inoculation to mice. After about 5 months of inoculation, the mice were killed and the infectivities of the materials were determined by bacteriological examination. The results obtained indicated that infective activity of Myc. lepraemurium could be maintained in vitro for even more than ten years by means of lyophilization, and that one of the most important factors influencing the maintainance of the infectivity of the bacilli seemed to be the dryness of the material at lyophilization, because the saline solution and serum water could be quickly dried and could maintaine the infectivity of the bacilli.
Cross precipitin reactions of type sera of the Klebsiella group could take place with K and O Klebsiella antigens. The cross precipitin reaction due to O antigen could be eliminated by diluting type antigens, because of low antigen titers. When diluted type sera were used, many cross reactions due to O and K antigens were removed, although it was difficult to remove cross reactions from all type sera. However, all the type sera became type-specific by absorbing with cross-reacting K and O antigens, and could be used for the identification of Klebsiella types. Thus, type 12 antigen was found to consist of type 29 and 41 antigens, and was doubtful to be regarded as an independent type. Type 37 antigen was very similar to type 22 antigen.
The mouse super-immunized with live vaccine of Salmonella enteritidis resisted intravenous injection of 1, 000 MLD of the same organism (Japan. J. Exp. Med., 30, 375-384). This anti-lethal resistance of mouse against the infection with S. enteritidis lasted for 16 months after the last injection of live vaccine (Japan. J. Exp. Med., 31, 187-190). The mononuclear phagocytes obtained from abdominal cavity, liver or subcutaneous tissue of mouse super-immunized with live vaccine did not allow the intracellular multiplication of bacteria regardless of the presence of antibody in the cell culture medium, whereas the cells obtained from normal mouse or the mouse immunized with dead vaccine did not (J. Bacteriol., 81, 863-868; J. Bacteriol., in press). This paper deals with the appearance and duration of cellular immunity of mouse when immunized with live vaccine of S. enteritidis. The mononuclear phagocytes were obtained from the abdominal cavity of mouse immunized with live vaccine of S. enteritidis and were maintained in cell culture for in vitro infection. It was found that the cells obtained from the mouse 3 weeks after immunization resisted the degeneration caused by intracellular multiplication of bacteria. Even after 13 months of the last injection of live vaccine the cells did not allow the intracellular multiplication of bacteria regardless of the presence of antibody in the cell culture medium.
In a previous paper of this series we reported that Staphylococcus aureus was reacted to anti-anthrax precipitin serum. This report describes a method of fractionation of crude common antigenic substance between Bacillus anthracis and Staph. aureus 209 p, and the results of the absorption test for the relation between antigenic structure of these crude common antigen. Each fraction was obtained as follows; The heat dried cells were suspended in physiologic saline solution and extracted at 37°C for 3 hours. HC1-fraction was precipitated from the extract by the addition to pH 3.8 with N-HCl. After removal of the HC1-fraction, the CuSO4 fraction was precipitated from the supernatant by the addition of CuSO4 to make the 10 per cent. After removal of the CuSO4 fraction, the supernatant was treated with Sevag method and Sevag-fraction was precipitated from the gel part by the addition of 4-fold of methanol and reprecipitated with trichloroacetic acid. The resultant supernatant was dialysed with distilled water and lyophilysed. It was polysaccharide fraction. The results of the absorption test in these fraction, the common antigenic substance was present in HCl-fraction and Sevag fraction.
It is a well-known fact that Bacillus anthracis produces in the culture medium a variety of substances, such as polysaccharide, protective antigen and toxin, and of these the in vitro antigenicity of the polysaccharide has been studied extensively, but that of the other substances has been rather few and not clear. In this study, the culture fluid of B. anthracis after the growth of the organism was separated from the cells by centrifugation and fractionated into both the acid-precipitating fraction (A-P substance) and the supernatant fluid fraction by adjusting the pH to 3.8, then the antigenicity of and antigenic relationships among the culture fluid and the fractions were examined. Parallel experiments were also carried out by the use of a strain of B. megaterium, the extract of which precipitates with anti-B. anthracis serum in a higher titer. The culture fluid of B. anthracis strongly precipitated not only with anti-B. anthracis serum but also with anti-B. megaterium serum. The supernatant fluid of the same organism also gave strong reactions with both antisera. Both the culture fluid and the supernatant fiuid of B. megaterium also gave strong reactions not only with anti-B. megaterium serum but also with anti-B. anthracis serum. The A-P substance of B. anthracis, however, precipitated only with anti-B. anthracis serum in a lower titer and it did not precipitated with anti-B. megaterium serum. The A-P substance of B. megaterium precipitated neither with anti-B. megaterium serum nor with anti-B. anthracis serum. The culture fluids of both B. anthracis and B. megaterium were positive to the biuret and Molisch tests. The A-P substances of both organisms were positive to the biuret test and very weakly positive or almost negative to the Molisch test, while the supernatant fluids of these organisms were negative to the biuret test and positive to the Molisch test.
Evans et al. in 1954 demonstrated that the early protection produced in mice by the injection of pertussis vaccine against the challenge with Haemophilus pertussiswas different from the immune phenomenon as mediated by antibodies and reported it as interference phenomenon. In fact, they were the first to take notice of interference phenomenon between becteria. The present authors observed the similar phenomenon with Bacterium tularense. The results of our tests revealed that when mice were challenged with virulent strains of Bacterium tularense within a short period after the injection of avirulent or inactivated strains of the organisms, they were alive for a longer period as compared with the control animals, or some of them could survive the challenge. This phenomenon was exhibited in guinea-pigs which have almost the same susceptibility with mice and also in rabbits which are higher in natural resistance than guinea-pigs and mice. In the test made using other species of becteria and viruses as interfering agents, brucella gave the phenomenon to some extent but viruses did not so, indicating that this was a non-specific phenomenon. In the mice which experienced the interference phenomenon, even if they were succumbed to the challenge, the pathological changes were macroscopically slighter than the control and the challenge organisms were hardly detectable in the lesion. The mice surviving the challenge looked almost the same with the mice immunized.
The idea that the lymphatic system, like the blood vessel system, may be affected by bacillus infection is quite reasonable. Thus we started basic investigations in determining the relationship between the lymphatic system and bacillus infection. To begin with, basic experiments were conducted to compare the bactericidal power of lymph with that of blood. As the first step, lymph was collected from ductus thoracicus of a rabbit; and several strains of Staphylococcus, Salmonella and Shigella, were chosen to test bectericidal properties of lymph against such bacilli, in comparison with that of serum. In our experiments, lymph proved, contrary to our expectation, equal to and even more powerful than blood in bactericidal properties against certain strain. Next, in order to determine the bactericidal substances, lymph and serum were inactivated by heating at 56°C for 30 min. Also, in order to investigate whether or not these bactericidal substances were the same as properdin system, RP was made from serum and from lymph, according to the method of Pillmer, with zymosan applied, to determine bactericidal effects. At inactivation of 56°C, 30 min. and in RP, bacterididal effects of serum and lymph showed marked reduction against Gram-negative bacteria. It is, therefore, presumed that properdin system plays an important role as bactericidal properties in lymph as well as in serum. On the other hand, in absorption experiments with the heated bacteria, the bactericidal effects of lymph and serum showed marked reduction, especially against the same bacteria, suggesting a factor like natural antibody. On comparing agglutination values and bactericidal effects of lymph with those of serum, after immunization by each strains of S. typhi Watson V and S. choleraesuis No.2, the increase in agglutination values was observed almost same as serum, but bactericidal power was inhibited specifically against immunized becteria, similarly to Neisser-Wechsberg's counter phenomenon in serum.
The present paper deals with the property of immunity induced by primary infection in malignant mouse leprosy, In a preliminary experiment of susceptibility of mice to subcutaneous inoculation with murine leprosy bacilli, it was found that hybrid mice between C3H and ddD responded with typical malignant leproma at the site of inoculation. Therefore, these mice were infected subcutaneously with murine leprosy bacilli and then subjected to subcutaneous superinfection with INAH-resistant bacilli 6 weeks later. In almost all cases, some delay in leproma onset was observed at the superinfected site, but the once appeared nodules showed gradual increase in size and reached to a large malignant leproma. In the later period, therefore, there was no significant difference in the leproma development of challenge infection between the two groups with and without primary infection. These findings show that this immunity manifests itself only by delayed development of leproma due to superinfection at the early period. However, an interesting observation was that exceptional one case showed a benign type leproma in the superinfected site, whereas malignant leproma was developing at the site of primary infection. Similar tendencies were observed in the subcutaneously challenged mice which had been previously infected intraperitoneally. But, as in the experiment of benign mouse leprosy, this experiment was inadequate to investigate sufficiently the immunity elicited by primary infection, because the severe visceral lesions of primary infection caused the hosts die within the observation period.
Cultures of Lactobacillus bulgaricus often fail to grow on media commonly used for lactic acid bacteria such as standard plate count agar. This is considered dus to deficiencies of some growth factors required by the organisms. To determine the factors deficient in these media, a survay of growth requirements of six cultures of L. bulgaricus in chemically defined media was done. All of six cultures required “tween 80” and enzymatic digest of casein for their growth. “Tween 80” could be replaced with “tween 40” plus oleic acid, lecithin or cephalin. Three cultures required pantethine in a medium containing 0.4γ/ml of calcium pantothenate. However, pantethine could be replaced with higher amounts (e.g., 1γ/ml) or calcium pantothenate. When cysteine level in a medium was reduced to 10γ/ml (one-tenth of usual synthetic media) and cystine was omitted, calcium pantothenate in a concentration of 2γ/ml failed to replace pantotheine. This suggests that “activity coefficient” pantethine/pantothenate is a function of cysteine level in a medium. One culture identified asThermobacterium jugurtOLRA-JENSEN did not grow in the absence of lactose even after prolonged incubation period. None required erotic acid or thymidine, and the latter seemed to be inhibitory to some cultures.
To analyse the role of endotoxin in the pathogenesis of experimental typhoid in mice, (1) lethal toxicities to mice of heat-killed bacteria, (2) the amount of lipopolysaccharide (endotoxin), and (3) the influence of endotoxin on infection of mice were investigated usingSalmonella enteritidisstrains with different virulence. Heat-killed cells of a rough mutant 11Rx were somewhat less toxic than those of its wild type strain No.11 (high-virulent) and an S-type strain ‘Jena’(almost non-virulent). A galactose-sentive (epimeraseless) mutant of No.11, 11-1-M, and its galactose-resistant mutant, 11-1-TW, were found to have extremely low toxicities and almost no virulence for mice. The yield of lipopolysaccharide obtained by Westphal's procedure following ultracentrifugation was poor in the strains of low toxicity, but no remarkable difference in the lethal effect for mice was found among endotoxins extracted from various strains. Influence of the endotoxin on infection with those strains was studied by observation of mortality and quantitative cultivation of the blood, peritoneal fluid, spleen and liver at various intervals after challenge and the simultaneous administration of endotoxin. Thein vivomultiplication of strains ‘Jena’ and 11-1-M was not enhanced by endotoxin, but strain 11-1-TW could markedly multiply in the peritoneal fluid and spleen when inoculated with endotoxin. Furthermore, strain 11 Rx, which was low-virulent compared with strain No.11, showed as high as the virulence that of strain No.11 when given with endotoxin.
It was reported by Gott; and her coworkers (1959), that Vibrio El Tor strain Denken grow in a semisynthetic media consisting of Casamino acids, salts, glucose, vitamins and purine. The author confirmed that Casamino acids in this composition could be substituted with 18 pure amino acids mixture, and tried to simplify the amino acids composition by the elimination test. This strain could grow in a medium containing 8 amino acids, Methionine, Asparatic acid, Leucine, Phenylalanine, Isoleucine, Lysine, Arginine and Glutamic acid. Stain Denken showed moderate growth in this medium, a little less than in 1% Casamino acids medium or 18 amino acids mixture, and in this media 5 serial transfer was possible. Hemolysine production in this medium was approximately on the same level as in 1% Casamino acids medium.
In the present paper it was attempted to make the comparative studies on the virulence of atypical acid-fast bacilli isolated in U. S. A. and Japan and various types of mycobacteria. At the different periods after the intravenous infection of these strains, three mice of each group were sacrificed. The fate of viable bacilli in organs was determined by culture and the pathological changes were histologically examined. The results obtained were summarized as follows.(1) Photochromogenic strains isolated in U. S. A. had high virulence for mice, showing almost the same grade of bacterial multiplication and pathological changes in organs of mice as those infected with human type (H2), bovine type (Ravenel) and avian type (Kirchberg) tubercle bacilli. (2) Mice infected with nonphotochromogenic and scotochromogenic strains and rapid growers showed a steady decrease in microbial population and slight pathological changes in organs. Therefore, the virulence of these strains was considered to be very low for mice. (3) A nonphotochromogenic strain, which had special affinity to the kidney of mice. This strain was isolated freshly from sputum of a patient. (4) The principal pathological changes of mice infected with atypical acid-fast bacilli was the granulom, consisting mainly of the proliferation of epithelioid cells and quite resembling to those of tuberculous lesions produced by tubercle bacilli.