Seven hundred fifty-four strains of mycobacteria were examined using 97 characters, and "Hypothetical Mean Organism" (HMO) was prepared for each species using numerical classification. The species could be defined as a group of strains showing a mean S-value of 90% or more to a HMO and showing mean S-values of 89% or less to other HMOs. The following species were recognized: (1) M. tuberculosis, combining M. tuberculosis and M. bovis into one species; (2) M. kansasii; (3) M. novum; (4) M. avium, combining M. avium, M. nonchromogenicum, M. gastri, M. intracellulare and M. scrofulaceum into one species; (5) M. marinum; (6) M. thermoresistibile; (7) M. chitae; (8) M. borstelense; (9) M. abscessus; (10) M. fortuitum; (11) M. phlei; (12) M. aurum; (13) M. parafortuitum; (14) M. lacticola; (15) M. smegmatis. Dendrogram of the species showed two main stems, indicating that the genus Mycobacterium be divided into two subgenera, subgenus Mycobacterium (from M. tuberculosis to M. chitae) and subgenus Mycomycobacterium (from M. borstelense to M. smegmatis). Some discrepancy was noted between the results of numerical classification using HMOs and that of the "proper" numerical classification, and this discrepancy is discussed.

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Interference phenomenon which reduces the number of transformed foci in the culture of chick embryo cell (CEC) exposed to Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) is described. The interfering substance(s) responsible for this phenomenon were detected and partially fractionated, and were also extracted from virusinfected chorioallantoic membranes (CAM). The amount of inhibitor(s) in the virus preparations was found to determine the number of cells registering as infective centers.Further, the inhibitor(s) we considered to consist mainly of inactivated virus particles and their degraded substances, suggesting that they are secreted from virus-infected cells into media during long culture.

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The growth activities of CoA², its precursors² (PA, P-PA, PAC, P-PAC, PAT, P-PAT), and CE² were determined for the strains of Bifidobacterium bifidum (Lactobacillus bifidus) isolated from the stools of breast-, bottle-, and mixed-fed infants. The activities of CoA and its precursors varied for each of the strains, while CE was active for most of the strains. The strains were divided into four groups. The first group was able to utilize PAT, P-PAT, and CoA, but none of the other precursors. The second group was able to utilize all of CoA and its precursors, while the third group neither of them, and the fourth group one or a few of them. However, these utilizabilities changed by subcultivation. These results indicate that the nutritional requirement of B. bifidum is complicated and that CE contains growth factors other than those hitherto recognized.

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The terminal α-galactosyl linkage was hydrolyzed by an enzyme preparation from either Clostridium maebashi or coffee beans. Reduction of blood group B activity of Salmonella milwaukee and Escherichia coli O80 lipopolysaccharides and of some oligosaccharides occurred consequently. The degraded lipopolysaccharides showed significant blood group O (H) activity indicating the presence of terminal α-fucosyl residues in them and the same activity was demonstrated with oligosaccharide A-3 after the enzyme treatment. α-Fucosidase derived from Bacillus fulminans caused liberation of fucose from the α-galactosidase-treated materials and abolishment of O (H) activity. The results of quantitative analysis, borohydride reduction, Morgan-Elson reaction and treatments with several kinds of enzyme preparations on a series of oligosaccharides indicated that the structure of O-somatic side chain of E. coli O80 is probably; α-D-Gal-(1→3)-β-D-Gal-D-Ga1NAc-(1→3)-D-GalNAc-(1→4)-Fuc-α-L-Fuc(β-D-Glc)It was evident that there is a similarity in the terminal structure of lipopolysaccharides of E. coli O86, S. milwaukee and human B blood group substance.

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In order to determine the antigenic determinant groups of the mannan of Candida albicans by the precipitation-inhibition test, several oligosaccharides were prepared by acetolysis of the polysaccharide. The manno-oligosaccharides, from biose to heptaose were separated by a charcoal-Celite chromatography and a subsequent cellulose column chromatography. The oligosaccharides thus separated were examined on the degrees of polymerization and the mode of the linkages, and evidence was obtained that the biose and triose were joined through α1→2 linkage only, while the tetraose, pentaose and hexaose contained α1→3 linkage in addition to α1→2 linkages. Heptaose was joined entirely through α1→2 linkage. In the precipitation-inhibition test, the inhibitory power of the oligosaccharides of acetolysis product was found to be the following order: hexaose>heptaose>pentaose>tetraose>triose>biose, and the amount for the 50% inhibitions were 0.025, 0.09, 0.12, 0.60, 3.96 and 5.84 μmoles respectively. On the other hand, the biose, triose and tetraose, which were isolated from the acid-hydrolysate of the mannan of Saccharomyces cerevisiae and joined through α1→6 linkage, showed poor or nearly no inhibitory power. The above facts provide an evidence that the consecutive α1→6 linkages were not located in a position that is responsible for antigenic specificity of the mannan of C. albicans.

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The strains of gram-negative rod bacteria which are resistant to α-aminobenzylpenicillin and do not harbor the R factors were selected from our stock cultures of clinical origin. It was found that all strains produced β-lactamases which are species-specific in their substrate profiles and classified into three groups; 1) Typical cephalosporinase in the strains of Escherichia freundii, Aerobacter aerogenes, Arizona, Proteus morganii, Proteus rettgeri, Proteus inconstans and a strain GN633 of the Serratia group. 2) Cephalosporinase in the strains of Proteus vulgaris and. a strain GN629 of the Serratia group, which has a property of penicillinase to some extent. 3) Penicillinase in the strains of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. It was found that cephalosporinase was generally inducible enzyme, penicillinase was constitutive, and the penicillinase synthesized by the strains carrying R factors belonged to the third group. Penicillinases of two R factors, RGN14 and RGN238 which were isolated in this laboratory and belonged to the penicillinase of the third group, were studied by comparing their substrate profiles and immunological properties. It was demonstrated that penicillinases of RGN14 and RGN238 differed each other, while the penicillinase of K. pneumoniae was quite similar to that of RGN14 both enzymologically and immunologically.

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The glucose dehydrogenase from vegetative cells of Bacillus subtilis PCI 219 strain was partially purified through two ammonium sulfate fractionations and a filtration on Sephadex G-200. The optimal pH for stability of this enzyme was 6.0 and the optimal pH for the reaction catalyzed was 6.8. The Michaelis constant for glucose was 5.1×10^-3M at pH 6.8. High concentrations of lutidinic acid and isocinchomeronic acid remarkably protected the enzyme from heat inactivation, while dipicolinic acid showed a moderate protecting effect, Other chemical analogues of dipicolinic acid, such as quinolinic acid, nicotinic acid and isonicotinic acid, had less effect than dipicolinic acid, and phthalic acid had no effect. These effects of dipicolinic acid and other chemicals on the activity of glucose dehydrogenase from the vegetative cells were same with those on the activity of glucose dehydrogenase from the spores.

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Eighty-five CF# 1 mice were inoculated with living leprosy bacilli into the left hind foot-pads, and twenty with heat-killed ones, using 10³ to 10⁴ cells per foot-pad. After three to seven and a half months the mice were sacrificed and both left and right hind foot-pads, lungs, livers, kidneys, spleens, and left inguinal lymphnodes were examined for presence of acid-fast bacilli. Multiplication of the leprosy bacilli in the left hind foot-pads of mice was confirmed in fifty-eight of eighty-five mice inoculated with living organisms. Acid-fast bacilli were also found in the tissues other than the inoculated foot-pads, regardless of viability of the leprosy bacilli. Discovery of acid-fast bacilli was 64.7 per cent in the mice inoculated with living organisms and 50.0 per cent with killed ones. Acid-fast bacilli were found most frequently in the lymphnodes draining the inoculated foot-pads, the number of bacteria being very small in most cases. The evidence presented suggests that the leprosy bacilli are carried from the site of inoculation to other sites of mice, regardless of the viability. However, the invasion or multiplication in those sites remains still doubtful.

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Cells of Clostridium botulinum type A strain 190 harvested at logarithmic growth phase rapidly autolysed in phosphate buffer and most of the cells were converted auto-lytically into spheroplasts in 0.5 M sucrose-phosphate buffer within 2-3 hr at 37 C. Electron microscope observations on the process of autolysis and spheroplast formation revealed that lysis of the cell wall commenced at one end of the cell and the cytoplasmic contents were released through such lesion. The rod cell was thusly transformed into a fragile spherical form in the hypertonic sucrose-buffer. The lysis of the cell wall pro-ceeded centripetally and finally morphological integrity of the cell wall was completely lost. From these findings it is suggested that the autolysis of the organism is preceded by autodigestion of the cell wall at one end of the cell. A crude cell wall fraction isolated from log-phase cultures by sonication and fractionation rapidly autolysed in phosphate buffer. Reducing sugars and amino sugars of the wall were released from the autolysing wall fraction. Electron microscopy of the residues obtained from wall-autolysates demonstrated that the rigid structure of the wall completely disappeared and only fragile membranous or amorphous fragments remained after autolysis of the crude wall fraction. Heated wall preparations digested with trypsin and nagarse were dissolved by a soluble wall-autolysate, but not by a soluble cytoplasmic fraction. It seems likely that autolytic enzyme(s) may exist at or near the cell wall.

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A virus, the Yamaguchi strain, was serially propagated in suckling hamsters, mice and rats, and hamster kidney BHK21-WI2 cells from a natural case in the 1966 outbreak of bovine epizootic fever, an acute febrile disease of cattle, resembling ephemeral fever, known in Japan since 1949. An acute phase blood from the natural case was first passaged in calves by intravenous inoculation, and a blood specimen at the second passage was used to initiate serial hamster passage. Infected hamsters died with nervous symp-toms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. The hamster passage line of virus was serially passaged by intra-cerebral inoculation in 1-or 2-day-old mice, and then in rats 1 or 2 days after birth, which developed fatal encephalitis. The hamster passage virus was also serially propa-gated with cytopathic effect in cultures of BHK21-W12 cell cultures. These viral lines were shown to be neutralized by, and to produce specific complement-fixing antigen reactive with, convalescent sera of calves infected with the original Yamaguchi strain, confirming the identity of these lines as the Yamaguchi strain. The hamster passage line, when inoculated intravenously in calves, induced an acute febrile illness which was similar to bovine epizootic fever; all the inoculated calves had viremia and developed neutralizing and complement-fixing antibodies against the virus. Serological evidence for infection with this virus was obtained in natural cases in the 1966 outbreak. These findings seem to justify this virus to be the causative agent of bovine epizootic fever.

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In view of the significance of the bacterial surface as an important determinant for the host cell-parasite relationship, the rate of the bacterial clearance from the blood circulation or in the peritoneal cavity of mice was compared between the wild type Salmonella typhimurium LT2 and its mutants which have different sugar components in their cell wall. The mutants which are devoid of the tetrasaccharide sequence of abequosyl-mannosyl-rhamnosyl-galactose in their cell wall were rapidly eliminated from the circulating blood and the peritoneal cavity of mice after the injection, whereas the wild type strain and other strains having the tetrasaccharide sequence(s) were resistant to the clearance. Bacterial concentration did not affect the rate of clearance. 32P-labeled bacteria heat-killed at 58 C for 30 min retained the characteristics of the living organisms of each strain in clearance tests, and use of the killed labeled bacteria clarified that the tetrasaccharide sequence(s) in the cell wall is a determinant for preventing phagocytosis of the bacteria.

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When mononuclear phagocytes (macrophages), were infected with Salmonella enteri-tidis and confined in a diffusion chamber to incubate with normal macrophages, they confer on the normal macrophages cellular immunity, as detected by inhibition to intracellular multiplication of a virulent strain 116-54 and resistance to cellular de-generation caused by phagocytosis of bacteria. An immune ribonucleic acid (RNA) was extracted from the peritoneal macrophages maintained in tissue culture bottles in a homogeneous cell population which had been infected with strains of S. enteritidis. When peritoneal macrophages, cultured in a homogeneous cell population, were treated in vitro with this agent, they developed cellular immunity and cellular antibody. The RNA preparation was not inactived by treatment with deoxyribonuclease, with pronase or with antibodies to a virulent strain 116-54 of S. enteritidis. These facts suggest that the macrophages constitute a cell line responsible for active antibody formation.

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An attempt to isolate cytomegalovirus (CMV) from specimens obtained from children with various diseases was carried out from June, 1966 thru August, 1967. Forty-five strains of CMV were isolated from 21 cases of 135 children examined. Success in isolation was most frequently observed in children ranging in age from 5 months to 4 years. Serological investigations revealed a rate of incidence of complement-fixing antibodies against CMV was over 90% in the newborn, decreased to 60% in infancy, and gradually increased after 10 years of age. The highest rate of primary infection incidence was in early infancy and it was noted that lower age infants showed a marked tendency to become persistent virus excreters.

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Oda (1965) reported the rescue of dermovaccinia (strain Dairen I) abortive infection by neurovaccinia virus (strain IHD-T) in mouse fibroblastic L cells. The present study, however, provided no evidence for such, but gave evidence for the occurrence of genetic recombination between the viruses in L and HeLa cells. HeLa and L cells were infected simultaneously with strain Dairen I which produces hemagglutinin (H⁺) and replicates well in HeLa cells but only poorly in L cells, and strain IHD-T which is H^- and multiplies well in both cells. Sixty-eight of 225 and 24 of 114 clone preparations from the mixed infection yields in L and HeLa cells, respectively, were mixtures of H⁺ and H^- viruses. The remaining 157 and 90 clones contained only one type of virus, H⁺ or H^-, and varied widely in the plaquing efficiency for L cells. This contrasted with the results from parent viruses. An interpretation is that the plaquing efficiency is controlled by multiple genes and genetic recombination between the two vaccinia viruses with differing plaquing efficiencies results in occurrence of recombinants demonstrating u variety of plaquing efficiency. Hemagglutinin production was an all-or-none character, no intermediates being found, and can readily be tested at the particle level by the plaque-hemadsorption technique. These points render hemagglutinin production as u suitable marker for the genetic studies in contrast to the polygenic plaquing efficiency.

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