The resistance of Staphylococcus aureus to penicillin G was studied quantitatively by the dilution method. A series of 338 strains obtained from patients fell into 3 groups according to degree of resistance; 150 highly resistant strains, 137 moderately resistant strains, and 51 sensitive strains. Furthermore, 287 strains resistant to 0.2 units per ml or more of penicillin fell into 2 groups according to antibiotic resistance pattern, 173 single resistant strains and 114 multiple resistant strains. The vast majority of the highly resistant strains belonged to phage-groups I and III, whereas the majority of the moderately resistant strains belonged to group II. Of the highly resistant strains, 65% were multiple resistant, and 35% single resistant. Of the multiple resistant strains, 86% belonged to the 52, 52A, 80, 81 complex within group I and to group III, but none belonged to group II. Of the strains that were highly resistant to penicillin only, 83% belonged to group I, and of these, nearly one-half were members of the 52, 52A, 80, 81 complex, and the other half belonged to the rest of phage-types within group I. In contrast, 88% of the moderately resistant strains were single resistant, and of these, 58% belonged to group II. Mercury resistance was observed in 77% of the highly resistant strains of the multiple resistance pattern, 29% of those of the resistance to penicillin alone and 3% of the strains moderately resistant to penicillin alone. Of the mercury resistant strains, 68% belonged to the 52, 52A, 80, 81 complex, 3% to the rest of phage-types in group I, 12% to group III, but none belonged to group II. The results suggest that there may exist a difference in the mechanisms of inactivating penicillin between strains of staphylococci highly resistant and moderately resistant to penicillin.

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A large epidemic of aseptic meningitis due to ECHO 6 virus swept over Japan in the summer months of 1965. In our studies on 89 cases of aseptic meningitis in the epidemic, primary culture of human embryonic kidney cells was shown to provide a highly sensitive host system for isolation of ECHO 6 virus from clinical materials. Virus was recovered from cerebrospinal fluid in 72% of 89 cases, from throat swabs in 64% of 36 cases, and from rectal swabs in 59% of 37 cases. Most significant is the finding that the rate of virus isolation from cerebrospinal fluid in this host system was considerably higher as compared with that obtained by other investigators in other host systems such as primary monkey kidney cells or human amniotic cells (primary or FL). This finding should be emphasized particularly because isolation of a virus from cerebrospinal fluid, in contrast to throat secretions or feces, is of much greater importance in establishing the etiologic relationship to the disease in the diagnosis of aseptic meningitis. The neutralization test was shown to be efficient in detecting ECHO 6 virus infection; acute serums should be taken preferably by the 4th day of illness and convalescent serums in the second week. Epidemiologic findings, such as the predominance of male patients and occurrence of the epidemic in summer months, generally coincide with the previous reports. However, our cases were in much younger age groups in contrast with the previous reports; 90% of our 89 patients were 6 years of age or younger, and 15 children or 17% were less than 1 year of age, including 3 less than 6 months of age. The clinical observations on our cases confirmed the previous reports.

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In order to determine the determinant antigenic group of the mannan of Saccharomyces cerevisiae, a series of inhibition tests were carried out employing oligosaccharides which separated from the acetolyzate and the hydrolyzate of the mannan. Tetraose, Manα1→3 Manα1→2 Manα1→2 Man², corresponding to the structure of the longer branching moieties of the mannan showed the strongest inhibition, while the isomer, Manα1→6 Manα1→6 Man, corresponding to the core moiety, produced only one-tenth the inhibition of the former. This provides evidence that the branching moieties of the mannan play important role in combining with antibody. The fact that the disaccharide, Manα1→3, showed significantly stronger inhibition than those of the other disaccharides, Manα1→2 Man and Manα1→6 Man, indicates that the most important part of the determinant group of the mannan isα1→3 linked D-mannose residue. The antigenic inactivity of the periodate-oxidized mannan containing unoxidized mannose residues indicates that the presence of 3-O-substituted-D-mannose residues adjacent to the n-mannose residues and joined with α1→2 linkages, are essential to fit the combining site of the antibody.

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Primary cultures of rabbit embryo (RE) cells were compared with that of African green monkey kidney (GMK) cells for sensitivity in the isolation of rubella virus from clinical materials. Virus was recovered in either or both RE or GMK cells from one or more sites in 82% of 38 children clinically diagnosed as having rubella. Virus was isolated from 50% of 32 throat swabs in RE cells, and from 53% of 38 throat swabs in GMK cells. Of 32 throat swabs inoculated into both cell systems, 24 yielded virus; 10 of these were positive in both cell systems, 8 were positive only in GMK cells, and 6 were positive only in RE cells. None of 19 blood specimens yielded virus in GMK cells, whereas of 18 of these same 19 specimens tested in RE cells, 10 (56%) yielded positive results. These results indicate that RE cells are more sensitive than GMK cells. Recognition of rubella virus in RE cells is achieved by direct observation of cytopathic effect, instead of the cumbersome interference technique as employed in GMK cells or certain other cell systems. RE cells are practically free of latent viruses, readily available, and easy to cultivate. All this indicates that the RE cells provides not only a highly sensitive but also a practical host system for the isolation of rubella virus from clinical materials.

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Marked cytopathic changes were induced by challenge with Newcastle disease (ND) virus in bovine testicle or kidney cell cultures which were previously infected with noncytopathogenic strains of bovine diarrhea (BD) virus. No cytopathic changes were induced by ND virus in similar cells not infected with BD virus. The development of cytopathic effect was shown to be associated with enhancement of ND virus replication. This exalting effect of BD virus appears to be dependent on infectivity, since the effect was inhibited when infection of the cells with BD virus was blocked by specific antiserum. Various factors involved in the phenomenon were investigated and an in vitro method (END) for the assay of BD virus and its antibodies was developed. The use of this method eliminates the difficulties in recognizing non-cytopathogenic strains of BD virus which hampered systematic investigations of the nature and behavior of BD virus as well as of the natural history and pathogenesis of the infection in cattle.

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The Sugiyama strain of measles virus adapted to FL cells produces plaques heterogeneous in size and morphology on FL and other stable human cell line monolayers making it difficult to assay by the plaque technique. A virus clone producing homogeneous clear plaques was established by repeated cloning of the strain. A practical reliable plaque assay method was established for this cloned line. FL cells were found to be well suited for this purpose, but Vero cells, a stable line of African green monkey kidney cells, were shown to be more sensitive than FL cells. HeLa cells could be used for the assay, but were less sensitive than the Vero or FL cell line. The initial step of measles virus adsorption onto host cells was shown to be reversible, dependent on electrolytes and almost temperature-independent, strongly suggesting adsorption is electrostatic in nature. At 36 C the initial stage of adsorption quickly passed into the next irreversible stage involving a temperature-dependent reaction; virus adsorbed at 36 C soon became resistant to the action of measles antibody, whereas little virus adsorbed at 4 C or 12 C became antibody-resistant. The time required for the adsorbed virus to move into the irreversible, antibody-resistant stage seems very short.

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Numerical classification of scotochromogenic mycobacteria revealed the presence of four groups: (1) Mycobacterium xenopei; (2) M marianum, M. scrofulaceum, M. aquae and M. gordonae; (3) M. acapulcensis and M. flavescens; and (4) M. aurum. Each of these groups was considered to be a species. Characteristics of these groups were described.

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Two adenovirus strains were isolated in calf testicle cell cultures from blood specimens of cattle in Japan. This is the first isolation of bovine adenovirus reported in Japan. The isolates were antigenically similar to each other and distinct from the hitherto described serotypes 1, 2 and 3 of bovine adenovirus. Unfortunately, bovine adenovirus types 4 and 5 were not available for comparison, and hence, until the matter is settled, the virus will be called "Bovine adenovirus type Nagano". Nagano virus was identified as adenovirus on the bases of the inhibitory effect of 5-iodo-2'-deoxyuridine on virus replication, ether-resistance, effect of temperature and pH on infectivity, and fine structure of the virus particle. The virus grew and formed intranuclear inclusion bodies, a characteristic of adenovirus, in bovine testicle cells but not in bovine kidney cells. The virus agglutinated rat erythrocytes very poorly, but not sheep, goat, cattle, horse, guinea pig, hamster, chicken, and mouse cells. The virus produced adenovirus group-specific antigen in cell cultures. Sero-negative calves were readily infected with the virus by the intravenous, subcutaneous, oral or intranasal routes of inoculation. The infected animals produced antibodies and showed u mild clinical reaction comprised of rhinorrhea, diarrhea and a degree of pyrexia; low-titered viremia of short duration and leukopenia were also observed. A serologic survey indicated wide-spread dissemination of the virus among Japanese cattle, but further studies are needed to determine the etiologic significance of the virus in the natural disease in cattle.

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Four strains of herpes simplex virus tested all showed inability to form plaques in chick embryo fibroblasts (CEF) at 40 C, while no such suppression of growth was observed with WEE, vaccinia or JE virus in the same cells. The suppression was not due to a complete inhibition of viral growth, because virus-infected CEF bottle cultures consistently showed a small but definite increase of virus titer in 24 hr. When CEF monolayers adsorbing herpes virus were placed at 40 C, the number of infective centers decreased gradually; however, this decrease was much slower than the degradation of free virus at this temperature. Transfer of virus-infected CEF bottle cultures from 35 C to 40 C at any time during a one step growth cycle promptly slowed down subsequent virus replication. When virus-infected CEF bottles were incubated first at 40 C for 24 hr and then transferred to 35 C, a new increase in virus titer took place following a short lag. What stage of virus replication is suppressed at 40 C remains yet to be determined.

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Growth of the pathogenic leptospirac, Leptospira canicola, strain Utrecht and L. icterohaemorrhagiae, strain Mikawajima, in modified Korthof's basal medium containing various lipid fractions obtained from Mycobacterium smegmatis and M. tuberculosis strain Aoyama B in place of rabbit serum, was examined. Growth of L, canicola, strain Utrecht was supported by all mycobacterial lipid fractions. The growth of L. icterohaemorrhagiae strain Mikawajima was supported by mycolic acid and mycolic acid-containing fractions, such as chloroform extract, purified wax, wax C, wax D, cord factor, bound lipid B and bound lipid D, but not by the fractions containing unsaturated fatty acids, such as alcohol-ether extract, and the bound lipids A and C. It is of interest that leptospiral growth was stimulated by a higher molecular fatty acid such as mycolic acid. Furthermore, distinct differences in Tween 80 requirement were found between L. canicola, strain Utrecht and L. icterohaemorrhagiae, strain Mikawajima.

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Two hundred and sixty six strains of Pseudomonas aeyuginosa isolated from various pathological or non-pathological materials were classified by serological and pyocine typing methods for the purpose of epidemiological investigation. The O antigenic groups detected were Verder and Evans' O1, 2, 3 (7a, 7b), 4, 5, 6, 8, 9, 10 and Habs' O4 and O10. In addition, three new O antigenic groups d0esignated tentatively as Ox11, Ox12 and Ox13 have been established. Among these groups, Verder amid Evans' O2, O3 (7a, 7b) and Ox11 were most common. No significant difference existed between the O groups of P. aeruginosa isolated from pathological materials and those obtained from non-pathological sources. Pyocine typing of Darrell and Wahba was considered to be useful in the epidemiological investigation. In a patient, P. aeyuginosa of a specific sero-pyocine type persisted for long period in an infected focus.

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