Genetic studies have shown that one of the transfer (T) factors, T₉₅, carries the following genetic properties: autonomous replication (rep⁺), conjugal transmission (tra⁺), transmission of the host chromosome (mat⁺), inhibition of F-mating (ifm⁺ or fi⁺), inhibition of superinfection with R factor (irs^-), and suppression of phage proliferation (spp^-). An Escherichia coli K12 strain carrying T₉₅ factor is insensitive to male phages (f1 and f2). These facts indicated that the T₉₅ factor is a sex factor but different from the F factors. By the interaction between T₉₅ factor and the nontransferable resistance determinants; r₂₁ (tet) for tetracycline resistance and r₃ (kan) for kanamycin resistance, recombinants, T-tet, T-kan, and T-tet-kan, factors were obtained. These are capable of conferring drug-resistance and are conjugally transmissible as a single genetic unit. E. coli K12 strains carrying T₉₅ or T-tet factor were insensitive to male phages and T-tet (probably T₉₅) factor was transmitted sexually at about 10^-5 transmission frequency after 1 hr of mixed culture. On the contrary, E. coli K12 strains carrying T-kan or T-tet-kan factor were found to become sensitive to male phages and their transmission frequency was 10² to 10³-fold higher than that of T-tet factor. As a result of these findings, we concluded that the pili formation of T₉₅ or T-tet factor is in a repressed state but the formation of a recombinant between T₉₅ (or T-tet) and r₃ (kan) determinants results in the derepression of pili formation.

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Replication of Ibaraki virus was not inhibited by 5-iodo-2'-deoxyuridine, indicating that the virus is an RNA virus. The virus was resistant to ether, chloroform and deoxycholate, sensitive to trypsin, very labile at acidic pH but stable at pH 6.4 or higher, and was resistant to repeated freezing and thawing. The virus was readily inactivated at 56 C or higher, was fairly stable at 37 C, and very stable at 4 C, while it rapidly lost infectivity when stored frozen at -20 C. The virus was readily sedimented by centrifugation at 40 000×g for 60 min. It readily passed through membrane filters of 200 mμ pore size, passed through 100 mμ filters but only with some titer loss and did not through 50 mμ filters. In these tests, the bluetongue virus used as a control behaved in the same manner as Ibaraki virus. These findings provide additional evidence for the similarity of Ibaraki virus to bluetongue virus which had been previously demonstrated on the basis of seasonal incidence, symptomatology and pathology of the diseases caused by these viruses and the behavior of the viruses in cell cultures, embryonated eggs and laboratory animals. The present study, however, provided no evidence for any serological relation between these two viruses. More information is needed to reach a final decision on the classification of Ibaraki virus, particularly regarding the morphology of the virion, the doublestrandedness of the viral RNA and other basic features.

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Germination of spores of Clostridium botulinum type E under varying conditions of Eh, pH, temperature, activation, and nutrition was studied. Germination in a complex medium consisting of thiotone, glucose and bicarbonate occurred at any level of initial Eh, regardless of the poising procedures employed. On the other hand, postgerminative development was affected by the initial Eh in the germination medium. Under an anaerobic condition, germination, outgrowth and division of cells were characterized by a peculiar profile of an Eh-time curve. The optimal conditions for germination of unheated spores in the complex medium were incubation at 37C and pH 6.6. The rate of germination increased with ageing time, the maximum rate being attained after storing an aqueous suspension of spores at 4 C for 20 days. The optimal heat activation was indicated when subjecting spores to heat-shock at 60 C for 10 min. Germination in a casamino acids medium was markedly stimulated by a supplementation of lactate. The minimum requirements of compounds needed for the germination of spores of C, botulinuin type E were L-alanine, glucose, lactate, and bicarbonate (CO₂). Germination occurred only in the following combinations of these compounds: r-alanine+glucose+lactate, L-alanine+glucose+bicarbonate, or L-alanine+lactate+bicarbonate.

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In order to provide further information about the immunochemical differences between two mannans of Candida albicans serotype A and serotype B, quantitative precipitation-inhibition tests of anti-C. albicans serotype B serum were carried out in the present study. Oligosaccharides were prepared by acetolysis of a homologous mannan, and α1→2 linked di-, tri-, tetra-, penta- and hexasaccharide were separated in chromatographically homogeneous states. The latter two oligomers contained a small amount of an isomer containing α1→2 and α1→3 linkages. In the precipitation-inhibition tests of anti-C. albicans serotype B serum with its homologous mannan and heterologous mannan of C. albicans serotype A, the inhibitory power of the oligomers was of the following order; hexa->penta->tetra->tri->disaccharide, and the amounts for 50%-inhibition of the former 4 oligomers were 0.02-0.03, 0.050.07, 0.1-0.2 and 0.3-0.4 μmoles respectively, whereas disaccharide was very poor inhibitor. The lower oligomers, α1→2 linked tri- and tetrasaccharide, showed considerably strong inhibitory activities. The results obtained in the present study confirmed that the antigenic determinants of the mannan of C. albicans serotype B is the hexasaccharide moiety corresponding to the longest branched chains of mannan, and moreover, the α1→2 linked tri- and tetrasaccharide moieties play an important factor in dominating immunochemical specificity.

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Investigation was made on the process of enteric infection with mouse adenovirus strain K87 in inbred DK1 mice and the intestinal resistance acquired through infection. The cells containing viral antigens were enumerated in most parts of the infected intestinal tract by a fluorescent antibody technique, and the infectivity titer of the virus in each part was examined in mouse kidney tissue culture. The virus was observed to grow in 3∼14 days (sometimes 3∼21 days) after oral challenge, and infectivity titers reached their peak after 7∼14 days, when a number of viral antigen-containing cells and cells with nuclear inclusions were detected. In the mice rechallenged 28 days after the initial challenge, the virus did not grow, and no viral antigen-containing cells were found. From these results it was concluded that the main sites where the virus grows in mice are the cells which are scattered in the epithelial layer of the mucous membrane of the small intestine, and which seem to be the usual epithelial cells and not Paneth's or goblet cells. As for intestinal resistance, experiments with inactivated vaccine and with passive transfer of serum-antibodies were performed in order to find out whether neutralizing antibodies in the serum had any influence on the growth of virus in the intestinal wall, and no influences were indicated. Eighteen days or more after challenge, K87 virus-neutralizing substances were detected in the intestinal wall and in the intestinal contents of the infected mice, but not in the serum-transferred mice, though both groups of mice had equal levels of serum antibodies. The substance continued to be found until 15 weeks after challenge in the intestinal contents, and until later than 34 weeks in the intestinal walls. The nature and the possible role of the substance is discussed, but actual data will be reported in subsequent papers.

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In the transduction of R factor with phage P1 from rec^- host, transduction frequency was less than that from the rec⁺ host but no segregation of resistance markers took place. However, a low percent of segregation was found in the transduction from rec⁺ host. In contrast, phage particles of small size contained in P1 lysate could not transduce R factor even from a rec⁺ host, although they could transduce chromosomal markers as did the usual P1 lysate. Recombination of the R factor took place in the rec^- host at a frequency similar to the rec⁺ host. Considering from the results that P1 is capable of transducing a DNA fragment of the same size as that of phage P1, it was concluded that the molecular weight of R factor DNA is smaller than that of phage P1 (6×10⁷ daltons) but larger than that of small P1 particle (2.4×10⁷ daltons). When transduced with phage P1, the R factor genome has to become a DNA fragment of the same size as the P1 particle by addition of other DNA following the recombination between R factor and P1 in rec⁺ host. Hence, the segregation of markers of the R factor takes place on occasion by this recombination. From the assumption that phage P1 cannot recombine with the R factor in rec^- host, it was predicted that the R factor genome becomes a DNA fragment of large enough size for transduction with P1 by polymerization of the R genome itself. This possibly could explain the decrease in transduction frequency from a rec^- host and no segregation of the resistance markers of R factor in transduction from rec^- host.

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Aluminum (A)-marker of 14 wild type 1 poliovirus strains (4 from the central nervous system of fatal cases, 10 from feces of paralytic cases) were studied. These strains were isolated in Japan from 1957 to 1961 before the implementation of the mass vaccination campaign with Sabin live poliovaccine. While an attenuated strain LSc, 2ab, and a virulent strain, Mahoney, were shown to be typically A^- and A⁺ respectively, only one of the wild strains examined was A⁺, 3 strains were A^- and the remaining 10 were apparently A^-, being thermoresistant in the presence or absence of Al³⁺ ions, when an ordinary stock virus was used. After three passages in primary monkey kidney cell cultures maintained with L-cystine-free synthetic medium No. 199 most of these strains became more or less thermosensitive in the absence of, but stabilized in the presence of Al³⁺. The results of the monkey neurovirulence test by intrathalamic inoculation with 2 A^- strains indicated that they are almost as neurovirulent as the reference A⁺ Mahoney strain. These results suggest that the A-marker is an attribute inherent to each strain of type 1 poliovirus without any correlation with neurovirulence in monkeys.

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A Japanese strain (Yamaguchi) of bovine ephemeral fever virus, propagated in BHK21-WI2 cells of hamster kidney origin, was used. Adult rats inoculated intracardially or intracerebrally remained healthy, but produced neutralizing antibody, suggesting actual viral growth in the animals. However, no clinical and serological evidence for infection with the virus was obtained in adult sheep, goats, swine and rabbits inoculated intravenously. One-day-old guinea pigs inoculated intracerebrally and adult guinea pigs inoculated intracardially remained healthy and produced no neutralizing antibody. The virus multiplied not only in BHK21-WI2 cell culture but also in cultures of stable line hamster cells (HmLu-1 and HmT), primary hamster kidney and embryo cells, long-term cultured bovine embryo cells (BEK1), primary calf kidney and embryo cells, and primary suckling rat kidney and embryo cells. However, no evidence for cytopathogenesis and viral growth was obtained in primary cultures of bovine leukocytes, bovine bone marrow, thymus, thyroid and adrenal cells, guinea pig, mouse and chick kidney cells, and mouse and chick embryo cells, and in culture of African green monkey kidney cells (Vero). The virus multiplied best and induced marked cytopathogenesis in BHK21-WI2 cells; lower temperatures such as 34 C or 30 C appeared preferable to obtain constantly better viral yields, although the virus grew more slowly. Similar effects of the incubation temperatures were also obvious in the other susceptible cell cultures, in which the cytopathogenesis was less marked than in BHK21-WI2 cells or absent, roughly coinciding with decreasing grades of viral growth.

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