Heat-resistant strains of Clostridium perfringens were isolated from normal feces samples of Japanese people more frequently than from European people. However, the predominant number of the C. perfringens cells in the human intestines were sensitive to the heat at 100 C for 10 min. This proved to be true of the feces samples which were demonstrated to carry the organism resistant to 100 C for 60 min. Further studies indicated that the more the number of the organism in the intestines, the higher was the recovery ratio of the heat-resistant strains. Concomitant estimation for α-toxigenicity of those strains isolated from unheated feces samples revealed that the α-toxigenicity ranged mainly between 0.05 and 0.9 α-antitoxin equivalents.

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Outbreaks of an acute febrile disease of cattle occurred in Japan in 1959 and 1960. Its occurrence was limited in late summer and autumn, and in Kyushu, Shikoku and Honshu roughly south of 37 degrees north latitude, suggesting a close correlation of the incidence with the climatic conditions, hence a possibility of the presence of arthropod vector. The disease was characterized by fever and lesions affecting the mucous mem-brane and skin, musculature and vascular system. Degeneration of striated muscles was observed in the esophagus, larynx, pharynx, tongue and skeletal muscular system. Edema and hemorrhage were marked in the mouth, lips, abomasum, coronets etc., occasionally followed by degeneration of the epithelium leaving erosions or ulcerations. Severe lesions affecting the esophageal and laryngopharyngeal musculature caused deglutitive difficulty which in turn resulted in dehydration and emaciation, and occa-sionally in aspiration pneumonia, constituting the major causes of death of the affected animals. These findings indicate that the disease resembles bluetongue in sheep and cattle. The clinical materials obtained from natural cases induced a clinical illness when inoculated into calves, and the disease was transmitted serially in calves by intravenous inoculation of the blood obtained at the height of febrile reaction. The experimentally produced disease was clinically and pathologically indistinguishable from the natural disease.

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Several strains of a virus, designated as Ibaraki virus, were isolated in bovine cell cultures from natural and experimentally produced cases of the disease. In some of these cases, antibody titer rises were shown against Ibaraki virus. The clinical materials used for virus isolation induced in calves a mild illness similar to the natural disease, as well as neutralizing antibody against Ibaraki virus. Antibody titer rises were also shown in a high percentage of natural cases clinically diagnosed as having the disease. Antibody was not detected in any of the cattle in non-epizootic areas, while many cattle in the epizootic areas had antibody after the outbreak. In the epizootic areas the antibody incidence after the outbreak was much higher among cattle diagnosed as having the disease during the outbreak than those not affected. These results leave little doubt that Ibaraki virus is the etiologic agent of these disease. Ibaraki virus became less virulent for calves upon serial passage in bovine cell cultures. The bovine cell cultures appeared less sensitive than the calf inoculation for the primary detection of the virus. The use of intermediary calf inoculation before inoculation into tissue culture may improve the isolation rate, but the tissue culture technique itself should be im-proved as calf inoculation is both expensive and cumbersome.

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It was found that the ruins of Bovine Epizootic Fever appeared to be RNA as its replication was not inhibited by 5-iodo-2'-deoxyuridine. It had a buoyant density of 1.196, was not filterable through membrane filter (Millipore Filter Corp. Mass.) of 100 mμ pore size, and was sensitive to ether, chloroform and deoxycholate, was inactivated by trypsin and ultraviolet irradiation, precipitated by protamine sulfate, readily inactivated at pH 3.0, fairly labile at 56 C but readily preserved at -80 C, not stabilized at 50 C by 1 M MgCl₂, and resisted repeated freeze-thawing. The virus appears not to require a DNA-dependent RNA synthesis in the host cell for its replication, as chromomycin A3 did not inhibit its replication. Sucrose density gradient centrifugation was unsuitable for purification because of a very poor recovery of infectivity. CsCl equilibrium density gradient centrifugation was successfully used for this purpose, and electron microscopy of the resulting fractions by phosphotungstic negative staining technique revealed virus-like bullet-shape particles, about 140 mμ in length and 80 mμ across, in the fraction of peak infectivity titer. The particle is probably the virion of the virus. These findings suggest relation of the virus to Rhabdoviruses [6], but further studies are necessary. The physicochemical properties of the virus provide additional evidence for similarity of the virus to bovine ephemeral fever virus, and emphasizes the desirability of further detailed comparative studies to decide whether they are one and the same or merely very closely related.

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Induction of chloramphenicol (CM) resistance in Staphylococcus aureus was investi-gated by using several CM derivatives. It was found that dl-threo-1-p-nitrophenyl-2-dichloroacetamino-1, 3-dichloropropane has high antibacterial activity but low activity of induction for CM resistance. In spite of low antibacterial activity, induction of CM resistance occurred after prior treatment with dl-threo-1-p-nitrophenyl-2-dichloro-acetamino-3-chloropropane-1-ol. It was found that dl-chloramphenicol di-acetate, dl-threo-1-p-nitrophenyl-2-dichloroacetamino-3-bromopropane-1-ol and dl-threo-1-phenyl-2-dichloroacetamino-1, 3-propanediol have induction ability in spite of the absence of antibacterial activity. Other derivatives were classified into two groups; (1) low anti-bacterial activity and induction of CM resistance and (2) loss of both activities.

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The Yamaguchi strain exhibited a rapid loss of pathogenicity for calves upon serial passage in suckling hamsters, suckling mice and hamster kidney BHK21-WI2 cells. Calves developed clinical symptoms after intravenous inoculation with the virus up to the fourth passage in suckling hamsters, the third passage in suckling mice, and the seventh or eighth passage in cell cultures, while the virus further passaged did not induce any clinical illness. Almost at the same time the virus lost the ability to produce antibody and resistance to challenge with virulent virus. The attenuated virus only stimulated production of antibody and immunity to virulent virus after repeated inoculations. These findings provide additional evidence for the similarity of bovine epizootic fever to bovine ephemeral fever.

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Shigella strains isolated in Japan from 1965 to 1967 were surveyed for drug resistance and distribution of R factors. Of 5, 875 strains tested, 5, 429 were resistant to either one or various combinations of four drugs, tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among these resistant strains, 80.3, 1.8, 2.2, and 15.7% were quadruply, triply, doubly and singly resistant, respectively. Fifty percent of these resistant strains were found to carry R factors which are capable of conjugal transfer. More than 50% of TC or CM resistant strains carried R factors, but in the remaining resistance type, R factors were rarely isolated. Only 3 and 17% carried R factors among SA and SM.SA resistant strains, respectively. The strains resistant to drugs other than the aforementioned four were very few; 0.9, 0.7 and 0.01% being resistant to aminobenzyl-penicillin (APC), nalidixic acid (NA) and kanamycin (KM), respectively. Conjugally transmissible NA resistance was not found among strains so far isolated and examined. About one third of APC resistance was transmissible. Among 9, 635 strains examined, one R factor possessing the KM resistance marker and 28R factors possessing the APC resistance marker were demon-strated. The R factor possessing the KM resistance marker was multiply resistant to 8 drugs; TC, CM, SM, SA, APC, KM, fradiomycin and paromomycin.

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Structure of TMV containing the base analogues such as 2-thiouracil, 8-azaguanine, and 5-fluorouracil was studied. TMV containing these analogues had the same anion-exchange chromatographic properties as had normal TMV, and 2-thiouracil was incorporated into TMV particles, probably into TMV-RNA. Cation-exchange chromato-graphic properties of TMV containing these analogues had no significant difference as compared with those of normal TMV. TMV containing 2-thiouracil was more stable to alkali degradation than normal TMV. Alkali-degraded TMV containing 2-thiouracil and normal TMV was characterized by chromatography. These results suggest that not surface structure but internal structure of TMV containing 2-thiouracil was different from that of normal TMV.

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Most of spores of B, anthracis introduced into the peritoneal cavity of rats could germinate when 5 mg of L-alanine and 5 mg of adenosine were injected by the same route shortly before the spore injection. However the spores hardly germinated in untreated rats. To develop vegetative cells after spore germination, 1 ml of an aqueous solution containing 1% casamino acid and 1% of yeast extract was injected into the peritoneal cavity of rats before injection of 55×10⁷ spores of B. anthracis. A few germinated spores and vegetative cells were seen in ascites, but their numbers were not enough to kill the rats. In order to produce a rapid germination and development to vegetative cells in a short time, 1 ml of solution containing 1% of casamino acid, 1% of yeast extract, 5 mg of L-alanine and 5mg of adenosine was injected before intra-peritoneal administration of spore suspension. Spore germination and vegetative growth were tremendous and all of rats died within 24 hours. From these results it was con-cluded that the natural resistance of the rat for B. anthracis is partly due to unfavorable internal conditions for spore germination and subsequent vegetative growth.

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