Bacteremia was observed during the late stage of experimental Tyzzer's disease in mice. The number of organisms in the blood in mice treated with cortisone increased markedly in the infection with highly virulent organisms, whereas bacteremia was of low incidence and less severe in infections with low virulence organisms. The number of organisms in the blood stream was shown to increase linearly during the course of fatal infection attaining a maximum level of 10⁷ organisms per ml blood. The number of organisms in the blood was found to be closely related to the number in the liver when bacteremia was observed. In the peripheral blood, organisms were first detectable when the number of organisms in the liver gained a level of 10⁷ per g tissue, and the subsequent increase in the number of bacteria in the blood was approximately 3 times more rapid than in the liver. The organisms in the blood were comparable to those in the liver morphologically as well as in pathogenicity. Histopathological examination frequently revealed liberation of organisms from liver cells into sinusoids. There was no evidence of significant multiplication of the organisms in organs other than the liver.

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Effect of Tween 80 on the growth of Leptospira canicola strain Utrecht and L. icterohaemorrhagiae strain Mikawajima was examined. The suspension of washed leptospira was inoculated into modified Korthof's basal medium containing varied amounts of Tween 80 and cultured at 30 C. Cell numbers were counted by using Petroff-Hausser counting chamber every other day. Optimum Tween 80 concentrations for L. canicola were 0.0125 and 0.025%. Cell counts in the second sub-cultures reached 108 per ml the same as the primary cultures. Generation time of L. canicola in 0.025% Tween 80 medium was about 13 hours. Growth of L. icterohaemorrhagiae was inhibited at concentrations greater than 0.0125 per cent. Cell numbers increased about 4 times at concentration of 0.0000125% Tween 80. L. canicola utilizes Tween 80 as a nutrient while L. icterohaemorrhagiae appears sensitive to it. A difference of more than 1, 000 times in maximal growth-supporting concentration between L, canicola and L. icterohaemorrhagiae exists. This difference appears to be caused by difference in surface structure and metabolic requirements.

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Type or representative strains of rapidly growing mycobacteria were tested in respect to 97 characters and submitted to a numerical classification. The results revealed that the following species be recognized: (1) Mycobacterium rhodochrous; (2) M. chitae; (a) M. phlei; (4) M. thamnopheos; (5) M. borstelense; (6) M. fortuitum; (7) M. vaccae; (8) M. parafortuitum; (9) M. smegmatis. Biologic and biochemical characters of type strains of these species were described. M. fortuitum was divided into two subspecies, M. fortuitum subsp. fortuitum and M. fortuitum subsp. abscessus. M. runyonii is synonym of M. abscessus and therefore has been included to the subspecies abscessus. M. parafortuitum was divided into two subspecies, M. parafortuitum subsp. parafortuitum and M. parafortuitum subsp. aurum, M. aurum being reduced to a subspecies. M. smegmatis was divided into two subspecies (or varieties), M. smegmatis subsp. smegmatis and M. smegmatis subsp. lacticola.

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In our previous studies, RNA phage strains were separated into 3 major groups on the basis of filtration efficiency through Millipore filters. In the present study, the strains of group III were shown to be further divided into 3 subgroups: (a) Qβ, NH, SG; (b) VK, SO; (c) ST. This subgrouping was found to be compatible with the serological grouping and pronase sensitivity with the exception of strain NM. Strain NM was classified in subgroup (a) by the Millipore filtration and in subgroup (b) by the other two methods.

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An attenuated virus strain (V423) showed lower multiplication in chick embryo (CE) cells than the original virus strain (Hotta) from which the V423 strain was derived. Both strains induced some amounts of interferon before their infectivity reached a maximum titer. Thirty-two units of interferon were detected between 24 and 48 hr after infection. While no significant difference was seen in the interferon production between the V423 and Hotta strains, the V423 strain was shown to be much more sensitive to interferon. The lower growth of the V423 strain, therefore, may be explained at least partially by the inhibitory effect of endogenous interferon. This hypothesis was further supported by evidence that actinomycin D (0.2 μg/ml) enhanced the virus growth, presumably as a result of inhibiting interferon formation. This enhancement, however, was revealed only after more than 24 hr postinfection. Within the first 24 hr some slight decrease in the virus growth was observed in actinomycin D-treated cells. This inhibitory effect of actinomycin D showed a dose response in a confirmatory experiment with different concentrations of the antibiotic.

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The cup method commonly used in antibiotic assay was applied to the serum neutralization with JE virus. A cup was fixed in the overlay covering chick embryo cells and filled with a test serum, and diffusion of antibody was allowed to take place at 20 C before addition of virus onto the agar surface. When the overlay contained 0.6% agar, 0.1 to 0.27% of the virus penetrated the overlay and formed plaques leaving a plaque inhibition zone around the cup. A clear inhibition zone was produced when the interval between the addition of serum and virus inoculation was 2 days, the amount of virus given per dish being 5×105 PFU, and the dishes were subsequently incubated at 20 C for one day and then at 35 C for 3 days before staining with neutral red. The plaque inhibition zone diameter was in linear relation to the logarithm of the antibody concentration used. The sensitivity of the test was such that positive results were obtained with sera possessing endpoint titers higher than 1:10 as determined by the ordinary 50% plaque reduction test. When sera of 83 children with different vaccination histories were tested by the present cup method and by the 50% plaque reduction method in parallel, a good correlation was observed between the two titers.

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Multiplicity reactivation (MR) seems to take place in leaves of Nicotiana glutinosa inoculated with ultraviolet (UV) light irradiated RNA from tobacco mosaic virus (TMV-RNA). A similar phenomenon was not observed with UV-irradiated TMV particles. Considering MR as resulting from genetic recombination between viral genomes, a recombination mechanism, which has been difficult to prove with plant viruses, is proposed as being operative during multiplication of TMV. From the pattern of MR of TMV-RNA, the location of the gene for the RNA replicase within a TMV-RNA strand is discussed.

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Complement-fixation tests of Rickettsia orientalis were performed by using antigens purified with sodium deoxycholate. Cross complement-fixation tests between antigens of the Gilliam, Ohzeki, Karp and Kato strains and immune mouse sera of these four strains and of the Kaneko strain revealed difference in antigenic structure among the Gilliam, Karp and Kato strains. The Ohzeki strain was identical with the Gilliam strain and the Kaneko strain was identical with the Karp strain. In the homologous system, complement-fixing antibodies of mice infected with R. orientalis began to appear on the 15th day after infection and rose rapidly in a few days, and reached maximum in 4-6 weeks after infection and 8 weeks at latest. Subsequently, the titers declined gradually and showed low titers on the 52nd week in spite of survival of rickettsia in mice. In the heterologous system among which antigenic structures were different, the titers rose somewhat later and declined earlier than those of the homologous system. It was confirmed that the antigen treated with sodium deoxycholate was utilizable in the specific serological diagnosis of scrub typhus, because convalescent sera from all ten patients with the disease showed significant rise of complement-fixing titers against the antigen.

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Virus strain No. 12, one of the new isolates from Japanese cattle described previously, was studied for its physicochemical properties. The new isolate was shown to be very small in size by centrifugation and filtration, being filtrable through Millipore filters of 50 mμ pore size. It appears to be an RNA virus as its replication was not inhibited by 5-iodo-2'-deoxyuridine. The virus was readily inactivated by ether and deoxycholate, and partially by trypsin; was labile at pH 3, not stabilized by 1 M MgCl2 at 50 C, was inactivated by ultraviolet, and withstood repeated freeze-thawing. Further it was readily inactivated at 56 C but more slowly at 37 C, and was stable at lower temperatures. These findings support the identification of the isolated virus as the bovine diarrhea (BD) virus. The properties of BD virus, i.e. size, type of nucleic acid, ether, chloroform and deoxycholate sensitivities, and acid lability, appear to be similar to those of arboviruses. The trypsin sensitivity of BD virus is similar to the B group of arboviruses, which, unlike the A group, sensitive to trypsin. For the classification of BD virus as well as hog cholera virus, which is closely related, further elucidation of properties, fine structure of the virion, etc., is needed.

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Polyribosomes have been isolated from tobacco leaves, Upon infection with TMV, a new polyribosome has appeared which is specific for infection, and TMV-antigenic protein is formed on this polyribosome. This new polyribosome has an S-value of 360-380. From these results, it is suggested that this TMV specific polyribosome is an aggregate of 60-80 ribosomes which is bound by TMV-RNA as messenger, and that TMV-RNA is translated polycistronically.

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Nine strains of an adenovirus serotype were recovered in bovine testicle cell cultures from Japanese cattle suffering with an acute febrile illness accompanied by rhinorrhea and diarrhea. The isolated virus was shown to have the physicochemical properties of the adenovirus group such as the nucleic acid type, the size and ultrastructure of the virion, and the resistance to ether and chloroform. The isolated virus produced eosinophilic intranuclear inclusion bodies characteristic of adenoviruses and the group specific antigen of adenovirus in bovine testicle cell culture. According to the results of crossneutralization tests the isolated virus represents a serological type distinct from bovine adenovirus types 1, 2 and 3 and from the Nagano virus. The isolated virus agglutinated erythrocytes of cattle, sheep, goat, guinea pig, rat, hamster and mouse, but not those of vervet monkey, horse, goose and chicken. HI test using cattle erythrocytes corroborated the results of serological typing by neutralization tests.

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The bovine diarrhea (BD) virus isolates from Japanese cattle induced an acute illness in experimentally inoculated calves. The disease was characterized by pyrexia, anorexia and depression; most characteristic were a diphasic temperature reaction accompanied by leukopenia and rarity of respiratory and alimentary symptoms and signs. The isolates also induced a similar disease in adult cows. The results of virus reisolation indicated the systemic nature of the infection and viremia was found to be a common feature. Rises of neutralizing antibody titer against BD virus were demonstrated. Infected lactating cows showed diminished lactation accompanied by virus shedding in milk. In infected pregnant cows the fetuses were also infected with virus. The analysis of the present data together with those in the literature indicates that although there are natural cases of the viral diarrhea-mucosal disease complex caused by BD virus, it seems necessary to evaluate the relative importance of BD virus in the etiology of the syndrome complex. Further the possibility exists that some other agent (s) may be involved in the syndrome complex or that BD virus may induce other clinical conditions.

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