Leptospira interrogans serovar canicola DNA was cloned into the plasmid pBR322 and introduced into E. coli. Eight out of approximately 10, 000 transformants were found to express antigens of canicola by ELISA including colony ELISA blot test using anti-canicola antiserum. The canicola antigens expressed in the transformants reacted with the antisera against the serovars belonging to Canicola serogroup and other serogroups of L. interrogans. They did not react, however, with the antiserum against L. biflexa (with only one exception) nor with the antiserum against Leptonema illini. Thus, the recombinant DNA technique may provide alternative possibilities for preparing antigens of leptospires.

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Cell wall, cytoplasm, polysaccharide, and peptidoglycan fractions prepared from Lactobacillus casei, L. plantarum, and L. acidophilus were examined for their efficacies to enhance resistance of host mice against Listeria monocytogenes infection. Intraperitoneal injections of those cellular fractions of L. casei led to elicitation of inflammatory cells in the peritoneal cavity and the efficacy was highest in the case of peptidoglycan. Macrophage ratio in the resultant peritoneal exudate cells was also highest in mice given peptidoglycan. Macrophages induced with cell wall fraction of L. casei showed the most potent phorbol myristate acetate (PMA)-triggered respiratory burst (chemiluminescence and O₂^- production determined on the basis of nitroblue tetrazolium reduction) followed by those elicited with peptidoglycan. All the macrophages induced with cell wall of L. casei (two strains) and L. acidophilus enhanced O₂^- production in response to PMA but L. plantarum did not enhance O₂^--producing ability in such a manner. The L. casei-cell wall also enhanced in vitro listericidal activity of mouse peritoneal macrophages, but such an activity was not noted in the case of L. acdiophilus-cell wall. When mice were intravenously given the cellular fractions 7 or 13 days before L. monocytogenes infection, cell wall fractions of L. casei caused the most potent protective activity. A weak protective activity was also found in peptidoglycan of L. casei. Therefore, the protective action of L. casei against L. monocytogenes infection in host mice may be attributed to cell wall compounds and partially to the peptidoglycan moiety.

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An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [³H]leucine in LLCMK₂ cells and of human red blood cells (RBCs). The efficiency of viral release from the virus-bound RBCs by incubation at 37C depended on the number of virus particles which had been used for adsorption onto the RBC at 4C. When 7.8×10² virus particles were previously adsorbed onto the RBC at 4C, most of the viruses were dissociated from the RBC at 37C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37C. Such RBC-bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK₂ cell-grown Sendai virus (LLCMK₂-Sendai) particles, but not released by treatment with hemagglutinin-neuraminidase protein (Sendai-gp) isolated from egg-grown Sendai virus.

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The biochemical characteristics of canine distemper virus (CDV) adapted to three human neural cells (glioblastoma, oligodendroglioma, and neuroblastoma cells) were compared with those of the unadapted original virus. The specific gravity of the virions and nucleocapsids of the original and the three adapted viruses were not different. The molecular weights of genomic RNA and messenger RNAs encoding H, F, P, and NP proteins of the adapted viruses as estimated by Northern blot hybridization were similar to those of the original virus. By T₁-resistant oligonucleotide analysis of the genomic RNA, the glioblastoma- and the neuroblastoma-adapted viruses gave two more spots than the original virus; the oligodendroglioma-adapted virus had a pattern identical to that of the original virus. By two-dimensional gel electrophoresis of virion proteins, we found a difference in the isoelectric point of the viral envelope proteins H and F between the original and the adapted viruses. These results suggest that viral genomic changes occurred during adaptation, resulting in the alteration of viral envelope proteins.

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Reoviruses (reos) were isolated from river water in various areas of Toyama Prefecture. The frequency of reo isolation was higher in the river water, the basin of which has a larger human population. The degree of river contamination with reo paralleled that with the Escherichia coli group of organisms, and reos were frequently isolated from sewage, too. The high antibody-positive (≥1:8 or ≥1:10) rates against reos in humans and other animals tested (swine, cattle, and field rodents) indicated their wide-spread infection with reos. These results suggested that the major source of reos present in the river water may be the excretion by humans and other animals, especially the former. Survival experiments in which reos were added into the filtered or centrifuged river water and kept at various temperatures, revealed that reos survived for more than 3 years at 5C. In the field experiment where reos suspended in cellophane tubes were kept in an agricultural water stream in winter (water temperatures below 10C), they survived for 6 months until the water temperature rose above 20C in summer.

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We studied the effects of breeding conditions on the development of immunological abnormalities in spontaneously hypertensive rats (SHR) with congenital T cell depression. The depression of T cell functions, the production of natural thymocytotoxic autoantibody (NTA), and the development of polyarteritis nodosa were more evident in SHR reared under a conventional (CV) environment than in specific-pathogen-free (SPF) SHR bred in a semi-barrier system. Enhancement of these immunologic abnormalities was also observed by the conventionalization of SPF-SHR. A high frequency of antibodies to mouse hepatitis virus (MHV), Sendai virus, and Mycoplasma pulmonis was detected in CV rat sera, whereas no antibodies were detected in SPF-SHR. The experimental infection of Sendai virus induced the enhancement of T cell depression and of NTA production in SPF-SHR. We interpret these results to mean that the natural infection of microorganisms causes an acceleration of immunologic abnormalities in SHR reared in a CV environment.

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Two immunization techniques that enable production of mouse monoclonal antibodies were evaluated in terms of small quantities of antigen. Various amounts of purified influenza A virus particles were applied either for in vitro sensitization in cultured splenocytes or for intrasplenic immunization, followed by hybridization of the immunized cells with mouse myeloma cells. Hybridomas producing specific antibodies for influenza viral proteins were detected by enzyme-linked immunosorbent assay when more than 50μg of antigens was used for the in vitro immunization method, and at least 5μg was necessary for a single intrasplenic immunization. On the other hand, as little as 60ng of antigen administered in two intrasplenic injections was sufficient to produce specific hybridomas. Two out of six randomly selected monoclonal antibodies obtained using the repeated intrasplenic immunization method were IgG and the other four were IgM. Immunoprecipitation analyses revealed that the recognized antigens involved a viral inner protein (nucleocapsid protein), as well as an envelope glycoprotein (hemagglutinin). We conclude that immunization by two direct injections of antigen into the spleen is the most effective method for sensitization with nanogram quantities of insoluble antigen such as influenza viruses.

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The effect of orally administered bacterial lipopolysaccharide (LPS) on host resistance against bacterial infections was studied. LPS orally given for 5 consecutive days prior to infection caused no apparent toxic effect and protected mice against Pseudomonas aeruginosa and Listeria monocytogenes infections.

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The number of rRNA genes in Mycobacterium smegmatis was examined by hybridization of BamHI and SalI digests of chromosomal DNA with 3'-end-labeled 5S, 16S, 23S rRNA and tRNA. Each RNA probe gave two hybridization bands. The PstI fragments of 6.6 kilobases were cloned to pBR322. The cloned DNA was characterized by restriction endonuclease mapping, DNA-RNA hybridization, and the R-loop technique.

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The stability of neurovirulence and in vitro phenotypes of canine distemper viruses adapted to neural cells was examined. Neurovirulence was estimated by the morbidity, mortality, and histopathological changes in the central nervous system of mice. After a single passage of the adapted viruses in Vero cells in which the unadapted virus had been passed, the neurovirulence of glioblastoma-adapted and oligodendroglioma-adapted viruses reverted completely to that of the unadapted virus. However, the neurovirulence of a neuroblastoma-adapted virus reverted partially. In vitro phenotypes such as the two-dimensional electrophoretic patterns of viral proteins and the cross-neutralization patterns also reverted to those of the unadapted virus. However, plaque sizes remained similar to those of the viruses adapted to neural cells.

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