Yuji Inaba

Isolation and characterization of mouse nasal-associated lymphoid tissue

A method for isolation of mouse nasal-associated lymphoid tissue (NALT), which is a principal mucosal lymphoid tissue of the respiratory tract in rodents, was developed. The paired lymphoid organs could be separated from the upper jaw by peeling away the palate where NALT was localized bilaterally on the posterior side. About 3 x 10(5) lymphocytes could be obtained from one NALT fragment. The NALT lymphocyte fraction from normal BALB/c mice contained T- and B-cells in about equal numbers, and contained about 4 times as many CD4+ T-cells as CD8+ T-cells when analyzed with a FACScan fluorescence analyzer. The composition of the NALT lymphocytes was similar to that of the lymphocytes from the portion of the nasal cavity remaining after isolation of the NALT. The NALT lymphocyte fraction from mice infected 7 days previously with influenza virus was also characterized. The numbers of NALT T- and B-cells from the infected mice were approximately 2 and 3 times higher than those of non-infected mice, respectively. In parallel with the cell increase, NALT lymphocytes produced IFN-gamma when cultured for 24 h and contained cells secreting influenza virus-specific IgA and IgG antibodies. The results suggest that this method can be successfully used for investigating cellular dynamics of mucosal immunology in the upper respiratory tract.

Characterization of mouse nasal lymphocytes isolated by enzymatic extraction with collagenase

A reproducible method for isolation of mouse nasal lymphocytes was developed. The cells were released from tissue fragments of dissected mouse nose by enzyme extraction with collagenase and separated by a stepwise Percoll gradient centrifugation. The partially purified nasal lymphocyte fraction from normal BALB/c mice contained CD4+ T cells (18-23%), CD8+ T cells (7-10%) and B cells (20-38%), when analysed with a FACScan fluorescence analyser. The ratio of T to B cells and that of CD4+ to CD8+ T cells in the nasal cell fraction were about twice as high as those in Peyers path lymphocytes. The nasal lymphocyte fraction from the mice infected with influenza virus was then characterized. The nasal lymphocytes contained a twice larger number of CD4+ and a three times larger number of CD8+ T cells than those of normal mice 7 days after infection. They produced IFN-gamma which increased after infection. They contained the cells secreting influenza virus-specific IgA and IgG antibodies 4 weeks after infection. Moreover, the nasal lymphocytes from infected C3H mice lysed the virus infected-target cells. These results suggest that this method can successfully be used for investigating cellular dynamics of mucosal immunity in the upper respiratory tract of experimental animals.

Effect of Heparin on Hemagglutinin of Herpes Simplex Virus Type 1

Heparin inhibited the hemagglutinin activity of herpes simplex virus (HSV) type 1. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of HSV ranged from 0.005 to 0.01 U/ml. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by HSV. Virus‐heparin complex formation was observed by sedimenting heparin with the virus particles.

Isolation of Fukuoka virus, a member of the Kern Canvon serogroup viruses of the family Rhabdoviridae, from cattle

Four virus strains with identical antigenic properties were isolated from blood samples of 4 sentinel calves having a fever and leukopenea in cultures of HmLu-1 cells derived from baby hamster lung. The virus was identified as Fukuoka virus, classified as a member of the Kern Canyon serogroup viruses of the family Rhabdoviridae, on the basis of its antigenic properties.

Effect of heparin on a haemagglutinin of porcine reproductive and respiratory syndrome virus

Heparin inhibited haemagglutination by porcine reproductive and respiratory syndrome virus (PRRSV) and by Aujeskys disease virus, but failed to inhibit haemagglutination by parainfluenza virus type 3. The minimal inhibitory concentration of heparin required to inhibit 8 HA U of PRRSV haemagglutinin ranged from 0.1 to 1 U ml-1. Mouse erythrocytes failed to combine with the haemagglutination inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by PRRSV. The formation of a haemagglutinin-heparin complex could be observed by sedimenting heparin with the haemagglutinin. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.

Isolation of a new serotype of bovine rhinovirus from cattle. Brief report

SummarySix strains of a new serological type of bovine rhinovirus were isolated in primary calf kidney cell cultures from nasal swab specimens of cattle with acute respiratory disease in Japan. They are serologically indistinguishable from one another and show no cross reaction with bovine rhinovirus types 1 and 2. We propose that the new isolates classified as bovine rhinovirus serotype 3.

Effect of heparin on hemagglutination by Akabane and Aino viruses belonging to the Simbu group of bunyaviruses.

Heparin inhibited the hemagglutinin activity of Akabane and Aino viruses. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of Akabane and Aino viruses was 10 U/ml. Goose erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, goose erythrocytes treated with heparinase had greatly reduced agglutinability by Akabane virus. Virus-heparin complex formation was observed by sedimenting heparin with the virus particles.

Hemagglutination with avian infectious laryngotracheitis virus

SummaryAvian infectious laryngotracheitis virus grown in primary chicken kidney cell cultures was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, 22°C, and 37°C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, swine, rabbit, guinea pig, chicken, and goose erythrocytes. A strain variation between mice in the agglutinability of their erythrocytes necessitated selection of mice to obtain erythrocytes. The HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual chicken sera had a significant positive correlation with their neutralizing antibody titers.

Virucidal Effect of Disinfectants on Some Viruses Coated on Dressing Gauze, Chicken Egg-Shell and Wooden Shavings and Its Durability

First, 8 disinfectants were tested for virucidal effect on three DNA viruses, bovine herpesvirus-1 (BHV-1), vaccinia virus (VV) and canine adenovirus, and three RNA viruses, Newcastle disease virus (NDV), bovine enterovirus and bovine rotavirus. To the viruses coated on a cotton dressing gauze, all the disinfectants used had a virucidal effect on both enveloped DNA and RNA viruses and the disinfectants on the chlorine and aldehyde groups had the same effect on non-enveloped DNA and RNA viruses. To the viruses coated on a chicken egg-shell, all the disinfectants used had a destructive effect on both enveloped DNA and RNA viruses. To the viruses coated on poplar-wooden shavings, the disinfectants of the chlorine and aldehyde groups had a virucidal effect on some enveloped DNA and RNA viruses, VV and NDV, and the disinfectant of only the aldehyde group had a virucidal effect on all viruses. Secondly, five disinfectants were tested for the durability of their virucidal effects on NDV and fowl adenovirus (FAV) after making their working solutions. The disinfectants of the orthodichloro-benzene-cresol and chlorine groups maintained their virucidal efficiency for 2-4 days on NDV, and the remainings for 2 days. In the presence of chicken feces as organic matter, the virucidal effect of the orthodichloro-benzene-cresol and chlorine groups were not influenced but that of the reverse soap group was strongly influenced and became invalid. Only the chlorine group maintained its virucidal effect on FAV for 2-4 days. ―J . Jpn. Vet. Med. Assoc. , 42, 877-882 (1989) .