Katsumoto Ueda

Variation of virulence and other properties among Sendai virus strains

The virulence of five Sendai virus strains (MN, Z, KN, Mol, and Hm) isolated from laboratory rodents was compared, using 3‐week‐old female Jcl‐ICR mice. The virulence of the strains was Mol, MN, KN, Z, and Hm in decreasing order. The 50% lethal dose and 50% lung consolidation inducing dose of the highest virulent strain differed by the order of more than 103 and 106, respectively, from those of the lowest virulent one. Other properties such as the growth rate in LLC‐MK2 cells, neuraminidase activities, and molecular weights of structural proteins also differed among the virus strains. These results indicate that Sendai virus prevailing in laboratory rodents is not homogenous with respect to virulence and some other properties.

HISTOPATHOLOGIC FEATURES OF NATURALLY OCCURRING HEPATOZOONOSIS IN WILD MARTENS (MARTES MELAMPUS) IN JAPAN

Nodular lesions containing Hepatozoon sp. schizonts or merozoite gametocytes were found in the tissues of 67 (96%) of 70 wild caught martens (Martes melampus) examined in Gifu, Japan, 1991 and 1992. The heart was the most commonly parasitized organ (96%), followed by the perirenal adipose tissue (36%); the diaphragm, mesentery, tongue, omentum and perisplenic adipose tissue generally had a prevalence of 10 to 15%. In the heart, two types of nodular lesions were differentiated based on developmental stages: nodules containing schizonts and nodules consisting of an accumulation of phagocytes containing merozoites or gamonts. Under electron microscopy, mature schizonts contained membrane-bound merozoites with a single nucleus and small scattered electrondense cytoplasmic granules in the schizont nodules; the merozoites and gamonts were engulfed in a phagosome-like vacuole of phagocytes with the nucleus compressed to one side due to the parasite in the merozoite-gamont nodule.

T Cell Subsets Responsible for Clearance of Sendai Virus from Infected Mouse Lungs

T cell subsets responsible for clearance of Sendai virus from mouse lungs determined by adoptive transfer of immune spleen cell fractions to infected nude mice. T cells with antiviral activity developed in spleens by 7 days after intranasal infection. Spleen cell fractions depleted of Lyt‐2+, Lyt‐1+, or L3T4+ cells showed antiviral activity in vivo, although the degree of the activity was lower than that of control whole spleen cells. The antiviral activity of the Lyt‐2+ cell‐depleted fraction was consistently higher than that of L3T4+ (Lyt‐1+)‐depleted cells. In vitro cytotoxic activity against Sendai virus‐associated, syngeneic lipopolysaccharide‐blast cells was detected in stimulated cells from intraperitoneally immunized mice but was lost after depletion of Lyt‐2+ cells. Multiple injection of anti‐Sendai virus antibody into infected nude mice had no effect on lung virus titer. These results indicate that L3T4+ (Lyt‐1+) and Lyt‐2+ subsets are cooperatively responsible for efficient clearance of Sendai virus from the mouse lung.

Detection of antibodies to Pneumocystis carinii by enzyme-linked immunosorbent assay in experimentally infected mice.

Pulmonary pneumocystosis, caused by Pneumocystis carinii is one of the most important opportunistic infections in man and animals under immunosuppressed state (Arean, 1972. Pathology of protozoal and helminthic disease. Williams & Wilkins Co., Baltimore, 291 p.; Frenkel et al., 1966, Laboratory Investigation 15: 1559-1577). In such immunosuppressive hosts, serum antibody to P. carinii is generally produced at a low level (Kagan and Norman, 1976, National Cancer Institute Monograph 43: 121125). Therefore the development of a highly specific and sensitive method to measure antibody would be required to elucidate the relationship between progression of infection and antibody response and to evaluate the plausibility of serodiagnosis (Pifer et al., 1978, Pediatrics 61: 3541) as well as for epidemiological survey. In the present study we developed an enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies to P. carinii, and the sensitivity was compared with an indirect immunofluorescence (IF) test routinely used in our laboratory (Furuta et al., 1984, Japanese Journal of Experimental Medicine 54: 57-64). The P. carinii, isolated from a naturally infected nu/nu mouse, has been maintained by the intranasal (i.n.) passage in nu/nu mice in our laboratory (Furuta et al., 1984, loc. cit.) and organisms from the sixth passage were used in this experiment. The standard ELISA technique for various viruses was modified for P. carinii as follows (Voller et al., 1976. Microplate enzyme immunoassays for immunodiagnosis of virus infection. American Society of Microbiology, Washington, D.C., 506 p.). Pneumocystis carinii cyst suspension was prepared from infected mouse lungs as described previously (Furuta et al., 1984, loc. cit.). The sample was sonicated at 200 W (Kubota Insonator, Model 200) for 50 min, and diluted to 5 ,tg/ml with phosphate buffered saline (PBS) pH 7.2. Fifty 1l of the cyst suspension was added to each well in a polystyrene microplate (Nunc-Immuno Plate 96-F, Nunc Inter Med, Roskilde) and the plate was incubated at room temperature for 1 hr. The plate was washed 3 times with 0.05% Tween 20 in PBS (Tween 20-PBS), and 50 ,ul of serum dilution was added to each well. After further incubation at room temperature for 1 hr, the plate was washed 3 more times with Tween 20-PBS. Subsequently, 50 ,l of 1:1,000 rabbit anti-mouse IgG or IgM serum which were both conjugated with horseradish peroxidase (specific to gamma or mu chain, respectively; Cappel Labs., Cochranville, Pennsylvania) was added to the well. After incubation at room temperature for 1 hr, the residual conjugate was removed by washing 3 times with Tween 20-PBS, and 200 ,dl of substrate solution consisting of 40 mg o-phenylenediamine dihydrochloride and 10 ,l H202 in 100 ml of 0. 1 M citrate buffer, pH 4.7 was added to each well. The plate was incubated in the dark for 1 hr at 37 C, and 50 ,1 of 6 N sulfuric acid was added to each well to stop the enzyme reaction. The absorbance of the reaction mixture was measured at 492 nm by a spectrophotometer (Titertek Multiskan; Flow Labs., Inc., Rockville, Maryland) and the reading of a given diluted serum higher than 1.5 times of equivalent dilution of normal serum was taken as a positive reaction. Outbred ICR mice were obtained from a commercial breeder (Charles River Japan Co., Atsugi). The colony had no detectable antibodies to P. carinii by our routine IF test. The mice were maintained in cages with a filter cap (Sankikagaku Co., Tokyo) and placed in laminar airflow racks. Twenty mice were inoculated i.n. with 104 P. carinii cysts in 0.05 ml PBS (Furuta et al., 1984, loc. cit.) and 5 mice were killed at weekly intervals. The number of cysts in the lung preparations stained with toluidine blue-O (Chalvardjian and Grawe, 1963, Journal of Clinical Pathology 16: 383-384) was counted under the microscope (Furuta et al., 1984, loc. cit.). As

Immunoglobulin Classes of Anti-Sendai Virus Antibody Detected by ELISA in Infected Nude Mouse Serum

Sendai virus‐infected nude mouse sera obtained on the seventh day after infection or later, in which anti‐Sendai virus antibodies were undetectable by hemagglutination‐inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase‐conjugated anti‐mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti‐Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti‐Sendai virus antibody activity of fractions from Sephadex G‐200 gel filtration was detected in the IgM fraction when anti‐mouse μ chain‐specific antiserum was used and in both IgG and IgM fractions when heavy and light chain‐specific anti‐mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A‐Sepharose affinity chromatography of Sendai virus‐infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti‐Sendai virus antibodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.

Hypertrophic Pulmonary Osteoarthropathy in a Raccoon Dog (Nyctereutes procyonoides) with Chronic Pulmonary Inflammatory Lesions

A female raccoon dog (Nyctereutes procyonoides) captured September 1992 in Nara prefecture, Japan, suffering from swollen joints of all four limbs was necropsied. Radiographically, there was a shadow in the right pulmonary caudal lobe and periosteal bone formation around several joints. Microscopically, there was extensive edema, along with one large and many small pyogranulomatous nodules and purulent bronchitis in the right lobe and left caudal lobe of the lungs. There were intermingled branching filamentous bacteria in the lesion, which stained positively with Warthin-Starry, Grocott, and Gram stains, and partially with periodic acid Schiff and Ziehl-Neelsen stains. Extensive exostosis with coralloid trabeculation had developed bilaterally at the carpal, stifle and hock joints in the surrounding well vascularized loose connective tissue. Degeneration of articular cartilage and villous proliferation of synovial membranes were apparent. This is the first report of hypertrophic pulmonary osteoarthropathy in the raccoon dog.

Cooperation between Humoral Factor(s) and Lyt-2+ T Cells in Effective Clearance of Sendai Virus from Infected Mouse Lungs

The mechanism of cooperation between the L3T4+ and Lyt‐2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long‐term‐cultured Sendai virus‐specific L3T4+ T cell line with L3T4+ cell‐depleted immune spleen cell (L3T4−) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4+ T cell line or concanavalin A‐stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4− fraction. The role of the Sendai virus‐sensitized L3T4− cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell‐deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL‐2), which was more than 12 times the IL‐2 activity present in the supernatants of the T cell line or concanavalin A‐stimulated spleen cells, failed to clear the virus in combination with the L3T4− fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL‐2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus‐sensitized Lyt‐2+ subset of T cells acts cooperatively with humoral factor(s) other than IL‐2 or Sendai virus‐specific antibody present in supernatants of the T cell line, of concanavalin A‐stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.

Recovery of Nude Mice from Sendai Virus Infection after Adoptive Transfer of Spleen T Cells or T Cell‐Depleted Spleen Cells

Sendai virus infection is one of the most prevalent diseases of mice and rats in animal laboratories (8, II). Vaccination is considered one of the protective methods against the infection (10, 18, 23). However, the mechanism of recovery from the infection still remains unclear. Sendai virus can cause persistent infection in the lungs of nude mice without producing detectable neutralizing (NT) and hemagglutination inhibiting (HI) antibodies after intranasal inoculation, in contrast to the infection in euthymic mice in which a transient virus growth followed by antibody production ensues (9). These findings indicate that T cells play an important role in recovery from Sendai virus infection in mice. Anderson et al (2) reported that both cellular and humoral immunity contribute to the recovery of mice from Sendai virus infection. In this report, we show that non-T lymphocytes as well as T cells, both from Sendai virusimmune nul-l-mice, can adoptively eliminate the virus from infected nude mice and that specific antibody is not sufficient for eliminating the virus. Congenitally athymic nude (both sexes) and nul+ (female) mice of the BALBI cA strain aged 4 to 5 weeks were obtained from the colony of the Animal Care Technology Laboratory of the Central Institute for Experimental Animals (Kawasaki). The egg-passaged MN strain of Sendai virus was used for intranasal inoculation under ether anesthesia with a microtiter dropper. Spleen cells were obtained from normal nul+ mice or convalescent nul+ mice inoculated intranasally with 103 TCIDso of the virus per 0.025 ml 8 weeks before. Dispersed spleen cells were fractionated on a nylon fiber (Wako Pure Chemical Industries, Ltd., Osaka) column. The nylon-passed cells were designated the T cell fraction, which was found to contain 91 to 97% brain-associated Thy-I positive cells when examined by the membrane fluorescein-labeled-antibody method (FA). Nylon-adherent cells were washed out and phagocytic and residual T cells were removed by treatment with carbonyl iron (Nakarai Chemicals, Ltd., Kyoto) and anti-Thy-1.2 antibody (anti-C3H mouse thymocyte AKR mouse serum) plus guinea pig complement, respectively. This fraction contained 82 to 89% surface immunoglobulin-positive cells, as determined by FA. T cell activity was considered to be

Antigenic Variation among Sendai Virus Strains Detected by Monoclonal Antibodies

Thirteen strains of Sendai virus isolated from various sources in the 1950′s and after 1976 were compared for their reactivities with monoclonal antibodies prepared against the prototype strain MN of Sendai virus. Results revealed that while the 5 strains isolated in the 1950′s reacted with all the monoclonal antibodies as the prototype strain did, the 2 strains isolated in 1976 and 1978 did not react with an F‐specific monoclonal antibody, and the other 6 strains isolated after 1978 lacked reactivity with an HN‐specific monoclonal antibody.

Gastric Adenocarcinoma in a Cougar (Felis concolor)

Diffusely invasive tumors occurred in the stomach of a 9-year-old female cougar (Felis concolor) from a zoo in Japan. The tumors consisted of tubular adenocarcinoma cells, and had infiltrative growth to the submucosa and muscularis propria. Tumor cells were positive for carcinoembryonic antigen (CEA), lysozyme, epithelial membrane antigen (EMA), gastrin, alpha-1-fetoprotein (AFP), keratin, and B72.3. Mucin-like materials occurred within cytoplasmic vacuoles.