Airborne particulates in Sagamihara city were collected on quartz fibre filters using a high-volume air sampler for 14 days in each month from July 1984 to June 1985. Organic components in airborne particulates were extracted by the ultrasonic extraction method using benzene-ethanol(3:1 v/v) as an extracting solvent. The mutagenicities of airborne particulates extract were measured by the pre-incubation method using Salmonella typhirnurium strains TA100 and TA98 with and without S-9mix. The concentration of airborne particulates was nearly the same level throughout the survey period, but the content of extract in particulates of winter was higher than those of other seasons. All the airborne partiuclates extracts showed positive mutagenic response to both strains with and without S-9mix. The average of mutagenic activities (revertants/m³ air) in winter was significantly higher than those in summer and spring. Furthermore, mutagenic activities fluctuated sharply in one month from several to ten times compared with the normal level, depending upon sampling days, and tended to be lower on Sundays and holidays in summer and new year holidays. In many days mutagenic activities without S-9mix were comparatively higher than those with S-9mix. The existence of nitroarene was surveyed using TA98NR strain. Nitroarene was found to be higher concentration in summer than in winter.

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Soluble antigens prepared from sporulated oocysts and second generation merozoites of E. tenella were used for enzyme linked immunosorbent assay (ELISA) to investigate antibody in sera of two breeds of chickens, i.e. commercial broilers and SPF single comb white leghorn layers, which were experimentally infected with E. tenella. In broilers inoculated with oocysts at 15 days of age, ELISA values increased rapidly after day 19 post inoculation (PI) and reached the maximum lebel on days 29 and 32 PI against both merozoite and oocyst antigens. The values against merozoite antigen were significantly higher than those against oocyst antigen. In SPF layers infected at 15 days of age, the values increased gradually after 7 days PI. There were no significant differences between values against two antigens. Generally, thc values in broilers tended to be higher than those in SPF layers, especially against merozoite antigen. In broilers inoculated with oocysts at 1 and 15 days of age, ELISA values increased rapidly and reached the maximum level on days 11 and 20 post second inoculation (PSI) against merozoite and oocyst antigens respectively and then the values against merozoite antigen decreased. The values against merozoite antigen were markedly higher than those against oocyst antigen. In SPF layers inoculated twice, the values reached the highest on day 11 PSI as in the case of broiler; however, after that day, the values against both antigens decreased. The sera reacted similarly against both antigens. The values against merozoite antigdn were significantly higher in broilers than in SPF layers. The values of maternal antibodies wdre combaratiVely high in broiler chicks and decreased gradually with the age of bird to the negligible level at 10 days of age.

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The dissemination of equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) among various horse pbpulations in Japan was investigated through the isolation and typing of virus strains from horses with respiratory diseases. Type specific monoclonal antibody pools were used for the typing of isolates. The 42 strains of EHV-1 and 64 strains of EHV-4 were isolated from 4593 nasal swabs and/or blood plasma samples collected from 3326 horses during a period from 1979 to 1990. All the strains of EHV-1 were isolated from racehorses only and during the winter season exclusively, when the epizootic of respiratory diseases occurred among racehorse populations at two Training Centers of the Japan Racing Association. In contrast, the strains of EHV-4 were isolated from horses irrespective of the season, facility, or horse population; foals and yearlings in breeding farms and our institute, rearing horses in rearing farms, and racehorses. Especially, 4 strains of EHV-4 were isolated from plasma samples containing buffy coat cells. We believe this is the first reported case of EHV-4 cell-associated viremia in the world. Al1 87 strains isolated from aborted fetuses were identified as EHV-1. The results suggest that EHV-1 is responsible for epizootic respiratory diseases in racehordes in the winter and abortion among mares at the late stage of gestation, and that EHV-4 causes respiratory diseases throughout the year among all horse populations.

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The fine structure of intercellular bridge (ICB) of goat germ cells was studied using testicular samples fixed by perfusion. In the seminifefous tubuled, the ICBs were observed between sister cells of spermatogonia, spermatocytes and spermatids. As a result of incomplete division of germ cells, the ICB first appeared as a midbody containing a remnant of the bundle of microtubules (spindle fibers). These microtubules then disappeared and were replaced by a shutter apparatus which was composed of multiple lamellar cisternae (bridge-paftitioning complex). The inndr part of the ICB was reinforced with a layer of electron dense mass (bridge density) which persisted up to the residual cytoplasm of spermiation. After complete reconstruction of the sister cells, the cisternae of the bridge-partitioning complex disappeared and the channel of the ICB was opened. Evidently (see electron micrographs), almost all of the cytoplasmic organelles could pass through the channel of the ICB. In the longitudinal section, the apperance of the ICBs between sister spermatogonia and between sister spermatocytes was observed as a double linear or drum shdpe, and that between sister spermatids was noticed as a horseshoe-like or concave formation. With the process of spermatogenesis, the ICBs gradually became widened and shortened. The functional significance of the ICB in the goat was discussed.

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Growth of the tumor autoinduced by 20-methylcholanthrene (MC) in rats was inhibited after administration of Toxoplasma lysate antigen (TLA). The antitumor activity of TLA was most obvious in the early stage of tumoral growth. When TLA was administered to rats before the appearance of tumor, tumor formation was delayed slightly. Histopathological studies revealed dense growths of spindle tumor cells in untreated control rat, while enlarged central necrosis with the infiltration of lymphocytes and neutrophils was apparent in TLA-treated rats. According to the immunohistological examination of tumor tissue with anti-Thy-1 antibody, the rats treated with TLA showed large Thy-1 positive granular cells, whereas the untreated rats indicated only a few small Thy-1 positive cells. These observations indicate that TLA is a useful modifier of biological responses to MC-induced tumors.

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Eight of 9 Mute swans (Cygnus olor) untied in the river acrossing the central part of Tottori-city died within the period of 40 days of summer in 1989. Seven of 8 Mute swans were pathologically examined. In all swans many yellowish-brown to greenish-brown granules were found in the cytoplasm of hepatocytes. The granules were intensely stained with rhodanine copper stain, schmorl method, and Berlin blue stain. Ultrastructurally, many lysosomes increased in size and density in the cytoplasm of hepatocytes. Other three swans, that died at other places, were served as controls. In control swans, many brown granules intensely stained with schmorl method and Berlin blue stain were also found in hepatocytes, but the number of rhodanine-positive granules were fewer than those of the affected cases. X-ray qualitative analysis showed three peaks corresponding to copper, zinc and sodium in the liver of the affected and control swans. Quantitative analysis demonstrated that mean hepatic copper concentration of the affected group was significantly higher than that of control group (P<0.01). From these findings, we concluded that all of 7 Mute swans died of copper poisoning.

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A serological survey was conducted on 4, 080 swine sera collected for the years 1985-90. The swine sera positive to A/New Jersey/8/76 (swine type H1Nl) strain were observed in annual (10-20%) and monthly (20-40%) incidences during the observation period except for occasional months. Antibodies to recent human HlN1 viruses in swine were recognized in relation to the human HlN1 influenza epidemics. Antibody responses of swine to human H3N2 strains appeared irrespective of human epidemics with the virus in the years 1985-87. However, in 1988 almost no antibodies to three human H3N2 isolates of 1983-88 were observed for this year except a few months though the human epidemic occurred in the area. Although in 1989-90 many swine had antibodies to the three strains in the percentage of 3 to 35, no antibody to the latest isolate, A/Hokkaido/20/89 (H3N2), was found for almost all the months of both years. These findings differed markedly from the possible relationship between the prevalence of H3N2 virus-antibodies in swine and the human influenza epidemics, which were described previously in many reports including our studies.

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Hemocytes of two marine neritid gastropods, Nerita albicilla and Heminerita japonica, were attracted chemotactically to live Vibrio parahaernolyticus and Escherichia coli strains. Chemotactic attraction of N. albicilla hemocytes was enhanced in the presence of N.albicilla plasma, while that of H.japonica hemocytes was not enhanced in the presence of H. japonica plasma. Chemotactic activity of the hemocytes seems to participate in the rapid climination of V. parahaemolyticus from these gastropods.

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An analytical method for measurement of tetrahydrofolate (THF) and N5-methyltetrahydrofolate (5MF) in pig plasma by the high performance liquid chromatography and electrochemical detection is described. The plasma sample was deproteinized by perchloric acid and the supernatant was analyzed using the following conditions; (a)phenyl bonded phase column as an analytical column; (b)mobile phase consisting of 20mM acetate buffer (pH 3.6) containing 0.1mM EDTA and acetonitrile (96.5:3.5, v/v); (c)an applied potentialof +300mV. Under the above condition, both peaks of THF and 5MF in the plasma were well separated. The detection limits of THF and jMF were 0.15 and 0.13ng/ml, respectively, at S/N=3. The recoveries of THF and 5MF from the plasma spiked with standard THF and 5MF were 77.6±2.1% and 83.0±1.7% (mean±S.D.), respectively.

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The growth-promoting activity in the culture supernatant of bovine lymphoblastoid B-cell lines (BL2M3 and BL312) were examined. BL2M3 cells proliferated well in response to conditioned medium (CM) obtained from BL2M3 and BL312 cell cultures. These BL2M3 and BL312 CM were used as sources of BL2M3 cell growth-promoting factor (BL2M3-GPF). BL2M3-GPF was sensitive to acid (pH 2) and alkali (pH 10) and was heat-labile. Proliferative responses of BL2M3 cel1s were not induced by human recombinant (r)IL 1, rIL 2, rIL 6, granulocyte-colony stimulating factor (rG-CSF) or tumor necrosis factor (rTNF)-α. Human low molecular weight B cel1-growth factor (LMW-BCGF) was, however, capable of augmenting the proliferation of BL2M3 cel1s. BL2M3 cells formed clusters in response to LMW-BCGF, whereas they showed single and discete appearance in the presence of BL2M3-GPF. These results suggested that bovine lymphoblastoid B-cell lines might release and respond to the growth-promoting factor for in vitro proliferation of its own cell line, BL2M3.

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The effects of three different types of breeding such as isolator, floor, and cage breedings on the bacterial flora of the respiratory tracts (nasal cavity, tongue, pharygolarynx, trachea and air sac) in chickens were determined. Total viable bacterial numbers on the nasal mucus of chickens in the isolator breeding as control group (Group A) aged of 14 days were 10^4.6/g of autopsy specimen (wet weight), 10^5.7/g of sample in the cage breeding (Group B) aged of 28days, and 10^7.O/g of sample in the floor breeding (Group C) aged of 28 days. Staphylococci and micrococci were predominant bacteria in the nasal cavities of all groups. Total viable numbers of tongue and pharygolarynx were from 10^5.4 to 10^6.5/g of autopsy specimen. Lactobacilli were the predominant bacteria in pharyngolarynx of chickens. The incidence of staphylococdi and micrococci in trachea was lower than those in the another regions. Staphylococci and micrococci dominated in the air sacs of two groups (B and C), but the number and incidence of lactobacilli in the air sacs of chickens were lower than those in the another respiratory tracts. The only clostridia isolation in the air sacs of Group A was observed. A total of 75 strains of Lactobacillus species was isoalted from all respiratory organs and intestine of chickens. These strains were divided into 19 groups. Lactobacillus salivearius subsp. salivarius was the predominant lactobacilli isolated from tongue and pharyngolarynx. Most of isolates from the chicken intestines were mainly identified as the L.acidophilus group and L.reutbri. These findings show that lactobacilli were Predominantly isolated from nasal cavity, tongue, and pharyngolarynx of chickens, but not from trachea and air sac and the difference at the species level of Lactobacillus is present.

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LEC strain rats (LEC rats), which have been known to develop hereditarily spontaneous fulminant hepatitis 4-5 months after birth, were highly sensitive to whole-body X-irradiation as compared to WKAH strain rats (WKAH rats). Radiation-induced acute intestinal death occurred at doses higher than 6.5 Gy in LEC rats, and at doses higher than 12.8 Gy in WKAH rats, respectively. By the probit analysis of survival data, it was shown that the LD_50/7 value of LEC rats was estimated to be 7.03 Gy which was significantly lower than that (12.99 Gy) of WKAH rats. Histopathological examinations of small intestines from LEC rats 2 days after irradiation at the dose of 8.5 Gy showed severe epithelial death together with edema, whereas little or no significant changes were noted in intestinal epithelium of 8.5 Gy-irradiated WKAH rats. These results suggest that the radiosensitivity of LEC rats to ionizing radiaton appears to be higher than that of other strains of rats.

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Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor α(rhTNFα) was examined by using mouse L929 cells derived from mouse fibroblast cel1s. The amount of DNA fragments derived from rhTNFα-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNFα caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNFα treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNFα-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNFα causes DNA-fragmentatioin but not DNA-protein cross-linking.

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The effects of several thiol compounds on the cytotoxicity induced by chromate (potassium dichromate) were examined. HeLa cells were incubated in Eagle's minimum essential medium (MEM) with or without the chromate alone, or with both chromate and any one of L-cysteine ethyl ester (LCysEE), L-cystcine methyl ester (LCysME), N-acetyl-L-(+)-cysteine, 2, 3-dimercaptosuccinic acid (DMSA), 2, 3-dimercapto-1-propanesulfonic acid (DMPS), or dithiothreitol. After a given period of incubation, the number of viable cells was counted using thc trypan blue exclusion test and the chromium content of the cells was cstimated by atomic absorption spectrophotometry. The results obtained were as follows. 1)Chromate-induced cytotoxicity evaluated by inhibition of cell growth at 3 days of incubation was diminished by all of the thiol compounds tested when thc cel1s were incubated in MEM with 2.5 to 10.0μM chromate and 25 to 100μM thiol compounds. 2)All of the thiol compounds produced a concentration-dependent reduction of chromate when a solution of the thiol compound (12.5 to 100μM) was mixed with a solution of chromate (10μM) in distilled water. 3)When cells werc incubated in MEM with both 10μM chromate and 25 to 100μM thiol compounds, the chromium content of the cells at 6 hr of incubation was decreascd in a concentration-dependent manner. 4)When these thiol compounds were added to MEM 1 hr before or after chromate, no or little protective effccts of these thiol compounds against chromate-induced cytotoxicity and chromium uptake by the cel1s were observed. The results of this study demonstrate that the thiol compounds, especially LCysEE, LCysME, DMSA, and DMPS are useful for treating chromate-induced cytotoxicity when they are given immediately after intakc of the metal and suggest that a part of this effect may be due to decrease in chromium uptake by the cells accompanying the reduction of chromate.

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The development process of a Cryptosporidium isolated in Japan in the chicken intestine was investigated by scanning (SEM) and transmission electron microscopies (TEM). The parasites were detected in the ileum; cecum, colon, cloaca and bursa of Fabricius (BF). The intensity of infection tended to peak later in the BF than ileum.Trophozoites and schizonts were detected in all the portions of intestine, and were dominant in the developmental stages. Although macrogamonts were the secondary dominant stage, they were absent in the ileum and cecum at 60 hr postinoculation (PI). A few microgamonts were detected in the ileum at 36 hr PI and in the BF on day 19 PI. Oocysts were observed in the ileum at 48 hr PI and in the BF on day 19 PI.

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The mechanism of immunosuppression induced by leukemic bovine serum was investigated with respect to lymphokine reactions using an interleukin 2 (IL-2)-dependent bovine T cell line generated from bovine peripheral blood lymphocytes (PBLs). The suppression of concanavalin A (con A)-induced PBL blastogenesis was observed at a high rate in leukemic cattle sera. The growth of IL-2-dependent bovine T cells and IL-2 production from con A-induced bovine PBLs were also inhibited by these sera, and particularly, the latter was correlated significantly to the degree of lymphocyte blastogenesis by the mitogen. Therefore, the lesser sensitivity of lymphocytes to IL-2 and the reduced IL-2 production by activated lymphocytes seem to play a role in suppressing the lymphocyte reaction.

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Changes in the intracellular and cell surface localization of Lewis x (Le^x) determinants in germ cells during fetal development in mice were examined by light and electron microscopy. Light microscopically, in undifferentiated gonads on day 12 post coitum (p.c.), the anti-Le^x monoclonal antibody (MAb) was specifically bound to the plasma membranes and to the cytoplasmic granule-like structures of germ cells. In the testes on day 13 p.c., most of the germ cells were enclosed within the testicular cords and showed an MAb-positive reaction which was restricted mainly to the cytoplasmic granule-like structures. The reaction on the plasma membranes almost disappeared. On the other hand, the ectopic germ cells still showed a positive reaction on their plasma membranes. In the ovaries on day 13 p.c., the germ cells also exhibited positive reactions both on the plasma membranes and on the granule-like structures. Immunoelectron microscopic observations agreed well with these light microscopic observations in such a way that both the plasma membranes and the "small dense bodies" (SDB) were positive in undifferentiated gonads on day 12 p.c. In the germ cells organized into the testicular cords, the reaction to anti-Le^x MAb became restricted to the SDB. These results may indicate that such intracellular changes in Le^x determinants during germ cell differentiation are associated with the enclosure of germ cells within the testicular cords.

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cDNA libraries of Theileria sergenti and T. buffeli piroplasms were constructed in λ gt11 and screened with rabbit anti-piroplasm sera. A major antigen of T. sergenti (33 kDa) and that of T. buffeli (34 kDa) was identified from the recombinant phages by using recombinant antigen-selected monospecific antibodies. The reactivities of the cloned proteins with rabbit antisera, infected calf sera and mouse monoclonal antibody suggested that the 33 and 34 kDa proteins expressed species-common and species-specific epitopes. The DNA probes from these recombinant clones showed species-specific hybridizatioins in Southern blotting with genomic DNA from piroplasms. These results indicate that the Japanese T. sergenti can be distinguishable from the Australian T. buffeli with regard to a polymorphism of the major immunodominant proteins of piroplasm.

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Porcine epidemic diarrhea virus (PEDV) was isolated in Vero cell cultures from the small intestine of a piglet experimentally infected with porcine coronavirus 83P-5, that had been isolated during outbreaks of porcine acute diarrhea and passaged in piglets. The isolation of the PEDV was successful only in Vero cells maintained in the maintenance medium (MM) containing trypsin. Infected Vero cell cultures exhibited CPE characterized by cell-fusion and syncytial formation, as well as cytoplasmic fluorescence when examined by the indirect immunofluorescent test using rabbit anti-83P-5 virus serum. The isolate was adapted to serial propagation in Vero cell cultures by adding trypsin to MM. Vero cell-adapted PEDV was successfully propagated in the MA104, CPK and ESK cell lines in the presence of trypsin in MM. Vero cell-adapted PEDV had morphologic and physicochemical characteristics similar to those of other members of the coronaviridae. The isolate differed serologically from porcine transmissible gastroenteritis (TGE) and porcine hemagglutinating encephalomyelitis viruses, and no antigenic relationship between the isolate and TGE virus could be detected by the indirect immunofluorescent test. Attempts to isolate PEDV in 6 types of primary fetal pig cell cultures and 6 of 10 established cell lines resulted in the failure, probably because these cells were damaged by the action of trypsin.

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Morphological profiles of porcine granulosa cells incubated with 10 mM Tris-HCl containing 1 M urea and 5mM EDTA (urea-EDTA solution) were investigated. The percentages of granulosa cells incorporating the dye, trypan blue on incubation with urea-EDTA solution did not change during the initial 30 min. Thereafter, granulosa cells gradually took up the dye throughout the incubation. The amount of protein released from granulosa cells increased dramatically during initial 15 min of incubation, but decreased during the following 15-30 min of incubation. Thereafter, the amount of proteins released from granulosa cells increased gradually again. The releasing profile of ⁵¹Cr-bounded substances in granulosa cells increased markedly during the initial 15 min of incubation, and decreased during the next 15 min of incubation. Subsequently the amount of ⁵¹Cr released was enhanced. The plasma membranes of granulosa cells remained intact at 30 min of incubation, although chromatin clusters of granulosa cells disappeared. Thereafter, a number of cells showed signs of degeneration, including broken plasma membrane and cytolysis. The present study revealed that urea-EDTA solution is useful in extracting materials from porcine granulosa cells. The majority of the materials extracted from granulosa cells during the initial 30 min of incubation with urea-EDTA solution is considered to be from the cell surfaces and/or intercellular matrix.

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To define the antigenic relatedness among unspeciated ureaplasma strains isolated from dogs, four canine ureaplasma isolates representing four serotypes were compared with the five type strains of the established species in Genus Ureaplasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Although all the strains showed distinct electrophoretic patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the Western blotting patterns were much more distinct. By Western blotting, the five type strains of established species reacted strongly with homologous antisera and showed slight cross reactions with heterologous antisera. However canine strains which showed little cross reactions with the established Ureaplasma species showed a variety of cross reactions among the four canine serotypes used.

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A case of aggressive fibromatosis (extra-abdominal desmoid) found in a 9-month-old male mixed breed cat is described. The right forearm was almost completely effaced by fibrous tissue and there were some tumours in the area from the shoulders to the mandible. These tumour-like tissues were composed of abundant collagen fibres and sparse numbers of well-differentiated fibroblasts, whereas their growing pattern was aggressive and non-encapsulated. There was dense growth of immature fibroblasts and multinucleated giant cells in some areas. Vimentin immunohistochemistry and electron microscopy suggested that the giant cells had close relation to the fibroblasts, and such areas may be the sites of cell prolifelation. This case is different from nodular fasciitis and may be a proliferative disorder induced by feline oncogenic retrovirus.

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Zona-intact and zona-free mouse embryos at the morula stage were exposed to Sendai virus in vitro, and then transferred to the uteri of recipient foster mothers. Both embryos developed into expanded blastocyst stage after 48 hr of culture. Zona-intact embryos were resistant to infection and subsequent transfer resulted in the development of fetuses, indicating that the zona pellucida plays a role of a barrier to virus infection. On the other hand, zona-free embryos were susceptible to infection and only one fetus out of 64 transfers developed to term. Implantation sites were scarcely observed in the uteri of the foster mothers that received zona-free embryos, suggesting that most of the embryos did not develop after embryo transfer. Sendai virus was shed in the culture fluid of the zona-free embryos indicating viral replication in the embryonic cells. By immunofluorescence assay, viral antigens were detected in the embryos, tissues of the fetus and implantation site derived from the zona-free embryos. These findings indicate that replication of Sendai virus in the embryonic cells interfere with early embryonic development and fetal growth of the embryo.

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