Partially purified vascular permeability (VP) factor (VPF) of Bacillus cereus induced fluid accumulation in the ligated intestinal loops of mouse (MIL) and rabbit (RIL), suggesting that the VP activity may correlate with fluid accumulation in ligated intestinal loops of these animals. Fluid accumulation was observed at 6-8 hr in 55-67% of mouse intestinal loops inoculated with 4-50 immunodiffusion units (IDU) of partially purified VPF, whereas it was found at 2hr in all loops with 400-600 IDU of partially purified VPF. In rabbit intestinal loops with 120-190 IDU of partially purified VPF, fluid accumulation was observed at 6 hr. From these findings, VPF produced by B. cereus can be easily detected in both MIL and RIL. The intestinal tissue of mouse intestinal loops was histopathologically damaged at different concentrations of the VPF to induce fluid accumulation. With 50 IDU of partially purified VPF, severe edema was found in the laminia proprial layer and submucosa. With 600 IDU of partially purified VPF, on the other hand, severe necrosis in the surface epithelium of villus and laminia proprial layer, and hyperemia in the submucosa were observed, suggesting that partially purified VPF may be cytotoxic and/or intestinecrotic.
Monoclonal antibodies were established for antigenic analysis of feline and bovine Chlamydia psittaci. The monoclonal antibodies recognized lipopolysaccharide (LPS), 56-64, 84 or 86 kDa antigens. At least 5 antibody-binding sites were detected on LPS with the monoclonal antibodies. The 56-64 kDa antigen was suggested to have both polypeptide and carbohydrate antibody binding sites. Immunoblotting analysis of cat and cattle sera indicated that the 56-64 kDa antigen is an important antigen in host immune response. The monoclonal antibodies are extremely useful tools to analyse the structure and function of chlamydial antigens.
Clinical and endocrinological responses to administration of gonadotropin releasing hormone analog (LH-RH-A) during the lactation period and postweaning in the sow were investigated. Plasma LH concentrations in lactating sows rose immediately after administration of LH-RH-A. However, in postweaning sows the increase of LH level was more slowly. Three of 5 postweaning sows came into estrus and ovulated after LH-RH-A treatment. One sow exhibited a distinct LH response, but her ovaries remained quiescent. The remaining one with feeble estrus for a short period became cystic ovaries. Thus, LH response to GnRH in the sow seems to be higher during early lactation than at 2 days postweaning.
Binding specificities of calcium-dependent and -independent bovine IgM and IgG reactive with unsubstituted Sepharose 4B were determined by competitive binding assays. The binding of 125I-labeled calcium-dependent bovine IgM to unsubstituted Sepharose 4B was most effectively inhibited by lactose which was about 100 times more reactive than galactose and melibiose. Other inhibitors were much less potent. With 125I-labeled calcium-independent bovine IgM and IgG, on the other hand, lactose was as potent as galactose. Melibiose was about 10 times less potent than lactose and galactose, whereas other mono- and disaccharides were much less potent. From these findings, the combining site of calcium-dependent bovine IgM reactive with unsubstituted Sepharose 4B may be specific for lactose, whereas those of calcium-independent bovine IgM and IgG specific for galactose.
Six sheep with lymphosarcoma induced by hypodermic inoculation of bovine leukemia virus (BLV) materials were examined to elucidate the relation between pathologic lesions and integration of BLV provirus in cellular DNAs. Antibodies to BLV gp-antigens had been detected since the 3rd week after the inoculation, and BLV was positive when checked 3 months later. Lymphosarcomas followed the leukemic phase in 4 sheep. The other 2 sheep showed initial lesions of lymphosarcoma and were aleukemic clinically. Five animals were killed by enthanasia and autopsied at 2.5 to 3.5 years postinoculation (pi) because of their diseased condition. One animal died 10 years pi following the 4th leukemic episode. Sarcomatous lesions were confirmed grossly and histologically, and the proliferating neoplastic cells were classified into lymphocytic, prolymphocytic, lymphoblastic and histiocytic types. Integration of BLV provirus in cellular DNAs of the peripheral blood lymphocytes (PBL) and neoplastic cells of sarcomatous lesions was examined by Southern blotting technique. BLV provirus was demonstrated in the PBL of all infected animals and in most of the sarcomatous lesions of the spleen, kidney and lymph nodes except 4 lymph nodes showing slight neopalstic infiltration. The results indicated that ovine lymphosarcoma could be caused by BLV and the cells carrying proviral information seemed to be disseminated and proliferate in the lesions.
Attachment of Vibrio parahaemolyticus strains to estuarine microalgae was examined in artificial seawater by viable counts of the organism and direct counts of the bacterial cells after immunoperoxidase staining. Thermostable direct hemolysin (TDH)-producing and TDH-non-producing strains of V. parahaemolyticus were found to attach to five estuarine strains of Navicula (diatom alga) in similar levels. The level of the bacterial attachment depended on salinity and temperature of the water, in which the maximum attachment was observed in 15%o artificial seawater at 25°C, a typical condition of Hashizu estuary in Japan during summer months. The attachment was inhibited by pectinase digestion of the algal cells. These evidences confirmed the participation of the microalgae to the ecological cycle of V. parahaemolyticus at the estuary.
The localization of α, βA and βB subunits of inhibin/activin polypeptides was studied in the ovary and testis of sexually mature, immature, and embryonic rats. Specific staining with these three subunits was also evident in the oocytes from embryonic to mature female rats. This result suggests that inhibin- and activin-like substances may be produced in the oocytes and these substances may play a role in the oocyte growth and differentiation. Judging from the intensity of immunoreaction in mature female rats, the three subunits should be produced more abundantly in luteal cells than in the granulosa cells. Immunoreactive α, βA and βB subunits were observed in the cummulus oophorous in the morning (11:00), but not in the evening (23:00) on proestrus. The results are in well agreement with the previous report that inhibin α and βA subunit mRNA signals decline on proestrus evening. It is supposed that the cyclic change may be related with physiological phenomena prior to the ovulation, such as primary gonadotropin surges, loss of cummulus-oocyte gap junctions, or germinal vesicle breakdown. In both germ cells and Sertoli cells of the testis, α, βA and βB subunits were more abundant in the embryonic rat than in the mature rat. Although clear reactions with βA and βB subunits were detected in Leydig cells, α subunit was not detectable in the cells throughout the developmental stages examined.
Cleavage of Oncorhynchus masou virus (OMV) DNA with restriction endonucleases BamHI, EcoRI, and XhoI resulted in 28, 26 and 17 fragments, respectively. Based on the molecular weights of digested fragments and those molar ratio, OMV DNA showed the molecular weight of about 100×106. Twenty out of 28 BamHI fragments of OMV DNA were successfully cloned into pBR322 vector. Restriction map of OMV DNA was constructed by blotting hybridization and double-digestion. The data suggested that terminal repeat of the end fragments of OMV DNA molecule was not existence.
From 1985 to 1989, a total of 129 mice was captured from 7 piers of Yokohama port. Of these, 9 (7.0%) were positive to lymphocytic choriomeningitis virus (LCMV) antigen in indirect fluorescence antibody test. Six out of 31 mice (19.4%) in 1985 and 3 out of 23 mice (13.0%) captured in 1986 were positive. All the mice (74) captured in 1988 and 1989 were negative. Although 7 out of 17 mice (41.2%) in Osanbashi-Shinko pier and 2 out of 23 (8.7%) in Honmoku pier were positive in 1985 and 1986, all mice captured in other piers were negative. This is the first report detecting LCMV antibody in wild house mice in Japan.
Three major fragments were generated by limited digestion of staphylococcal enterotoxins A (SEA) and E (SEE) with papain, whereas five major fragments were generated by limited digestion with staphylococcal protease V8 (V8). All of these fragments were detected by immunoblotting with polyclonal anti-SEA and -SEE sera. Some of generated fragments were detected by monoclonal antibodies (MAbs) with specificities for SEA (A-111 and A-211), SEE (E-142), or both (AE-32, AE-37, and AE-53). This indicates that fragments of SEA and SEE containing the type-specific and cross-reacting epitopes may be generated by digestion of the toxins with either papain or V8.
To test the interaction between various species of bacteria and Salmonella serovar typhimurium (S. typhimurium), the population of S. typhimurium was measured in the cecum of gnotobiotic chickens in the presence of Escherichia coli (E. coli) and one of the four intestinal bacteria; Lactobacillus acidophilus, Clostridium perfringens, Bifidobacterium thermophilum and Bacteroides vulgatus. Competitive exclusion of S. typhimurium by di-flora chicken was not demonstrated. But the population of S. typhimurium was temporarily suppressed in di-flora chickens with E. coli and L. acidophilus. In penta-flora chickens with E. coli and these four intestinal bacteria, the population of S. typhimurium was suppressed for only 2 days. In normalized chickens, the population of S. typhimurium was markedly suppressed.
The morphological features of the testicular artery and the pampiniform plexus in the boar spermatic cord were evaluated by light microscopy, corrosion cast technique combined with scanning electron microscopy and by transmission electron microscopy. The testicular veins could be subdivided into 4 types according to their perivascular elements and their location to the testicular artery. Type I vein consisted of large veins and gave rise to type II and III veins. Type II vein was composed of a single layer of veins and ran along the testicular artery, while type III vein consisted of several layers of veins and was located between type II veins. Type IV vein, which was regarded as a venous portal system, was composed of small muscular and pericytic venules and was located in the tunica adventitia of type II vein and the testicular artery. Occasionally, type IV vein penetrated deep into the tunica media of the testicular artery, accompanied by a fenestrated endothelium in its thin portion. The direct arterio-venous anastomosis between the pampiniform plexus and the testicular artery was not found. Type IV vein was considered to be the most important vessel in reducing the capacity of the barrier between the testicular artery and the veins (type II and III). It is, therefore, suggested that type IV venous network may play a role in transferring the substances between the artery and the veins.
Development of the testis, epididymis and prostate in 53 male beagles was examined histologically with PAS-hematoxylin stain from birth to sexual maturity. The diameter of the seminiferous tubules of the testes was less than 100μm until 20 weeks of age, however, it increased markedly between 22 and 28 week of age, reaching 180±7 (mean±SD) μm at 28 weeks of age. Only Sertoli cells and gonocytes (or spermatogonia) were detected in the seminiferous tubules until 16 weeks of age. Spermatocytes and spermatids appeared in the tubules at 20 and 22 weeks of age, respectively. Spermatozoa were first observed in the testes of 2 of the 5 dogs at 26 weeks of age and were found in the testes of all the 3 dogs at 28 weeks of age. The diameter of the ducts in the cauda epididymidis was 146±4 μm at 20 weeks of age. Thereafter it increased markedly, reaching 341±14μm at 28 weeks of age. The height of the epithelium and stereocilia in the ducts of the caput epididymidis increased markedly at about 28 weeks of age. A large number of spermatozoa was seen in the lumens of the ducts of the corpus and cauda epididymidis after 32 weeks of age. The shape of the lumens in the glandular alveoli of the prostate became irregular as a result of projection of the glandular epithelium into the enlarged lumens and the epithelial cells of the alveoli became PAS-positive at 24 weeks of age. The projection of the glandular epithelium into the lumen became more marked after 32 weeks of age. Thus it was demonstrated that histological development of the testis, epididymis and prostate of the dog is most marked between 20 and 32 weeks of age.
In order to study the regulation of food intake and correlated body energy metabolism, the effect of restricted feeding during the light period in female IVCS mice was investigated. Access to food and water was restricted (RF group) for 3 weeks only from 10:00 hr to 17:00 hr, and that in the control group remained ad libitum. After starting food restriction (day 1), mean food intake decreased to 10% of the control value, then rose sharply, over the next 3 days, to reach 70% of the control value. Then, it decreased gradually to about 50% of the control value and remained at this low level thereafter. There was no significant difference between mean body weights for the two groups. Feed-efficiency was considered, therefore, to be higher in the RF than in the control group. RF-treatment increased plasma corticosterone levels and decreased locomotor activity. However, the diurnal patterns of plasma corticosterone levels and locomotor activity observed suggest that the circadian rhythm, synchronized with the light-dark cycle, persisted during RF-treatment. These findings suggest that restricted feeding during the naturally inactive phase (light period) induces a decrease in food intake. Animals seem to adapt to underfeeding, at least partly, by increasing feed-efficiency and plasma corticosterone levels and decreasing locomotor activity.
The inhibitory effects of a prescription of herbal medicine, tentatively named P-3, were studied pathologically in an experimental model of the glomerular lesion induced by purified snake Agkistrodon acutus venom proteinase (Ac1-P) in mice. Ac1-P was intravenously inoculated at a single LD50 dose. In the treated group, mice were intraperitoneally injected with an extract of P-3 at a designated time once every two days from 2 days before to 1 week after Ac1-P inoculation. The control group mice were injected with saline instead of P-3. In the control group, the main pathologic changes within 48 hrs after inoculation were pulmonary and gastrointestinal tract hemorrhage and renal petechiae with hematuria. The kidney microscopically showed cystic transformation of the glomerular capillary tufts, followed by occlusive thrombosis. One week after inoculation, the glomerular lesions were mostly replaced by proliferative or proliferative-sclerosing changes with occasional crescent formation. Early signs of tubular atrophy accompanying the glomerular changes were observed. In the P-3 treated mice surviving 48 hrs and 1 week, the changes observed in the controls were markedly inhibited, although P-3 treated mice dying earlier than 30 hrs exhibited hemorrhagic changes similar to controls. This indicated that the herbal medicine had efficacy against the tissue injuries induced by Ac1-P as a proteolytic enzyme.
Prevalences of thermostable direct hemolysin (TDH)-producing strains in communities of a gastropod mollusc, Clithon retropictus, and a bivalve mollusc, Corbicula japonica, and levels of the strains in attached microalgae and muddy sediments were investigated at a brackish-water area along Hashizu Creek and Togo Pond in Japan. V. parahaemolyticus was detected from attached microalgae at Hashizu Creek in summer months with the highest level of 1.4×105 cfu/g. Levels of the organism among 20 animals of C. retropictus and C. japonica at the area varied betwen non-detectable level and 103 per mollusc in summer months. TDH was detected from culture supernatants of 11-16% of strains isolated from the algae, sediments and C. japonica and 28% of those isolated from C. retropictus at Hashizu Creek. These evidences suggest that C. retropictus would get TDH-positive strains from the algae.
A newly cloned serotype 2 Marek's disease virus (MDV), strain ML-6, was inoculated via the nasal cavity in specific-pathogen-free chicks to examine early virus replication and the expression of Marek's disease (MD)-related antigens. Following inoculation, viral intracellular antigens (VIAs) were detected in lymphoid organs (bursas and spleens) between 5 and 14 days post inoculation (PI), in feather follicles between 14 and 30 days PI, and in lungs at 3 days PI by the immunohistopathological staining of avidin-biotin-peroxidase complex method. But, very few VIAs were expressed in the thymuses between 5 and 14 days PI. However, MD tumor-associated surface antigens were not detected in any organs. Viruses were isolated from separated spleen cells at 14 and 30 days PI. Fluorescent antibodies of convalescent sera were also detected after 10 days PI. AS most of the VIAs were detectable in B-cells in bursas and spleens, B-cells were considered to be the main first target cells for the serotype 2 MDV infection.
BALB/cA, DBA/2N and CDF1 (BALB/cA x DBA/2N) mice were inoculated intranasally with the Mol strain of Sendai virus (SV), and their mortality and histopathological lung lesions were compared. BALB/cA and CDF1 were resistant and DBA/2N was susceptible in terms of mortality. The lung lesions of the resistant strains were mild and focal, and limited to the bronchial regions, whereas those of the susceptible stain were severe and diffuse, extending to the alveoli. SV antigen was found mainly in the bronchial epithelium in the resistant strains, but in the susceptible strain, the antigen was found also in many alveolar epithelial cells and alveolar macrophages. SV antigen was detected in neither regenerated bronchial epithelium nor endothelial cells. Tumor necrosis factor (TNF) was detected immunohistologically in edematous perivascular regions and in some mononuclear cells infiltrating to the regions, suggesting that TNF is involved in the development of lung lesions by SV infection in the three mouse strains.
Purification of an extracellular protein exhibiting the vascular permeability activity produced by Bacillus cereus was performed by ammonium sulfate precipitation followed by chromatography on DE-32 cellulose, Sephadex G-100, and Sephadex G-75. The purified protein was found to be electrophoretically and antigenically almost homogeneous although it contained a trace of contaminant. The molecular weight of the protein was calculated to be 45, 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified protein showed vascular permeability activity and mouse lethal toxicity, and caused fluid accumulation in ligated mouse intestinal loops, whereas it did not show any hemolytic and lecithinase activities. From these findings, the purified protein is suggested to be an enterotoxin (or a diarrheagenic toxin) responsible for diarrhea caused by B. cereus in a diarrheal-type food poisoning.
A total of 691 normal embryos were recovered from 183 superovulated donor cows on the 5th and 6th days after the first insemination, and were examined for their morphology and size in relation to their developmental stage. There was no significant difference in the thickness of the zona pellucida, the diameter of the cell mass, and the overall diameter of the embryos among zygotes, 2-, 4-, 8- and 16-cell embryos, and morulae. In the blastocyst stage, however, the diameter of the cell mass and the overall embryo diameter were significantly greater and the zona pellucida was significantly thinner than in the earlier-stage embryos. The volume of the blastomere significantly decreased from zygote to morula in proportion to the increase in the number of blastomeres. The volume of the cell mass of 2-cell embryos was decreased by about 30% compared with that of zygotes and no increase in the volume of the cell mass was observed during the progression from 2-cell stage to morula. The diameter of the cell mass and the overall diameter of morulae recovered on the 6th day after the first insemination were significantly greater than those of morulae recovered on the 5th day.
Spontaneous amelanotic melanocytic tumors of the pinna were found in six females of 960 male and 960 female albino (F344/DuCrj) rats which had been used in three different 24-month chronic toxicity studies. The age when the pinnal tumors were detected ranged from 37 to 59 weeks. The tumors were located unilaterally in the pinna and observed as subcutaneous spherical to irregular, solid white masses measuring 7 to 25 mm in diameter. The pinnal tumors were histologically classified into spindle cell and pleomorphic cell types. The spindle cell type was observed in four rats and composed of fusiform cells arranged in interlacing bundles. The pleomorphic cell type was observed in the remaining two rats and composed of pleomorphic large cells arranged in sheets. One tumor of the latter type metastasized to the submaxillary lymph node and lung. Melanin pigments were not demonstrated in any of the tumors. In immunohistochemistry, nuclei and cytoplasm of tumor cells in all the tumors were slightly positive for S-100 protein. Ultrastructurally, tumor cells contained a considerable number of premelanosomes in the cytoplasm. Desmosomes were occasionally observed between the cell membranes of the adjacent tumor cells. No distinct basal lamina was seen around tumor cells.
Vibrio parahaemolyticus D-3 was observed to attach to hemocytes of a marine gastropod mollusc, Nerita albicilla, regardless of the presence of N. albicilla serum. The organism attached to hemocytes of an estuarine gastropod, Clithon retropictus, in the presence of C. retropictus serum while the attachment to the hemocytes was decreased significantly in the absence of the serum. These evidences suggest that N. albicilla hemocytes would facilitate the clearance of V. parahaemolyticus from the alimentary tract of the mollusc and that C. retropictus hemocyte would protect C. retropictus against the invasion of V. parahaemolyticus to hemocoel of the mollusc.
Visual evoked potentials (VEPs) recorded from the scalp in guinea pigs were compared with those from the dura. The study was performed with ten adult male guinea pigs weighing 350-750g. VEPs recorded from the scalp had large negative components (N40 and N75) and d large positive component (P6<55>). The waveform of the VEP in the scalp recording was similar to that in the dural recording in that N40 was a major early negative component. Great differences between the scalp and the dural recording were observed in the late negative components (N6<75> and N140). In the dural recording, the peak N75 was a very small component, and the peak N140 was very large. There was no significant difference between the peak latencies of the two kinds of VEPs except for the peaks P55 and P100. Peak-to-peak amplitudes of VEPs recorded from the scalp were smaller than those from the dura except for P55-N75. The peak-to-peak amplitude in the scalp recording compared to that in the dural recording varied from a ratio as low as 1.0:2.9 to as high as 1.0:36.2, and was markedly variable in each component. The scalp recording correlated with the dural recording as regards the early component.
To establish the usefulness of the bovine clinical renal function tests, experimental glomerulonephritis was experimentally induced in calves and some renal clearance tests were performed. Two (No. 1, 2) of three calves were injected intravenously with anti-bovine kidney rabbit serum (antiserum) and the other (No. 3) with normal rabbit serum (control serum). The early stage of proliferative glomerulonephritis was observed in the kidneys of Nos. 1 and 2. The degree of lesions in No. 1 was severer than that in No. 2. No remarkable change was observed in the kidneys of No. 3. Endogenous creatinine clearance value (CCRE), thiosulfate clearance value (CTHIO) and maximal tubular secretion of para-amino hippuric acid (TmPAH) of all calves did not show remarkable changes after the injection of antiserum or control serum. In the phenolsulfonphthalein (PSP) test, PSP excretion of Nos. 1 and 2 was disposed to delay after the injection, and in No. 3 there was no significant change after the injection. PAH clearance value (CPAH) of No. 1 decreased from 10.24 to 6.96 ml/min/kg (-32%). A small change was noted in the CPAH of Nos. 2 and 3. These results suggest that the simplified method for measuring CPAH performed in this study could assess the lesions formed in No. 1, which was pathologically diagnosed as the early stage of proliferative glomerulonephritis.