To clarify the features of the seminiferous epithelium of the Japanese rat snake, Elaphe climacophora, the identification of spermatogonium and the examination of features of cell to cell junctions were performed in the present study. As for the identification, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined immunohistochemically to mark spermatogonia. The seminiferous epithelium was observed throughout a year at the electron microscopic level. BrdU immunoreactivity was detected not only in the cells of the first layer of the seminiferous epithelium but also in the second and/or third layers. The cells immunoreactive in the first layer did not seem to attach to the basement membrane and were recognized throughout a year. To investigate cell to cell junctions, we performed actin filament detection by phalloidin staining. Distribution of actin filaments was different from that in mammalian species. At the ultrastructural level, Sertoli-Sertoli cell junctions were observed. Sertoli cells formed junctional complexes. Tight junctions were clearly found, but lacked the backing by actin filaments. These results indicate that the blood-testis barrier of the Japanese rat snakes was structurally different from that of mammalian species. In conclusion, the seminiferous epithelium of the Japanese rat snake is intermediate in morphology between amphibians and mammals.
The role of Bordetella bronchiseptica sialic acid-binding hemagglutinin (SBHA) in bacterial colonization was studied by testing the ability of a phase-I strain carrying SBHA and its spontaneous mutants deficient in SBHA to colonize mouse tracheae. The mutants exhibited a remarkable reduction in colonizing ability, approximately 1,000-fold, when compared with that of the parent strain, suggesting that the possession of SBHA and the effective colonization by B. bronchiseptica organisms are closely related.
In efficacy tests, 7 primary specific-pathogen-free piglets vaccinated with the Bordetella bronchiseptica and type D Pasteurella multocida bacterin-toxoid were challenged with B. bronchiseptica and type A P. multocida. Severe or moderate nasal turbinate atrophy was produced in the non-vaccinated pigs, whereas, only one of the 4 pigs in the vaccinated group had slight turbinate atrophy. Other immune sera against crude toxin of P. multocida type A or D were cross neutralized. The results of the present study show that the P. multocida serotype D bacterin-toxoid is effective against atrophic rhinitis caused by toxigenic P. multocida serotype A as well as toxigenic P. multocida serotype D.
Bacterial isolation from slaughtered pigs with endocarditis was carried out from 1985 to 1994. A total of 495 (0.025%) out of 2,006,127 pigs were diagnosed as having endocarditis. Though bacteria were significantly isolated from 399 of the 495 pigs, bacteria could not be isolated in 96 pigs (19.4%). In 11 pigs, 2 bacterial species were isolated from heart lesion. Streptococcus suis was isolated from 127 cases (25.7%), Streptococcus dysgalactiae from 75 (15.2%), Erysipelothrix rhusiopathiae from 63 (12.7%), Actinomyces pyogenes from 39 (7.9%), Pasteurella multocida from 11 (2.2%), Staphylococcus aureus from 10 (2.0%), and Streptococcus porcinus from 8 (1.6%). Among the 99 isolates biochemically identified as S. suis, the major serotype was S. suis type 2 (35.4%). The remainder of the typable isolates were identified as serotypes 1/2 (2.0%) and 9 (1.0%). A total of 61 isolates (61.6%) were untypable.
To estimate canine interleukin-8 (cIL-8) levels in blood plasma samples, a sandwich enzyme linked immunosorbent assay (ELISA) was established. For the development of the sandwich ELISA, polyclonal anti-cIL-8 (capturing), biotinylated anti-cIL-8 (developing) antibodies and glutathione-S-transferase/cIL-8 (GST/cIL-8) fusion protein as an antigen were used. cIL-8 in the fusion protein of GST/cIL-8 was detected in a dose dependent manner. The lowest limit of GST/cIL-8 detectable by this method was 2 ng/ml of GST/cIL-8 (containing; 0.470 ng/ml of cIL-8). IL-8 levels in the plasma samples from apparently healthy dogs were less than 0.470 ng/ml. Higher levels of IL-8 were detected in the plasma samples of dogs with cystitis, dermatitis, and gastric cancer. These results suggest that the determination of cIL-8 by the sandwich ELISA is useful in diagnosis of inflammatory diseases in dogs.
This paper deals with blood levels of calcium (Ca), inorganic phosphorus, parathyroid hormone and 1,25-dihydroxyvitamin D in 6 cows treated for milk fever. Four of the cows stood within 1 day after Ca therapy, whereas 2 other cases showed an unsatisfactory response to Ca therapy and did not rise. The necropsy revealed microscopic necrotic myocardial lesions scattered in the heart of these 2 unrecovered cows. The degree of hypocalcemia and hypophosphatemia were similar in the 6 cows. However, the recovery from hypophosphatemia was markedly delayed in the cows with an unsatisfactory response.
Our seven-storey laboratory animal facility was damaged during the 1995 earthquake of magnitude 7.2. The seismic intensity experienced at our facility was estimated to be around 5 on the Japanese version of the Richter scale (a very strong shock). However, no damage was sustained to the water, gas or electricity supplies. In many of the offices, the upper drawers of the steel cabinets fell to the floor. In the animal-rooms, while the floor-fixed cage-racks were hardly damaged, most of the movable cage-racks were dislodged by the shocks. We describe here our countermeasures, including those taken prior to the earthquake.
After UV-irradiation, no difference in the survival curves was observed among cell lines derived from LEC strain (LEC) rats and WKAH strain (WKAH) rats. The dose-response curves for inhibition of DNA synthesis in WKAH-derived cells showed a sharp decline at lower doses and a mild decline at higher doses of UV-rays. In contrast, the dose-response curves in LEC-derived cell lines had no sharp component, and were almost identical to the mild component of the curves in WKAH-derived cells. These results show that DNA synthesis in the cell lines of LEC rats was more resistant to UV-irradiation than that of WKAH rats.
Nucleotide sequence analysis within the unique long segment of the infectious bovine rhinotracheitis virus (IBRV) genome identified an open reading frame of 1461 base pairs whose deduced polypeptide of 487 amino acids exhibited homology to alkaline deoxyribonucleases of other herpesviruses. To determine whether this IBRV gene product has nuclease activity, the gene designated UL12 was inserted into the vector pET-28a(+) and expressed in Escherichia coli as an oligohistidine-tagged protein. Upon induction with isopropyl β-D-thiogalactopyranoside E. coli BL21 (DE3)[pLysS] cells harboring this recombinant plasmid produced a 57-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using metal affinity chromatography resulted in a homogeneous preparation of this recombinant protein. The purified protein exhibited both exonuclease and endonuclease activities, each with an alkaline pH optimum.
We constructed a cDNA clone of canine distemper virus (CDV) encoding an entire nucleoprotein (NP) gene, by means of the reverse transcription-polymerase chain reaction (RT-PCR). The cloned NP gene was inserted into the eucaryotic expression vector, pRVSV. After transfection of the plasmid into Vero cells, we examined the expression of CDV-specific NP antigen by means of indirect immunofluorescence assay (IFA) and Western blotting, using various antibodies against NP of CDV and an antiserum against NP of measles virus. The CDV-NP specific antigen was detected in the nuclei of the cells transfected with pRV-ON, by means of IFA with antibodies specific to the NP.
Theileria parva parasites have been isolated from different locations in Zambia where malignant theileriosis has been recorded. A total of 16 bovine lymphocytic cell lines infected with T. parva schizonts were characterized using a panel of anti-schizont monoclonal antibodies (MAbs). Comparison of the Theileria stocks isolated before (old) and after (new) the Muguga cocktail of T. parva from Kenya was used to vaccinate cattle against theileriosis in Zambia revealed differences in their reactivity against MAbs. The new isolates are showing MAb profiles similar to that exhibited by the Muguga cocktail which was used to vaccinate cattle in these areas between 1983 and 1989. These results suggest that the use of the Muguga cocktail to vaccinate animals against theileriosis in Zambia may have introduced Theileria stocks of different antigenic properties.
Toxocara spp. eggs were detected from 30 (75%) out of 40 sandpits of parks, in Osaka city. This prevalence was higher compared to those reported other areas of Japan. Since we examined a large quantity of sample, this could have resulted to higher prevalence. The number of eggs recovered decreased following fence construction around sandpits, but it did not sufficiently prevent the contamination of eggs. Improvement of fence design and education of sandpit users are necessary when contemplating fence construction around sandpits as a measure against contamination with Toxocara spp. eggs.
Fusarenon-X (FX) 1.5 mg/kg was administered intraperitoneally (ip) to 6-week-old male Wistar rats for examination of pathologic effects on the glandular stomach. Rats ip-treated with sterilized physiological saline were used as control. FX-administered rats showed dilatation of the stomach with increased fluid contents after 1-4 hr. Light microscopically, a few apoptotic karyopyknosis were seen in chief cells in the basal region at 1 hr postadministration (PA) and mitotic inhibition was evident after 2 hr PA. Marked apoptosis of nuclear pyknosis and cytoplasmic inclusions in both zymogenic and oxyntic cells developed from basal to middle regions of the gastric mucous membrane at 2-4 hr PA with a peak at 3 hr. Apoptotic changes of differentiating neck cells and surface epithelia were less evident. Electron microscopy revealed that the chief cells were the main target of FX-induced apoptotosis. The parietal cells were secondarily involved because they phagocytosed chief cell-derived apoptotic bodies. In situ detection of DNA breaks by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction revealed the positive nuclei after 1 hr PA, which increased with time and reached a peak at 3 hr PA, in accordance with apoptotic changes in histological study. Agarose gel electrophoresis of DNA isolated from the gastric mucosae of FX-administered rats showed ladder pattern of DNA fragments after 1.5 hr PA with the maximum distinctness at 3 hr PA.
Taste signal transduction occurs in the microvillous membrane of taste cells. Previously, we hypothesized that c-GMP may mediate sweet taste transduction. Some data indicated that IP3 may have a role in vertebrate bitter taste transduction. Here we report that the different second messengers are activated by different tastes. We used techniques designed for radioimmunoassay measurement. The results indicate that sucrose triggers an increase in c-GMP concentration and quinine increases the IP3 concentration in mouse taste cells. These results support the sweet and bitter taste transduction hypotheses.
Clostridium perfringens enterotoxin (CPE) was found to possess interferon (IFN)-producing and mitogenic activities to human peripheral blood mononuclear cells. Both activities were demonstrated only in the T lymphocyte-rich fraction from healthy volunteers. The IFN produced appeared to be γ-type since the activity of the IFN was neutralized by antiserum against human IFN-γ. With formalin-treated CPE, the IFN-producing and mitogenic activities were weakly found. Similar findings were also obtained in the mouse lethality and cytotoxicity to Vero (African green monkey) cells, suggesting that the biological activities of the CPE molecule may be existing on the similar (or the same) sites. From these findings, human peripheral T cells may be one of useful reagents to study the mode of action of CPE since CPE was found to be a T cell mitogen which is supposed to be a superantigen.
Campylobacter jejuni in chicken feces was detected by PCR and Southern blot hybridization (SBH). The detection limits of C. jejuni in chicken feces were 34,000 cells by PCR and 340 cells by SBH. Some cecal contents of chickens up to 3 weeks old were C. jejuni positive by SBH whereas all of them were negative by PCR. Two of 51 cecal contents of 18-day-old chicken embryos were C. jejuni positive by PCR and SBH; but, C. jejuni were not isolated from the samples by conventional culture with selective enrichment.
The acute effects of aspirin on auditory functions were examined electrophysiologically in conscious rats with chronically implanted electrodes for auditory brainstem response (ABR) recording. A single intravenous injection of aspirin at a dose of 225 mg/kg caused a reduction in the amplitude of the ABR P1 wave evoked by a 2 kHz tone pip 1 and 24 hr after dosing at almost all sound intensity levels, while the P1 amplitude at 4 kHz was reduced mainly 1 hr after dosing, and the P1 amplitude at 8 kHz was not significantly affected at middle and high intensities even 1 hr after dosing. The audiogram obtained from the P1 amplitude showed a significant increase in the sound threshold 1 and 24 hr after dosing at 2 kHz, and 1 hr after dosing at 4 kHz, but not at 8 kHz. The peak latency of the P1 wave was also prolonged. Furthermore, reduction of the P2 and P4 wave amplitude and prolongation of the P1-P2 and P2-P4 interpeak latency were also observed at 2 kHz but not at 4 or 8 kHz. These results suggest that the rat auditory function for low frequency is vulnerable to the effects of aspirin. This paradigm, i.e., frequency selectivity, in rats may be useful to further assess the different outer hair cells along the cochlear duct and provide additional evidence for the mechanism(s) or site underlying aspirin ototoxicity.
The hypothesis has been proposed that cell proliferation, or replicative DNA synthesis (RDS) in S-phase cells, is a nongenotoxic (Ames-negative) mechanism involved in tumorigenesis, providing a very useful conceptual basis for carcinogen testing. In this present study, hepatocyte RDS experiments were conducted using 5-bromo-2'-deoxyuridine (BrdU) labeling in combination with histopathological observation, comparing our results with earlier findings for in situ [3H]thymidine (TdR) labeling. The present BrdU data proved to be consistent with the previous TdR data in all but one case. Hepatocyte RDS induction was observed for some chemicals without hepatotoxicity. BrdU labeling in combination with histopathological observation is therefore a reliable approach to assessment of test compound effects in vivo.
To investigate whether the teratogenicity of ethylene thiourea (ETU) is due to ETU per se or to its metabolites, rat embryos cultured in vitro were exposed to 10 and 30 μg/m l of ETU in the presence or absence of rat liver microsomal fraction (S9mix). In the absence of S9mix, the incidence of morphological abnormalities increased dose-dependently. In the presence of S9mix, abnormal morphogenesis was almost absent in all embryos. These findings suggest that teratogenicity of ETU is due to ETU per se.
In order to evaluate and enhance the sensitivity of the neutralization (NT) test for detecting antibody in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus, the effect of altered incubation conditions and complement use on neutralizing (NT) antibody titer were investigated. Higher NT antibody titers were consistently obtained by addition of 20% guinea pig fresh serum to virus-serum mixtures in NT tests. Furthermore, the complement-requiring NT antibody titer increased in many serum samples when the virus-serum mixtures, rather than being incubated at 37 °C for 60 min, were incubated first at 4°C for 48 hr and then with a complement at 37°C for 60 min. The slow-reacting and complement-requiring NT antibody was detected as early as 8 days post-inoculation. It was detected in sera collected at 8 to 28 days post-inoculation and was sensitive to 2-mercaptoethanol treatment. Sera collected at 35 to 44 days post-inoculation contained 2-mercaptoethanol resistant NT antibodies. These results indicate that the modified NT test is useful for early serodiagnosis of PRRS virus infection through detection of higher NT antibody titers, and in detecting them earlier.