Distribution patterns of corticotropin-releasing factor (CRF), [arginine8]-vasopressin (AVP) and oxytocin (OXY) neurons were examined immunohistochemically in the female goat hypothalamus. The majority of the CRF immunoreactive (-IR) cells were located in the parvocellular part of the paraventricular nucleus (PVN) with smaller population found in the magnocellular part of the PVN. CRF-IR cells were also found in the suprachiasmatic nucleus, the preoptic area and around the fornix in the caudal part of the hypothalamus. AVP- and OXY-IR cells were similarly distributed in the hypothalamus. The majority of AVP- and OXY-IR cells were observed in the magnocellular part of PVN and the supraoptic nucleus. Smaller numbers of AVP- and OXY-IR cells were found in the parvocellular part of the PVN and lateral hypothalamic area. AVP-IR but not OXY-IR cells were located in the suprachiasmatic nucleus. CRF-IR fibers were concentrated in the external palisade zone of the median eminence (ME) with a few fibers found in the internal palisade zone of the ME, whereas AVP- and OXY-IR fibers were concentrated in the internal palisade zone of the ME with a few fibers found in the external zone. These results support the view that not only CRF but also AVP and OXY are released into the hypophysial portal blood and involved in the control of pituitary endocrine function in ruminant species.
The pancreas of a striped hyena (Hyena hyena) was examined by the naked eyes and light microscopy. The distribution of A, B, D and PP cells in the islets of Langerhans was investigated immunohistochemically. The pancreas, located in the mesoduodenum, consisted of three lobes. It was about 420 mm in total length, about 10-30 mm in width and about 10 mm in maximum thickness. The smallest lobe was caudally separated from two cranial lobes. This characteristic lobe was named as a caudal lobe. Two pancreatic and one accessory pancreatic ducts opened into the duodenum. The pancreatic acini, secretory ducts and endocrine islets were arranged as shown in other carnivores. A cells were located in the peripheral region of the islets, whereas B cells were equally distributed in the islets. Both D and PP cells were also discerned in the islets.
Two monoclonal antibodies capable of inducing granulosa cell apoptosis were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were dissected from the ovaries of gilts, and then granulosa cells were isolated. BALB/c female mice were immunized with the isolated granulosa cells. Antibodies against the granulosa cells were detected by immunofluorescent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulosa cells were fused with Sp2/O-Ag14 mouse myeloma cells by standard hybridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which produced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies specifically recognized cell-membrane proteins with molecular weights of 55 and 70 kD and isoelectric points of 5.9 and 5.4, respectively. The monoclonal antibodies immunohistochemically reacted with granulosa cells of healthy follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 or 10 μg/m l PFG-1 or PFG-2 antibodies, respectively, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulosa cells.
The localization of carbohydrates in rat livers with fibrosis induced by heterologous serum was examined by lectin histochemical and biochemical techniques. Twenty-four lectins were used to visualize the different carbohydrates in paraffin sections of normal and fibrotic liver tissues. No differences in staining patterns of these lectins were observed between normal and fibrotic livers in hepatocyte cell membranes including bile canaliculi, sinusoidal endothelial, or bile ductal cells. Kupffer cells strongly stained with Vicia villosa agglutinin (VVA) were seen only in the periportal zone of the normal liver, but they were observed in the periportal zone and scattered throughout the pseudolobular zone in the fibrotic liver. The cytoplasm of some hepatocytes was strongly stained by Bandeiraea simplicifolia lectin-I (BSL-I). BSL-I positive hepatocytes in normal liver were localized in the periportal zone, but those in the fibrotic liver were scattered in the periportal and perifibrous zones. After polyacrylamide gel electrophoresis of liver glycoproteins, differences in molecular sizes of BSL-I positive glycoproteins (79 and 81 kD) were detected by lectin blotting. Cell density of perifibrous BSL-I positive hepatocytes may be useful as a diagnostic parameter for liver fibrosis and/or cirrhosis. Two distinct staining patterns with twelve lectins were observed in fibrotic septa of the fibrotic liver. The fibrotic septa were stained with six characteristic lectins, and the centrilobular septa were stained with all these twelve of lectins. Histopathological assessment of the centrilobular fibrotic septa stained with these characteristic lectins may contribute to the diagnosis and prognosis of hepatic fibrosis.
The concentrations of apolipoprotein B (apoB)-48 and apoB-100 in triglyceride-rich lipoproteins (TRL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) fractions separated by gel permeation chromatography were determined in Holstein and Japanese black cows by enzyme-linked immunosorbent assay (ELISA). A significant correlation (p<0.01) was observed between apoB-48 in TRL and plasma triglyceride (TG) levels in both Holstein and Japanese black cows. Additionally, apoB-48 in TRL and plasma TG levels in Holstein cows were significantly lower (p<0.01) than those in Japanese black cows. These results suggested that TG derived from intestinal (exogenous) TRL rather than from liver (endogenous) TRL was the major source of milk fat.
Holstein bullocks were used in this study to compare the effectiveness of five commercial parenteral fluids (saline IS, Hartmann's IH, 5%-glucose 5G, Ringers IR, and 1/2 Ringer's and 2.5% glucose combination solutions RG) in correcting the disturbances associated with dehydration induced by fasting for 48 hr. These five commercial fluids (30 ml/kg) were given to bullocks with dehydration induced by fasting for 48 hr. Arterial and venous blood samples were taken before fasting, and at 0, 15, 30, 45, 60, 90, 120, 240, 360 min, and 24 hr after initiation of fluid administration. Fasting for 48 hr induced significant reductions in body weight and relative plasma volume (rPV), of approximately 7.72 and 21.93%, respectively. During the administration period, rPV showed a progressive increase from approximately 88.1% after fasting to 113.0% with no significantly differences between groups. A rapid decrease of rPV when fluid administration has been finished was observed in the 5G and RG groups. The results of the fluid administration trial showed that the 1/2 Ringer's and 2.5% glucose combination solution inhibited the acidification of the blood, produced no change in the electrolyte balance of serum, and induced a proper reabsorption rate of glucose in the renals, and was therefore considered the best choice for the rehydration of adult cattle which have had no appetite for over 2 days.
Neurogenic muscular atrophy was examined using computed tomography (CT) in dogs induced by crushing the sciatic nerve. The CT number and cross-sectional area in denervated muscles decreased in 1 to 2 weeks after denervation. Those were significant after 3 weeks. The examination with CT might be useful to diagnose canine neurogenic muscular atrophy.
Genomic DNA encoding bovine CD14 was isolated from a bovine (Holstein) genomic library. Utilizing PCR fragment of mouse CD14 gene as a probe, we screened 9 × 10 5 plaques and obtained 3 clones containing the bovine CD14 gene. DNA sequencing showed that bovine CD14 gene encodes 373 amino acids, and the coding sequence was separated by a 90 nt intron. The identity of the deduced amino acid sequence of bovine CD14 was 61-73% to those of mouse, rabbit and human. Northern blot analysis revealed that CD14 mRNA (1.5 kb) was expressed in the lung. Expression of CD14 mRNA was stimulated about 2-fold in bovine peripheral blood macrophage activated with LPS in vitro.
From June 1993 to November 1996, a total of 977 gobiid fish consisting of three species (Tridentiger brevispinis, Chaenogobius urotaenia, and Rhinogobius brunneus) collected from eastern Aomori Prefecture, were examined for Gnathostoma nipponicum larvae infection. Only one species, C. urotaenia was infected with advanced third-stage larvae (AdL3), and a total of 22 larvae were recovered from 17 (3.4%) of 500 fish. The infected fish were larger than 12 cm in body length and collected in the May-June and September-November seasons. Experimental studies confirmed that C. urotaenia was susceptible to both the early third-stage larvae (EaL3) obtained from Eucyclops serrulatus and AdL3 from Misgurnus anguillicaudatus. Eight of 10 C. urotaenia inoculated orally with 10 EaL3 were positive, and 36 AdL3 were recovered from them (recovery rate: 36.0%) at 30 days postinoculation (PI). All 10 C. urotaenia inoculated with 10 AdL3 were also positive, and a total of 63 AdL3 were recovered (recovery rate: 63.0%) at 10 days PI. The main location of the larvae was the body muscles of the fish. No morphological alterations or death of the larvae were observed in this study. From these results, it seems that the C. urotaenia has characteristics suitable to be the host to the larvae and they may serve as the second intermediate and paratenic host in the natural life cycle of this nematode.
Monoclonal antibodies (MoAbs) secreted by 12 hybridomas and reactive with the surface antigens of E. tenella sporozoites were produced. These MoAbs designated as KC-1 to KC-12 were characterized as IgG3k. In Western blot analysis, these MoAbs reacted with only one polypeptide band of sporozoite antigens having a molecular mass of 25 kDa. All the MoAbs were reactive with sporozoites, trophozoites, immature and mature first generation schizonts, first generation merozoites, and the interior structure of sporulating oocysts of E. tenella, however, not with any of the methanol-fixed sporozoites of E. acervulina, E. mitis, E. maxima, E. brunetti, E. necatrix and E. praecox. Invasion of primary culture of chicken kidney cells by E. tenella sporozoites was significantly inhibited by several of the MoAbs. These results suggest that these MoAbs recognizing the E. tenella-specific surface molecule are involved in the invasion of sporozoites into host cells.
We conducted the survey on intestinal nematode, mainly Toxocara canis, and cestode infections in stray puppies aged less than 5 months in Ibaraki Prefecture for 8 years from 1988 to 1995. The rate of T. canis infection stayed almost unchanged for the 8 years and ranged from 72.2 to 85.4%. Of the total 916 dogs, 732 (79.9%) harboured with T. canis. The average rates of other helminthic infections showed 24.6 (Diphylidium caninum), 2.5 (Trichuris vulpis), 1.2 (Ancylostoma caninum) and 0.7% (Spirometra erinaceieuropaei) , respectively.
Beige rats, a model animal of Chediak-Higashi syndrome (CHS), frequently developed the skin lesions consisting of crust formations and alopecia in the skin around the neck from about 4 months of age. Erosion and ulceration were also observed in advance of the skin lesions. In severe cases, the lesions spread to all of the dorsum of the trunk. Skin tissues with or without lesions were studied histopathologically in 41 beige rats comparing with normal skin from 26 age-matched DA rats. Microscopically, epidermal lesions consisted of spongiosis, pustules and erosions with crust. Inflammatory cells in pustules consisted predominantly of eosinophil, and colonization of gram-positive cocci was occasionally observed in the surface area. Mites on the epidermis were also seen in some cases. Dermal lesions were superficial perivascular inflammatory cell infiltrations of eosinophils, neutrophils and mastocytes, and edema under the epidermal lesions. Follicles in the alopecic area showed resting stage and atrophic hair germ, but inflammatory changes were slight. Morphologic characters were very similar to those of chronic eosinophilic dermatitis or spongiotic dermatitis.
Gastric carcinoid tumors were found in seven of 135 striped field mice (Apodemus agrarius) by routine histopathologic examination. All these carcinoids occurred in mature striped field mice aged 72-100 weeks. Six animals were females and only one was male. Only two of seven tumors were detectable by gross examination. Grossly, tumors were located in the fundus of the glandular stomach. All seven tumors were microscopically single in the stomach and two mice exhibited extragastric metastasis. Tumors from all the mice were characterized by densely packed sheets of round to polygonal cells, subdivided into packets by a fine fibrovascular stroma. The cytoplasm of all tumor cells from all the mice contained argyrophil granules when stained by Grimelius and Sevier-Munger silver procedures. All seven mice with gatric carcinoids exhibited positive immunoreactivity to neuron specific enolase. Psammoma bodies, concentrically laminated microcalcification, were characteristic findings in gastric carcinoids from five mice. There were also a concomitant and independent hepatocellular adenoma in one case and hepatocellular carcinoma in two cases. The present cases provide the first description of spontaneous gastric carcinoid tumors in the striped field mice.
Bacterial translocation occurs when viable bacteria pass through the mucosa of the gastrointestinal tract to the mesenteric lymph nodes (MLN) and other organs. The ability of a diet containing 1% culture condensate of Bifidobacterium longum (MB) to inhibit bacterial translocation from the gastrointestinal tract was tested using antibiotic-decontaminated specific-pathogen-free (SPF) mice and germ-free mice, both of which were monoassociated with Escherichia coli C25. Feeding of MB diet decreased the number of E.coli C25 translocating to MLN to about half the number of the control diet group, but did not reduce the incidence of translocation to MLN. MB diet ingestion also decreased the number of E.coli C25 translocating to MLN in the SPF mice injected with zymosan, but it could not prevent bacteria translocation in mice receiving a 30 % thermal injury.
A sensitive and rapid high-performance liquid chromatographic (HPLC) method was developed and used for the simultaneous determination of bilirubin and biliverdin in pericardial fluid samples collected from broilers at a poultry inspection site. A photodiode array detector distinguishing the bilirubin (UV 450 nm) and biliverdin (365 nm) was used as an analytical detector for HPLC system. An internal-surface reversed-phase silica support column was used, and the mobile phase consisted of acetonitrile: 0.5 M Tris HCl buffer (20:80, pH 7.2). Bilirubin was detected from all of the jaundiced pericardial fluid samples, and a small amount of biliverdin was detected with bilirubin in some samples. These jaundiced broilers had hepatic or bile duct lesions similar to those found in edible animals. From these results, a working definition of jaundiced broilers for poultry inspection sites was suggested: bilirubin is detectable from pericardial fluid and the carcass is in a state of yellow color change.
The pars intermedia of the pituitary gland, and plasma ACTH and cortisol levels in the pony, which was first diagnosed in Japan as indicating equine Cushing's disease, were examined by immunohistochemistry and radioimmunoassay, respectively. The pars intermedia was greatly enlarged and most of its cells were immunoreactive for antisera to both adenocorticotropic hormone (ACTH) and β-endorphin (β-End). The plasma ACTH level was elevated when clinical symptoms appeared. The present results reveal that equine Cushing's disease in this pony was induced by the hypersecretion of ACTH and β-End from the enlarged pars intermedia of the pituitary gland.
We attempted to measure the gestagen concentration in the feces of pigs by using a commercial bovine milk progesterone quantitative test EIA kit, and investigated the possibility of applying of this method of gestagen concentration measurement to early pregnancy diagnosis in the sow. Feces were collected from the rectum of the pig, and 0.5 g of the feces was placed in 20 ml of distilled water, stirred, and centrifuged. The supernatant was used as the fecal solution for measurement of gestagen. The procedure used for measuring gestagen in feces was the same as that for the measurement of progesterone in milk, except that a standard fecal gestagen solution (0.5-30.0 ng/m l) was prepared by the authors in the laboratory. The sensitivity of measurement using this method was 0.80 ng/ml, or 32.0 ng/g of fecal weight. The recovery was 105.2-105.6%. Intra-assay coeffecients of variation (CVs) were 2.8-8.5%. The inter-assay CVs were 7.4-10.2%. Gestagen concentrations in feces measured by the present method and progesterone concentrations in peripheral plasma, collected at the same time as the feces, were highly correlated (r=0.98, p<0.001). The criteria for diagnosis of pregnancy based on the fecal gestagen level was positive for a gestagen level of ≥ 200 ng/g and negative for a gestagen level of < 200 ng/g. When fecal gestagen measurements were applied to early pregnancy diagnosis in 149 sows, the accuracy of diagnosis from day 21 to day 25 after the last mating was 96.2% for positive cases (102/106) and 95.3% for negative cases (41/43). Thus, the results of this study show the quantitative measurement of the fecal gestagen concentration in the sow using a bovine milk quantitative test EIA kit is a practical method for early pregnancy diagnosis.
Marek's disease virus (MDV) serotype 2 (MDV2) gene homologous to the glycoprotein H (gH) gene of herpes simplex virus type 1 was identified and sequenced. The predicted region encoding for the MDV2 gH gene was 2436 nucleotide and the primary translation product was 812 amino acids with a molecular weight of 89.4 kDa. The protein encoded by MDV2 gH gene has a number of features characteristic of a membrane-associated glycoprotein. First, there are 9 potential N-linked glycosylation sites and 11 cysteine residues, and 6 of the sites and 8 of the residues were conserved among all of the three MDV serotypes. Second, this protein had N-terminal and C-terminal hydrophobic regions, which were a signal sequence and a transmembrane-anchor domain, respectively. From the northern blot analysis, it was suggested that a transcript encoding MDV2 gH and a poly-cistronic transcript encoding MDV2 thymidine kinase, gH, and possibly other genes of downstream on this strand existed. Alignment of the amino acid sequences of the gH homologues among the three MDV serotypes showed 57.5% (MDV1 and MDV2), 56.2% (MDV1 and HVT), and 50.1% (MDV2 and HVT) identities.
A trial vaccine containing pseudorabies virus (PRV) glycoprotein gC as the main component showed excellent protection against virulent virus infection in pigs. Glycoprotein gC-rich antigen was prepared by heparin affinity chromatography from PRV- infected cell lysates. The preparations were mixed with mineral oil adjuvant as a water-in-oil emulsion. Six-week-old pigs were immunized twice at two-week intervals with trial vaccines containing 128,000, 12,800 and 1,280 HA units per dose of gC antigen. They were then challenged with a virulent PRV at day 7 after the final immunization. Neutralizing (NT) antibodies were produced with increase of antibody titers after challenge. Pigs immunized with 128,000 HA units per dose of gC survived and showed no virus shedding during the 2-week experimental period after the challenge. The role of cell-mediated immunity was examined using BALB/c mice, and induction of gC-specific cytotoxic T lymphocytes (CTLs) was detected by 51Cr release assay. From these results with mice, it is inferred that cell-mediated immunity, especially CTL, may play an important role in the effectiveness of our trial vaccine in addition to humoral immunity.
The number of plaques formed by equine arteritis virus (EAV) and Aujesky's disease virus (ADV) was reduced to 14% and 5% of the untreated control(100%), respectively, by 10 U/ml of heparin, but could not be reduced below to 13 and 4%, respectively, by use of concentration up to 100 U/ml. An inhibitory effect of heparin, at concentration up to 100 U/ml, was not observed on parainfluenza virus 3 (PIV-3). Heparinase treatment of RK13 cells reduced the number of EAV-, as well as ADV-induced plaques. On the other hand, the number of PIV-3 induced plaques did not decrease after treatment of RK13 cells with heparinase.