Effects of a triple enzyme digestion method using glycosaminoglycans-degrading enzymes upon a diamine reaction have been tested for electron microscopic histochemical detection of glycosaminoglycans in extracellular matrix of the rat aorta. The triple enzyme digestion method consists of a sequence of chondroitinase B, testicular hyaluronidase and heparitinase. The results obtained by the present experimental and control studies indicated that dermatan sulfates, chondroitin sulfates (A and/or C) or heparan sulfates were apparently observed in various ultrastructural features of aortic extracellular matrix, such as bundle of collagen fibers and soluble matrix of interstitial space. Particularly, we found that both heparan sulfates and chondroitin sulfates (A and/or C) were detected in association with the basal lamina of smooth muscle cells and the external surface of elastic lamina, and in the latter heparan sulfates were frequently recognized as a mass, whereas chondroitin sulfates (A and/or C) were found intermittently along the external surface of elastic lamina. This suggests that the triple enzyme digestion method which combines the glycosaminoglycans-degrading enzymes with the diamine reaction can be postulated to represent efficient and useful technique for precise electron microscopic histochemical detection of the glycosaminoglycans in the extracellular matrix of the rat aorta.
Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatozoa. In the present study, to determine the expression pattern of MCS mRNA in the hamster testis, northern blot and in situ hybridization analyses using digoxigenin-labeled riboprobes for the hamster MCS were performed in the testes of 10-week-old golden hamsters. According to the northern blot analysis, hamster MCS was detected as a single transcript of about 1 kb in the testis. During spermatogenesis, the hamster MCS mRNA first appeared in step 6 spermatids, gradually increased in round spermatids during spermiogenesis, reached a peak in step 8 spermatids, and persisted a high level until step 13 spermatids. After step 14, the signal began to show a progressive decline in the spermatids and was weakly detected in the tails of step 17 spermatids. However, the signal was not observed in spermatogonia, spermatocytes, Sertoli cells, peritubular myoid cells, and interstitial cells. These findings indicate that hamster MCS is mainly related to the spermiogenesis during spermatogenesis.
Serum samples collected from 739 free-living wild birds of 44 species from Gifu, Mie and Hyogo Prefectures in Japan during the period 1989 to 1997 were tested for antibodies to infectious bursal disease virus (IBDV) serotypes 1 and 2 by a virus neutralization test. Serological evidence of infection with serotypes 1 and 2 was found in 15 (2%) of the sera of 6 species and 36 (4.9%) of the sera of 11 species, respectively. Antibodies to IBDV were detected from both sedentary and migratory species. These findings suggest that free-living wild birds have an important role in the natural history of IBDV. These findings raise the possibility that the IBDV prevalent in the breeding grounds of these birds in other countries could be imported by the migratory species. This is the first report of an extensive serological survey of IBDV in wild birds.
The production of exfoliative toxins A and B (ETA and ETB) by Staphylococcus aureus isolated from mastitic cow's milk and farm bulk milk was examined by the reverse passive latex agglutination method (RPLA). ETA was detected in 2 (1.2%) of 162 isolates from mastitic cow's milk and in 1 (0.6%) of 166 isolates from farm bulk milk. RPLA titers of these isolates were much lower than in human isolates. No ETB was detected in any of the isolates tested. These ETA-positive isolates belonged to bovine ecovar. They were non-typable using the international phage set for human strains. When these ETA-positive isolates were subcutaneously inoculated into neonatal mice, general exfoliation of the epidermis accompanied by the so-called Nikolsky sign was not recognized. By the immunoblotting and PCR methods, however, ETA and eta gene were recognized in the ETA-positive isolates from mastitic cow's milk and farm bulk milk. These data suggest that ETA is also produced by bovine isolates of S. aureus, but in smaller quantities.
Porcine tumor necrosis factor alpha (TNF-α) was produced using a baculovirus system in Spodoptera frugiperda (SF21AE) cells. Cytotoxic activity was detected in supernatant of sonicated SF21AE cells infected with recombinant viruses. The recombinant protein was also demonstrated to be functionally active by its ability to cause apoptosis in Wehi 164 cells. Three distinct bands of 26, 17 and 14 KDa were revealed by Western blot analysis using anti-human TNF-α antibody. Moreover, the anti-human TNF-α antiserum significantly neutralized the cytotoxic activity of the supernatant of sonicated SF21AE cells infected with recombinant viruses.
The reactivity of native bovine conglutinin (Kg) with antibody against recombinant Kg (rKg), with deletion of the N-terminal and collagen-like regions of the native Kg molecule, was studied by sandwich enzyme-linked immunosorbent assay. With anti-recombinant Kg antibody as the coating antibody, rKg reacted with biotinylated homologous anti-rKg and heterologous anti-Kg antibodies as probing antibodies, while native Kg did not. With anti-native Kg antibody as coating antibody, native Kg reacted with biotinylated homologous antibody as probing antibody, while recombinant Kg reacted weakly with both biotinylated homologous and heterologous antibodies. Consequently the N-terminal and collagen-like regions of native Kg molecule are essential to express the complete immunogenicity and/or antigenicity of the native Kg molecule.
Interleukin-12 (IL-12) is an important cytokine for Th1 response which stimulates the T-cell population to produce cytokines for cellular immunity. Interleukin-10 (IL-10) is a pleiotropic cytokine capable of suppressing cytokine production from macrophages and T-cells and participates in Th2 immune response. The present study was carried out to examine the effect of these cytokines on virus replication and apoptosis in T-cells infected with feline immunodeficiency virus (FIV). Infection of a feline T-lymphoid cell line (Fel-039) resulted in an increase of the reverse transcriptase (RT) activity in the culture supernatant accompanied by cell death from apoptosis. Addition of human recombinant IL-12 significantly inhibited the virus replication and apoptosis in Fel-039 cells in a dose dependent manner. Furthermore, the antiviral activity of IL-12 was associated with the expression of IFN-γ in the FIV-infected Fel-039 cells. In contrast, human recombinant IL-10 did not show any inhibitory effect on the virus replication and apoptosis in the Fel-039 cells infected with FIV. These results suggest that the inhibitory effect of IL-12 on both virus replication and apoptosis has potential implications for the design of immunotherapy strategies using IL-12 in FIV infection.
Infection of feline immunodeficiency virus (FIV) has been shown to induce apoptosis that might be associated with the lymphocyte depletion in the infected cats. To investigate the inhibitory effect of antioxidants on FIV-induced apoptosis, we examined the effect of N-acetylcysteine (NAC) and ascorbic acid (AA) on apoptosis and virus replication in feline lymphoblastoid (Fel-039) and fibroblastoid (CRFK) cell lines infected with FIV. The treatment with NAC or AA induced a significant inhibition of viral replication and apoptosis in Fel-039 cells and tumor necrosis factor α (TNF-α) - treated CRFK cells infected with FIV. Both cell lines in the presence of noncytotoxic concentrations of NAC or AA showed an increase of intracellular glutathione (GSH) level, which might protect the cells against oxidative stresses exerted by FIV infection and TNF-α treatment. On the basis of these in vitro results, we suggest that antioxidant therapies aimed at restoring depleted GSH level might be effective for inhibition of viral replication and cell death associated with the development of immunodeficiency.
Electrogastrography (EGG), in which the electrical activity of the smooth muscular layer of the stomach is recorded percutaneously through the abdominal wall, has been applied in recent years to humans as a non-invasive method. In acute abdominal disease in horses, it is considered diagnostically useful to analyze digestive activity using EGG. Electrocecography (ECG) was examined to determine its effectiveness in evaluating equine digestive motility through comparison, after xylazine administration, between the results of the percutaneous ECG method and the results obtained using a strain-gauge force transducer (Force Transducer) chronically attached to the serous membrane of the cecum. As subjects, the test used six male thoroughbreds (average weight: 457.5 ± 9.2 kg). The test showed a reduction in both the percutaneous electrical potential of the cecum in ECG and in cecal contractions measured with the Force Transducer. After xylazine administration, an average rates of decrease of the amplitude from the control period were 17.8 ± 3.4% and 20.0 ± 4.6% respectively, demonstrating a significant correlation (r=0.90) between the two methods. On the other hand, power distribution centered around 6 cycles per minute in a Fourier transform (FFT) analysis of ECG, thought similer to the contraction frequency of 5.4 ± 3.0 per minute observed with the Force Transducer. After xylazine administration, the total frequency band (1.8-12 cycle per min) in the running spectrum total power in ECG decreased to 37.0 ± 5.1% of the pre-xylazine value. Based on these findings, it appears that the ECG potential reflected electrical activity of cecal origin, suggesting high clinical applicability of ECG to the percutaneous evaluation of equine cecal motility.
Experiment was designed to determine whether heat stress suppresses neutrophil function and injections of selenium and vitamin E prior to heat stress prevent suppression of neutrophil function in goats. Twelve female goats were divided into 2 groups of 6 each and were kept at 25°C. Goats in the treatment group were injected intramuscularly with 0.1 mg/kg of selenium and 2.72 IU/kg of vitamin E at 8 and 1 day prior to the initiation of heat stress. The other group was kept as control. All goats were exposed to hot environment at 38°C from day 0 through 8. Decreased tendency in plasma cortisol concentrations and temporary increase in plasma glucose concentrations were shown in both groups. In the control group, plasma selenium concentration gradually increased and a-tocopherol concentration decreased during the first 2 days. After the second injection with selenium and vitamin E, plasma selenium and a-tocopherol concentrations significantly increased and remained higher than those in the control group. Whole blood glutathione peroxidase (GSH-Px) activity in the treatment group tended to be greater than that in the control group, but no significant difference was observed between 2 groups. The nitroblue tetrazolium (NBT) reduction by activated neutrophils significantly decreased on day 6 in the control group but not in the treatment group. The NBT reduction by resting neutrophils significantly decreased in both groups. These data suggest that heat stress depresses neutrophil function, and selenium and vitamin E injection prior to heat stress has no apparent effect on neutrophil function during the stress.
The process of hepatic fibrosis, and the changes in contents of hepatic hyproxyproline (HYP) and serum procollagen type III peptide (PIIINP) were examined in two rat models for hepatic fibrosis, i.e. bile duct ligation/scission (BDL/s)- and dimethylnitrosamine (DMN)-induced models. In addition, an expression of type III collagen mRNA in the liver of BDL/s model was also examined. In BDL/s model, hepatic fibrosis started at 2 weeks after operation (WAO) and cirrhosis with prominent bile duct hyperplasia was detected at and after 5 WAO. Serum PIIINP content measured using a modified double armed inhibition enzyme-linked immunosorbent assay (ELISA) method proposed by us started to increase at 1 WAO and continued to increase thereafter. Hepatic HYP content measured colorimetrically started to increase at 3 WAO and it continued to increase until 7 WAO. An expression of type III collagen mRNA in the liver was enhanced at and after 2 WAO, especially at 4 and 5 WAO. In DMN model, marked hepatic fibrosis was detected at 1 week after the last DMN administration (WAA), and the degree of fibrosis was apparently reduced at 4 WAA. Serum PIIINP content prominently increased at 1 WAA and decreased at and after 3 WAA. Hepatic HYP content showed a marked increase at 1 WAA and decreased thereafter. The present results indicated that the sequences of hepatic fibrosis, hepatic HYP content and serum PIIINP content were well correlated with each other in both BDL/s and DMN models. In conclusion, ELISA system for the detection of serum PIIINP content is considered to be reliable method for assessment of cirrhotic liver, and the present two rat models for liver fibrosis/cirrhosis seems to be a good tool for researching antifibrotic agents.
A mobile right-ventricular mass dynamically occluding the right ostium atrioventriculare in the systolic phase was detected in a 3-year-old male Tosa dog by echocardiography. At necropsy, multiple tumor masses of various sizes were observed in the heart base, right ventricular lumen, myocardium, lung and liver. Dysplasia of tricuspid valve characterized by irregular shape of leaflets, upward malposition of large papillary muscles, and shortened and stout chordae tendineae was also detected. Histopathologically, the tumor cells, arranged in sheets or nests, were polyhedral with lightly eosinophilic and finely granular cytoplasm, and contained a hyperchromatic round or oval nucleus. By Grimelius' silver stain, tumor cells had cytoplasmic positive granules. Ultrastructurally, tumor cells contained characteristic small membrane-limited granules. This is the first report of metastatic intracavitary cardiac aortic body tumor in a dog.
Rare case of patent ductus arteriosus (PDA) was observed in a 2-year and 9 month-old Miniature Dachshund which had been diagnosed as severe cardiac failure. Pulmonary artery from the right ventricle connected to dilated ductus arteriosus, and pulmonary artery-ductus arteriosus-descending aorta formed a continuous duct, which seemed to be the main route of bloodstream. Ascending aorta from the left ventricle was hypoplastic, and connected to the dilated ductus arteriosus. Glomerular mesangiolysis due to heart failure was also observed in the kidney.
Effect of neurotensin (NT) on carbachol(CCh)-induced tension development due to Ca2+ release from intracellular stores was investigated in β-escin-skinned smooth muscle of guinea-pig ileum. NT (10 nM) increased the tension development in response to CCh. NT also increased the tension response to caffeine, another stored-Ca2+ releaser. NT did not exert such an effect in pertusis toxin (PTX)-treated preparations. Treatment with isoprenaline to elevate endogenous cyclic AMP levels or with dibutyryl cyclic AMP did not affect the effect of NT. A nonpeptide NT antagonist, SR 48692, failed to block the effect of NT. NT shifted the pCa-tension relationship in the lower direction of Ca2+ concentration. NT was incapable of releasing Ca2+ from intracellular stores. The results suggest that NT may cause an increase in Ca2+ sensitivity of contractile elements to potentiate the CCh-induced tension development due to release of stored Ca2+ and that the effect is mediated by SR 48692-insensitive NT receptors linked to a PTX-sensitive G protein which works with no relation to a change in cytosolic cyclic AMP levels.
Urine samples of cats and dogs collected for 24 hr after a subcutaneous injection of orbifloxacin (OBFX) were analyzed. The metabolites were examined using HPLC. In the dog urine, 87% of total was the parent compound and 13% glucuronide compound of OBFX and 96% was parent and 4% metabolite in the cat urine. The metabolite of cat urine was identified as N-hydroxy OBFX, determined by comparison of the extraction of urine with chloroform with the standard compound of N-hydroxy OBFX, using LC/APCIMS. N-hydroxy OBFX had a weaker antibacterial activity against fluoroquinolone sensitive bacteria than the parent compound.
The present study was performed to assess the prevalence of Salmonella in chickens in Thailand. In 1997, 22 serovars of Salmonella were isolated from 72 of 100 chicken meat samples purchased from 10 retail markets in Bangkok and 20 of 200 chicken meat samples from one slaughterhouse for export and 19 of 285 chicken feces obtained from three farms located in the east region of Thailand. The most predominant serovar was S. Enteritidis, which was isolated from 28% of the retail chicken meat, 4.5% of the chicken meat from slaughterhouse, and 6.6% of chickens feces samples examined.
The purpose of this study was to investigate the potential for circulatory arrest during surgery under systemic hypothermic anesthesia, using the abdominal cavity cooling method. Eighteen beagles, each weighing 10.5 ± 2.3 kg, were cooled by filling the abdominal cavity with crushed ice. Just after the esophageal temperature reached 30°C, the heart was exposed, and a left-heart bypass from the left atrium to the aortic root was created. At 20-23°C, the heart was arrested by infusing cooled Young's solution into the aortic root. After a period of cardiac arrest, resuscitation and rewarming were initiated simultaneously. Throughout these procedures, an electrocardiogram (ECG) and the arterial blood pressure (ABP) were monitored continuously. Hematocrit (Ht), total protein (TP), and arterial blood pH and gases were measured every 30 min. The recoveries after surgery were divided into three types as follows, 1) recovery without any complications-11 dogs, 2) not extubated with spontaneous breathing-4 dogs, 3) no reappearance of heart beat-3 dogs. PaO2 during resuscitation was significantly higher in dogs which recovered completely than in the rest of the dogs. These results suggest that hypothermia induced by the abdominal cavity cooling method could be useful for organ-protection during open-heart surgery, and that successful recovery may be attained through protection of the lung as well as the myocardium.
The blood concentrations of endotoxin in dogs with pyometra (n=45) were compared with those in healthy dogs (n=17). The blood endotoxin concentrations in the healthy dogs (n=17), in those with good prognosis (n=41) and those with poor prognosis (n=4) were 3.4 ± 2.8 pg/ml, 9.5 ± 11.3 pg/ml and 74.2 ± 18.3 pg/ml, respectively. The concentrations in the dogs with good prognosis and poor prognosis were significantly (p<0.01) higher than those in the healthy dogs. The dogs with poor prognosis had significantly (p<0.01) higher endotoxin concentrations than those with good prognosis. Blood endotoxin concentrations were measured in 9 dogs after surgery, and were found to be decreased. These results suggest the possible involvement of endotoxin in the pathophysiological changes due to pyometra in dogs, and also that the blood endotoxin concentration could be used as a marker to determine prognosis.
Vitamin D3: 1-α,25(OH) 2D3 (calcitriol), 22-oxa-1,25(OH)2D 3 (OCT), cholecalciferol (vitamin D3), and retinoids: all-trans retinoic acid (ATRA) and 9-cis retinoic acid, induced morphological changes in POS canine osteosarcoma cells into elongated, spindle or fibroblast like-shaped cells, and apoptotic like cell death characterized by cell shrinkage, condensation and margination of the nucleus for all drugs at 10-6M-10 -9M after 72 to 120 hr culture. Apoptosis as shown by DNA laddering was induced at 48 hr by all drugs at 10-6M, 10-7M at 96 hr, 10-8M and 10-9M at 120 hr respectively. These vitamins are suggested to adjunct antineoplastic agents in canine osteosarcoma therapy by induction of apoptosis.
A seroprevalence study of bovine lentivirus, known as bovine immunodeficiency virus (BIV), was conducted in 12 different dairy herds in Hokkaido, where some herds were a high prevalence of bovine leukemia virus (BLV) infection. Amongst 611 cattle, 28.6% of cattle were BLV-seropositive, and 11.7% of cattle were seropositive for BIV, while 4.2% of cattle were seropositive for both BIV and BLV. For the isolation of BIV, 19 samples of peripheral blood mononuclear cells (PBMC) and one sample of milk-derived leukocytes were prepared from BIV-seropositive cows. These PBMC and leukocyte preparations were then co-cultivated with cc81 cells, a cat cell line transformed by mouse sarcoma virus. BIV was isolated from 17 PBMC and one milk-derived leukocyte samples. The isolated viruses showed slow replication and syncytia formation. Major core antigen, p26 from these isolates were reacted with anti-BIV (American isolate R-29) serum. In addition, proviral DNA was detected in blood and milk samples by nested polymerase chain reaction and subsequent Southern blot hybridization. Nucleotide sequence analysis of the amplified pol gene products showed its 99.0 to 99.7% homology to that of BIV R-29. These results indicate that the Japanese BIV isolates appear to be antigenically and genetically similar to the American R-29. Since BIV was isolated from milk samples, BIV could possibly be transmitted through milk. This is the first report of BIV isolation in Japan.
Restriction fragment length polymorphism analysis was used to differentiate recent field viruses of canine distemper virus (CDV) from vaccine strains. Virus genomes were amplified by using reverse transcriptase-polymerase chain reaction in part of the haemagglutinin gene. After digestion with EcoRV, the PCR products of recent field isolates were cut into two fragments that differ from the uncut form of old strains including all of vaccine strains. This method could be applied to fresh or stored brains, spleens and peripheral blood mononuclear cells of infected dogs. This molecular approach is useful for determining the causative agent of post-vaccinated CDV infection.
The prevalence of infections with three feline retroviruses (feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and feline syncytial virus (FSV)) was examined in northern Vietnam in 1997. We collected a total of 77 blood samples from 69 domestic and 8 leopard cats, and examined the presence of anti-FIV and FSV antibodies and FeLV p27 antigen in the plasma samples by the indirect immunofluorescence and/or two commercial kits. None of the samples was positive for FIV and FeLV. The overall positive rate of FSV was 31% and the positive rates among the domestic and leopard cats were 29 and 50%, respectively. We isolated FSV from peripheral blood mononuclear cells of 6 domestic and one leopard cats.