Feline herpesvirus type 1 (FHV-1) is a causative agent of feline viral rhinotracheitis and belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae. Since first isolated in 1958 by Crandell and Maurer, FHV-1 is distributed worldwide and is the most clinically significant agent for respiratory infections in cats. In this review, we describe the recent findings with properties and functions of FHV-1 glycoproteins, especially hemagglutinins.
The distribution and entire shape of olfactory receptor cells were investigated by means of whole-mount preparations of the nasal mucosa. Whole mucosa isolated from the nasal septum of rats was processed, as "a free-floating section", and examined by the avidin-biotin complex (ABC) method using antisera against protein gene product 9.5 (PGP 9.5) and calbindin. Essentially all receptor cells were immunolabeled with the PGP 9.5 antiserum, but only half of PGP 9.5-immunoreactive cells were calbindin-immunoreactive. In the immunostaining of whole-mount preparations, pretreatment of tissues by freeze-thawing and dipping in ethanol and xylene greatly improved the permeability of antibodies. Overview of the nasal septum showed that the dorsal and ventral portions of the rostral olfactory area extended deeply into the respiratory area, making a "semi-lunar" shape. The boundary between the two areas was clearly demarcated, although several receptor cells were scattered in the respiratory area near the boundary. Observation at higher magnification clearly demonstrated that several axons derived from perikarya gathered to form nerve bundles showing a dendritic pattern. Proximal axons close to perikarya displayed beaded structures with intense immunoreactivity. They were electron-microscopically identified as swollen portions of axons which might be formed in association with the axonal flow. The present study showed that whole-mount preparation of the nasal mucosa for immunohistochemistry is a useful tool to analyze the morphology of olfactory receptor cells and axons.
Inhibin is believed to play roles in the pituitary secretion of FSH and in the paracrine regulation of testicular function. Although it has been generally accepted that inhibin is produced in Sertoli cells, there was a recent evidence for the localization of inhibin in Leydig cells of primates, rat and sheep. However, there is no report on the expression of inhibin in the adult horse testis. Therefore, using immunohistochemistry, western blotting and in situ hybridization techniques, the present study examined inhibin α-subunit (Ih-α) expression in the adult horse testis. For the detection of Ih-α protein, we used anti-porcine Ih-α antibody in immunohistochemistry and western blotting. Furthermore, digoxigenin-labeled complementary RNA probes were prepared to detect intracellular messenger RNA (mRNA) of Ih-α. Immunostainings for Ih-α were found not only in Leydig cells but also in Sertoli cells. The intensity in Leydig cells was stronger than in Sertoli cells. Immunoreactivities for Ih-α were found at approximately 46 kDa, 56 kDa and 90 kDa in the homogenates from testicular interstitial tissues. The bands at 56 kDa and 90 kDa agree with previous report, but not at 46 kDa. Signals for mRNA of Ih-α by in situ hybridization were detected in Leydig cells and in the basal region of seminiferous epithelium including Sertoli cells. These results suggest that Ih-α is expressed in Leydig cells and Sertoli cells of horse testis, and the expression level should be higher in Leydig cells than Sertoli cells.
Lectins are sensitive probes which bind carbohydrate structures specifically. In this study, we modified the lectin staining procedure for sensitive detection of carbohydrate structures in formalin-fixed, paraffin-embedded sections of normal and heterologous serum-induced fibrotic livers. The liver sections were heated in hot distilled water at 100°C for 10 min (thermo-treatment: TT), and then stained with 24 different lectins. In comparison with the results from sections without TT (nonTT), enhanced and/or alternated staining patterns of 19 lectins were demonstrated in sections with TT, and enhanced staining of Vicia villosa agglutinin seen in Kupffer cells was noted. Interestingly, no positive staining was seen with Dolichos biflorus agglutinin, peanut agglutinin or soybean agglutinin (SBA), which recognize O-linked carbohydrate chains, in Kupffer cells of non-TT sections, but strong positive staining was demonstrated in those of TT sections. SBA-positive staining in the cytoplasm of some scattered hepatocytes located in the periportal and perifibrous zones and central zone of pseudolobules was demonstrated only in the fibrotic liver sections with TT. Such findings indicate the heterogeneity of hepatocytes in the liver with fibrosis. Formalin fixation causes masking of lectin binding sites, especially O-linked carbohydrate chains, and TT may recover such masking reactions. TT improved the staining reactions for many lectins in formalin-fixed, paraffin-embedded liver sections, and new staining patterns appear after TT. Modified TT staining procedures may be useful for the diagnosis and prognosis of liver fibrosis.
This work was conducted to determine whether the angiotensin-converting enzyme inhibitors (ACEIs) (enalapril and captopril) administered to mother rats prenatally can potentiate a re-opening of the neonatal ductus arteriosus (DA) induced by prostaglandin E2 (PGE2) after postnatal closure. A subcutaneous injection of PGE2 (4 μg) was administered to newborn rats 3 hr after a Cesarean delivery from females which had been orally given 0.1, 1 or 10 mg/kg/day of enalapril or 15 or 150 mg/kg/day of captopril from day 14 to day 20 of gestation. The ratio of the DA to the pulmonary artery (PA) was determined at intervals after the injection. The DA/PA ratio was significantly higher in the newborn rats of mothers who were transplacentally administered these agents compared to the controls, except at the low dose (0.1 mg/kg) group of enalapril. We found that the level in the neonatal lungs of 15-hydroxy prostaglandin dehydrogenase, a key enzyme that catalyzes PGE2 to convert it to its inactive metabolite 15-keto-PGE2, was not affected after maternal treatment with enalapril or captopril. These results indicate that the increased ductal responsiveness to PGE2 in newborn rats was a common response after maternal ACEI treatment, but the catabolism of PGE2 in the lungs did not contribute to this response.
The antibodies to B. (S.)hyodysenteriae in experimentally infected mice were detected by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA). The reactions in MAT were serotype specific while those in ELISA were common to both strains. A further investigation with immunoblotting technique demonstrated that 22- and 17-kDa proteins reacted strongly with the sera. The proteins in ATCC 27164 strain strongly reacted with the serum from ATCC 31212 strain-infected mouse and vice versa. These proteins were sensitive to proteinase K.
Changes in iron and ferritin in calves infected with Theileria sergenti were investigated to elucidate iron metabolism in animals with extravascular hemolytic anemia. During severe anemia, serum iron was remarkably elevated while the total iron-binding capacity remained relatively unchanged or decreased slightly in the infected calves, resulting in elevated transferrin saturation. The serum ferritin concentration gradually increased with the progress of anemia. The erythrocyte ferritin content drastically increased when mean corpuscular volume was elevated. The concentration of non-heme iron and ferritin in the liver, spleen, and bone marrow of the infected calves was markedly higher than that in the respective tissues of the control animals. In particular, the liver of the anemic calves was found to contain 23 and 35 times as much non-heme iron and ferritin, respectively, as that of the non-anemic healthy cattle. The liver type (L) to heart type (H) subunit ratio of liver ferritin was significantly higher in the protozoa-infected than in the non-infected cattle. On the other hand, the L/H ratio of marrow ferritin was significantly reduced by the anemia. These results indicate that the anemic calves infected with T. sergenti apparently present symptoms of iron overload.
To understand the relationship between novel parainfluenza virus 3 (PIV-3), which has recently been isolated from the lungs of guinea pigs, and other PIV-3 strains, we determined the complete nucleotide sequence of the novel PIV-3 (GPv) genome. A comparison of the nucleotide sequence among PIV-3s, including bovine PIV-3, revealed that GPv is closely related to human PIV-3. The results of the phylogenetic analysis clearly showed that GPv is a lineage of human PIV-3, suggesting that GPv has probably been introduced into guinea pig colonies via infected humans.
A concentration of ellipticine, an inhibitor of topoisomerase II, required to reduce cell survival to 37% (D37) is used as an index to compare the cellular sensitivity. D37 values of LEC and WKAH rat cells were 1.2 and 2.2 μM, respectively. Thus, LEC rat cells were approximately 1.8-fold more sensitive than WKAH rat cells to ellipticine. There was no significant difference between the topoisomerase II activities in nuclear extracts of LEC and WKAH rat cells. These results suggested that the high sensitivity of LEC rat cells to ellipticine is not associated with the level of topoisomerase II activity.
To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD), lymphoid leukosis, reticuloendotheliosis, and field tumors, some portions of p53 cDNA corresponding to core and C-terminal domains (nucleotide positions 277-1104 in the p53 open reading frame (ORF)) were sequenced. Several mutations were identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been reported as the site for transformation-activating mutations. Moreover, partial cDNA clones with a 122-bp deletion in the p53 ORF were identified in two cell lines, MSB1 and MTB1 derived from MD tumors. Southern blot analysis showed that no deletion occurred in the genome of p53 in MSB1, indicating that deletion occurred at the transcriptional level. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. These mutations of the p53 gene and deletion in the p53 transcript may be ones of molecular changes specific to the transformation induced by MD virus.
Crude or dehydrated bulbs of autumn crocus (Colchicum autumnale L.) were fed to eleven calves. All the calves developed severe diarrhea and died or euthanized within 63 hr. At necropsy, the gastro-intestinal mucosa was edematous and hemorrhagic. Histologically, necrosis and degeneration with karyopyknosis and karyorrhexis were shown in the basal cell layer of the tongue, esophagus, forestomach, renal pelvis, urinary bladder, neck cell layer of the abomasal gastric glands, and intestinal cryps. These findings were also seen in Kupffer cells, renal tubular epithelial cells, and lymphocytes in the lymphoid and hemopoietic systems. The lesion of the present acute crocus poisoning of cattle closely resembled those reported in humans with colchicine intoxication. Refined acetone extract of organs of poisoned cattle proved to contain colchicine and demecolcine by high performance liquid chromatography.
The tumor of the thoracic cavity, which arose from the ribs, was diagnosed as mesenchymal chondrosarcoma. No distant metastasis was observed. Histologically, the tumor was characterized by the nests of well-defined cartilaginous tissue within a proliferation of primitive mesenchymal cells. Additionally, the deformed blood vessels compressed by the proliferating mesenchymal cells exhibited clear stag-horn appearance. Immunohistochemically, most neoplastic cells that formed multifocal cartilaginous islands were positive for S-100 protein, while the surrounding mesenchymal cells were negative. This is the first report of canine mesenchymal chondrosarcoma of the ribs.
Seven sika deer (Cervus nippon) died in a park where 30 deer were kept. One adult female deer died suddenly was necropsied. Severe hemorrhages were noted beneath the serous membranes of the forestomach and abomasum. Hyphal proliferation with neutrophil infiltration was observed in the mucous membranes of the stomaches, and the hyphae showed characteristics of order Mucorales. Catarrhal enteritis with hemorrhages was also observed. A large number of Clostridium perfringens was isolated from the contents of the abomasum and small intestine. The case was diagnosed as gastric mucormycosis associated with proliferation of Clostridium perfringens. The incidence occurred during breeding season and incorrect management was considered to be a predisposing factor for the infection.
Based on the recent findings that show how recombinant human tumor necrosis factor (rh-TNF)-α has potent antitumor activity on human cancer patients when it locally administrated, we have tested the cytotoxicity of rh-TNF-α on 3 canine cultured cells: (1) canine kidney carcinoma (CKCa-1), (2) mastocytoma and (3) Mardin Darby canine kidney cells (MDCK). The cell surface expression of TNF-α receptors on these canine cells was also determined with anti-human TNF RI and RII polyclonal antibodies. Our data shows that on CKCa-1 which has TNF RI receptors rh-TNF-α induced cytotoxicity. By contrast, it exhibited no toxicity on canine mastocytoma which has mainly RII receptors. The data also suggest actinomycin D (ACT-D), an anticancer antibiotic, enhanced the cytotoxicity of rh-TNF-α. Combined with ACT-D, rh-TNF-α showed the cytotoxicity on MDCK which possessed both TNF RI and RII receptors. The results indicate that the cytotoxicity of rh-TNF-α depends on the presence of TNF RI receptors on canine tumor cells.
A new cell line designated FRM was established from pleural effusion of a 13-year-old female cat with mammary adenocarcinoma. The cell line exhibited irregular round and polygonal shaped epithelial cells and demonstrated cell growth in a monolayer fashion with a doubling time of 22.4 hr. It possessed a modal chromosome number of 79. The immortality of this cell line was demonstrated using the TRAP assay which revealed a high telomeric activity of these cells. Scatchard analysis revealed quite low levels of estrogen receptors in both tumor mass produced in nude mice and FRM cells. Subcutaneous transplantation of the cells produced localized palpable masses in athymic nude mice within two weeks. This cell line may provide a good model for in vivo and in vitro studies on feline mammary tumors.
The effect of largest follicle aspiration or hCG administration before induction of superovulation on the ovarian response of Japanese Black beef cows was investigated using a crossover design in which induction of superovulation was attempted in every cow. The superovulatory response of cows whose largest follicle had been aspirated from the ovaries by ultrasound-guided follicular aspiration 1 day before induction of superovulation, did not differ from the response in non-treated control cows. In contrast, in cows given 5,000 IU of hCG 3 days before induction of superovulation, the proportions of fertilized ova and transferable embryos significantly decreased compared with the other groups.
An electroejaculation technique was applied to the Hokkaido brown bear (Ursus arctos yesoensis) for semen collection and characterization of their seminal traits. Ten captive sexually mature bears were anesthetized and subjected to 21 electroejaculation trials during their mating season in 1995 and 1996. Spermic electroejaculates were recovered from 6 of the 10 bears (14 of 21 trials). The semen was characterized by serous fluid of semitransparent white color and a neutral pH. The mean values of ejaculate volume, sperm concentration, percentage of sperm motility, percentage of live spermatozoa, and percentage of pleiomorphic forms were 2.7 ml, 471.6 × 10 6 cells/ml, 80.2%, 89.7% and 21.8%, respectively. Although there was considerable variation among the seminal traits of the individual bears, the electroejaculation technique was effective in obtaining ejaculates from captive bears.
The wild-type pseudorabies virus (WT-PRV) produced a round-type cytopathic effect (CPE) in PK-15 cell line of porcine kidney origin, while PRVgCs lacking in gC-transmembrane-anchor region and PRVgC- defecting in gC gene produced a syncytium-type CPE. The mouse embryo cell line (BALB/3T3 clone A31) were transfected with recombinant plasmid of pcDNA3 which incorporated with gC gene. The transfected A31/gC cells were stably expressing gC. Only a round-type CPE was observed in these cells infected with WT-PRV, while a syncytium-type CPE was observed in the cells infected with each of the PRVgCs and PRVgC-. Any viruses described above induced a syncytium-type CPE in A31/pcDNA cells transfected with a plasmid without gC gene. By WT-PRV infection, PK-15 cells generated about 2- or 8-fold more gC than the A31/gC and A31/pcDNA cells when gC was measured by hemagglutination test. Flowcytometric analysis revealed that amount of gC on the cell surface of A31/gC and PK-15 cells increased after infection with WT-PRV. Round-type CPE was observed with the increase of gC. These results suggest that the type of CPE formation induced by PRV is dominated by the amount of gC on the infected cell surface.
The clinical effects of recombinant feline interferon-ω (rFeIFN-ω), produced in silkworm by recombinant baculovirus, were examined in 3-4 month-old beagle dogs given an experimental canine parvovirus type-2 (CPV-2) infection. Clinical symptoms, such as pyrexia, vomiting, anorexia and diarrhea, were observed on day 4 after oral inoculation of 107 TCID 50 of CPV-2 (cc 238 strain) in almost all the inoculated dogs. From day 4, rFeIFN-ω (1 mega units/kg/day) or physiological saline was administered intravenously to infected dogs for 3 consecutive days. Seven out of 17 dogs treated with physiological saline showed hemorrhagic diarrhea and continuously expressed severe clinical enteritis; one dog died with a large amount of hemorrhagic rice-water stool on day 6 after viral exposure. In contrast, 4 out of 12 dogs treated with rFeIFN-ω showed severe clinical enteritis associated with intermittent diarrhea. Scoring of fecal condition revealed that treatment with rFeIFN-ω significantly shifted the enteritis from a severe to mild form. Furthermore, rFeIFN-ω administered in the morning decreased the number of dogs expressing clinical enteritis in the evening suggesting a rapid effect. Vomiting and anorexia were also improved by treatment with rFeIFN-ω. These results suggest that rFeIFN-ω can reduce severe enteritis caused by CPV-2 infection in dogs.
Previously, we reported that a feline T lymphoid cell line, FL74 cells, was very sensitive to feline parvovirus (FPV) infection. In the present study, we developed new quantitative methods for detection of FPV and virus neutralizing antibody against FPV using FL74 cells. The methods presented here were very simple and applicable to both canine parvovirus and feline panleukopenia virus.