Monoclonal antibodies (mAbs) were prepared against the 8597/CV94 strain of turkey rhinotracheitis virus (TRTV). These mAbs were used to investigate antigenic relationships among three strains (8597/CV94, 1162/92 and CVL14/1 strain) of TRTV, together with polyclonal chicken and rabbit antisera to 8597/CV94 strain, and guinea pig antisera to each of the three strains. Thirty mAbs to the glycoprotein (G:3 clones), fusion (F1:6 clones), phosphorylated (P:6 clones), nucleocapsid (N:12 clones), and matrix (M:3 clones) proteins of viral antigen were obtained by cell fusion. Among these, two mAbs to F1 protein showed virus neutralizing activity. The results of ELISA test indicated that some mAbs only reacted to the 8597/CV94 strain, some reacted to 8597/CV94 and 1162/92 strains, and others reacted to all three viral strains. In neutralization tests with the three virus strains, polyclonal chicken and rabbit antisera against the 8597/CV94 strain showed the same antibody titers. Results with four neutralizing mAbs including two previously reported mAbs [Ref. 21] indicated the titers of two mAbs (Pn2-2E and Pn3-2F) to 8597/CV94 were much higher than those to the other two viral strains. No differences were observed in the titers of the other two mAbs (Pn01-8E and Pn06-4D) against any viral strains. In cross-neutralization tests with polyclonal guinea pig antisera, there was some variations among viral strains. This work demonstrated that the Japanese isolate 8597/CV94 of TRTV is somewhat different in antigenicity from two British isolates from chickens and turkeys.
The nucleotide sequences of the 16S rDNA in 17 strains of 16 taxa of the genus Staphylococcus were determined. The sequences were compared phylogenetically together with the gene sequences of 10 (including 7 other species) Staphylococcus species retrieved from the DNA Data Bank of Japan. Although the primary and secondary structures of most of Staphylococcus species were very similar (homology values 96.4% or more) except for S. caseolyticus MAFF 911387T (homology values 95.4% or less), the 23 staphylococcal species were divided into 10 groups based on similarity, evolutionary distance and phylogenetic tree analysis. Nucleotide stretches in several variable domains in the 16S rDNA sequence appeared to be specific for the bacterial groups or the species. By comparing such characteristics in the sequence and phylogenies of 5 staphylococcal clinical isolates from bovine mastitis, canine and feline pyoderma, and feline urogenital syndrome with the information obtained in this study, the species level of each organism was identified.
We evaluated the feasibility of using the two-step polymerase chain reaction (PCR) in determining the withdrawal time of antibiotic treatment for Ehrlichia platys infection. We also present experimental evidence of a dog remaining a carrier after treatment with tetracycline. Canine infectious cyclic thrombocytopenia (CICT) was induced in 3 dogs by intravenous inoculation of blood infected with E. platys. Tetracycline was administered to one of the dogs for 2 weeks when parasitemia appeared. Although the hematologic abnormality of cyclic thrombocytopenia soon disappeared, a few parasitized platelets reappeared after the withdrawal of treatment, and the dog thus remained as a carrier. The other dogs were treated with doxycycline when parasitemic episodes first developed. The durations of antibiotic regimens were determined by the results of two-step PCR in which the 16S rDNA of E. platys was amplified from blood samples. Doxycycline was withdrawn after 8 days of treatment, and the follow-up monitoring continued for 3 weeks. The platelet counts of the 2 dogs remained within the normal range, and the etiologic agent of CICT was not found either by Giemsa staining or by the two-step PCR, indicating complete elimination of the agent.
A polymerase chain reaction (PCR) was developed for the detection of the hemolysin (alpha toxin) gene of Clostridium septicum. The PCR primers were designed from the sequence of the hemolysin gene and synthesized. A DNA fragment of 270 bp was amplified from10 strains of C. septicum, but was not from strains of C. chauvoei, C. perfringens, C. novyi, or C. haemolyticum. When the PCR product was digested with Sau3AI, two DNA fragments of the expected 148 bp and 122 bp were recognized. The lowest detectable threshold of PCR for the hemolysin gene was 3.8 × 10 3 cells/ml. The PCR technique may be useful for rapid detection or identification of C. septicum associated with malignant edema.
Alterations of T-cell subsets in the lymph nodes from FIV-infected cats in various clinical disease stages were examined histologically. In the early stage of infection (AP stage), follicular hyperplasia accompanied by expansion of the paracortical area was observed. Follicular involution and depletion with reduced paracortical area was observed in the ARC and AIDS stage nodes. The maximum section area of the entire popliteal lymph node was expanded significantly in the AP nodes. The paracortical area expanded in the AP nodes and decreased in the ARC and AIDS stage nodes. The cell density in the paracortical area in the AP nodes did not show a significant increase, while there was a significant reduction in the ARC and AIDS stage nodes. The lymph node CD4/CD8 ratio in the AP and ARC stages significantly decreased as compared with that of uninfected control cats, but conversion of the ratio was not seen. The estimated total numbers of CD4+ and CD8+ cells in the maximum section were increased in the AP stage but significantly decreased in the ARC and AIDS stages. Our study indicated that the lymphocyte depletion in the terminal ARC and AIDS stages of FIV infection was associated with both CD4+ cells and CD8+ cells. Findings obtained in this study might provide useful information for studying the pathophysiology of FIV infection.
Erythrocyte volumes of thoroughbred horses were measured. The volumes of splenectomized horses and sham-operated horses 2 hr after injection of 50Cr- tagged erythrocytes (at rest) and during maximal exercise were measured using the non-radioactive isotope 50Cr. Because splenic erythrocytes are released into circulation during exercise, it was estimated that the erythrocyte volumes of the sham-operated horses during maximal exercise are larger than those of the horses at rest. However, the erythrocyte volumes of the sham-operated horses at rest were about equal to those during maximal exercise. In the splenectomized horses, furthermore, erythrocyte volumes at rest and those at exercise were nearly equal. From these results, blood stored in the equine spleen is gradually mixed with circulating blood, and it was clarified that the phenomenon was completed within 2 hr. Although it is basically impossible to measure the circulating erythrocyte volume at rest using the erythrocyte tagged method, we observed that it is possible to measure the total erythrocyte volume using the 50Cr method. Also, the plasma volumes of the splenectomized horses during maximal exercise were found to be slightly smaller than those at rest. On the other hand, in the sham-operated horses, the plasma was decreased by a large quantity after maximal exercise. Therefore, it was suggested that the spleen participates in the phenomenon involving the disappearance of plasma from circulation due to exercise.
Since somatosensory evoked potentials (SEP) by hindlimb nerve stimulation are known to ascend bilaterally in the spinal cord, it was investigated whether or not simultaneous bilateral stimulation causes facilitation or inhibition of the stimuli. In an experiment using 36 adult Beagle dogs, the difference between simultaneous bilateral stimulation and unilateral stimulation was studied as to latencies and amplitudes. No significant difference was noted. However, the detection of far-field potentials by bilateral stimulation was not effective in which case far-field potentials did not recorded by unilateral stimulation. It was therefore confirmed that simultaneous bilateral stimulation does not cause facilitation or inhibition in the relay pathways, and does not affect the latency.
The effect of X-irradiation of cell lines from LEC and WKAH strain rats on a progression of cell cycle was investigated. When WKAH rat cells were exposed to 5 Gy of X-rays and their cell cycle distribution was determined by a flow cytometer, the proportion of S-phase cells decreased and that of G2/M-phase cells increased at 8 hr post-irradiation. At 18 and 24 hr post-irradiation, approximately 80% of the cells appeared in the G1 phase. On the contrary, the proportion of S-phase cells increased and that of G1-phase cells decreased in LEC rats during 8-24 hr post-irradiation, compared with that at 0 hr post-irradiation. Thus, radiation-induced delay in the progression from the G1 phase to S phase (G1 arrest) was observed in WKAH rat cells but not in LEC rat cells. In the case of WKAH rat cells, the intensities of the bands of p53 protein increased at 1 and 2 hr after X-irradiation at 5 Gy, compared with those of unirradiated cells and at 0 hr post-irradiation. In contrast, the intensities of the bands were faint and did not significantly increase in LEC rat cells during 0-6 hr incubation after X-irradiation. Present results suggested that the radioresistant DNA synthesis in LEC rat cells is thought to be due to the abnormal G1 arrest following X-irradiation.
A canine metallothionein cDNA obtained from the liver of a cadmium-treated beagle was cloned and sequenced. Asn at position 4 conserved among all mammalian metallothionein-1 and metallothionein-2 is replaced by Asp in the canine metallothionein cDNA clone. Because the acidic amino acid doesn't exist at either position 10 or 11 in the deduced amino acid sequence, it is supposed that this cDNA is derived from canine metallothionein-1 mRNA. Northern blot analysis using the cDNA as a probe revealed the induction of the canine metallothionein mRNA expression in the liver and kidney of a cadmium-treated beagle. Thus, the canine metallothionein cDNA obtained in the present study should provide an useful tool for the molecular investigation of metallothionein in dog.
A polymorphism in the experimentally successful peptide vaccine sequence (EVVWKEKKEVKDLDA, amino acids 134-148) derived from the 33 kDa piroplasm major surface antigen (p33) of Theileria sergenti was examined. The vaccine sequences obtained by PCR amplification and sequencing of the p33 gene from a total of 15 parasite-infected cattle blood samples collected from 4 prefectures through Hokkaido to Kumamoto revealed the two major sequences (Ikeda and Chitose stock types) either of which was identified in all samples. Since the peptide vaccine develops the parasite species- or stock-specific immunity in the animals, an application of the two major peptide sequences as cocktailed vaccine should be evaluated for a practical use of this strategy to controlling T. sergenti infection in Japan.
Apoptosis induced by high doses of Galactosamine (GalN) was investigated in mice hepatocytes in vivo. In mice intraperitoneally (ip) treated with GalN 3 g/kg, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were first observed at 6 hr postadministration (PA). Both acidophilic bodies in hematoxylin and eosin (HE)-stained sections and TUNEL-positive cells were markedly found at 24 hr PA. At 48 hr PA, cellular degeneration and necrosis of hepatocytes were prominently observed, and TUNEL-positive cells were scarcely found. In the mice ip treated with GalN 1.5 g/kg, the lesion was milder than that in those treated with GalN 3 g/kg. Acidophilic bodies and TUNEL-positive cells were scarcely found at 24 hr PA, whereas they were markedly seen at 48 hr PA. In addition, a ladder-like DNA fragmentation pattern by agarose gel electrophoresis was observed most remarkably at 24 hr PA with GalN 3 g/kg and at 48 hr PA with GalN 1.5 g/kg, and less distinctly at 48 hr PA with GalN 3 g/kg. On the other hand, sGOT and sGPT activities increased prominently at 48 hr PA with GalN 3 g/kg. These results suggest that the cell death induced by high dose of GalN may be caused by apoptosis, and subsequently by necrosis in vivo.
When verotoxin (VT)-producing attaching and effacing Escherichia coli (AEEC, serotype O5: H-) were inoculated perorally into 10-day-old rabbits, attaching of the E. coli to enterocytes and effacing of their microvillous portion were observed extensively from the ileum to the colon. Subsequent apoptotic changes of the infected enterocytes were demonstrated by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction and electron microscopy. Apoptosis was also induced in cultured Vero cells by inoculation of VT extracted from the AEEC. This study clarified that VT-producing AEEC induce apoptosis of enterocytes, causing mucosal damage.
A spontaneous pituitary gangliocytoma with abundant, immature neuronal cell elements was found incidentally in a 109-week-old female Fischer 344 rat. The pituitary parenchyma was largely occupied by a tumor nodule with necrotic and hemorrhagic foci and cyst. The tumor was composed of mature ganglion-like (M) cells, small immature ganglion (I) cells and transitional (T) cells, with a fibrillar matrix. The I and T cells were intermingled with the M cells or were arranged in compact clusters, in which the I cells formed perivascular rosette-like structures, sometimes with mitotic figures. Immunohistochemically, all types of tumor cells were positive for neuron-specific enolase, and only the M cells was positive for chromogranin A. This result may be correlated with the degree of cytodifferentiation.
Gymnema sylvestre (GS) is one of the Asclepiad strains that grows in South-east Asia. Their therapeutic effects for treating diabetes mellitus, rheumatic arthritis and gout have been well known for a long time. However, the problem is that GS suppresses sweetness and tastes bitter. For this study, we chose Gymnema inodorum (GI) instead of GS, since it has an advantage that it does not suppress sweetness nor is it bitter in taste. In this paper, effects of glucose availability of some saponin fractions (F-I to F-IV) extracted from GI leaves, which were obtained by high-performance liquid chromatography were studied on a high K+-induced contraction of guinea-pig intestinal smooth muscle, O2 consumption on guinea-pig ileum, glucose-evoked transmural potential difference (ΔPD) of guinea-pig everted intestine and blood glucose level in glucose tolerance tests on rats. The extracts of GI leaves suppressed the intestinal smooth muscle contraction, decreased the O2 consumption, inhibited the glucose evoked-transmural potential, and prevented the blood glucose level. Our studies suggest that the component of GI inhibits the increase in the blood glucose level by interfering with the intestinal glucose absorption process.
Disposition profile of ampicillin (ABPC) among honeybees, larvae, honey and royal jelly in a hive after oral dosing to adult bees was studied. Four honeybee colonies were administered the single dose of ABPC at the rate of 30 mg/hive by addition to sugar syrup or pollen substitute (paste) for 1 day intake. The colonies received ABPC in syrup showed high drug residue levels in honey and it lasted over 14 days beyond the detection limit of residual analysis. In the hives given ABPC in paste, relatively low honey residues were found, however, the distributions of the drug in young larvae and jelly which was the food of the larvae were very low. ABPC was considered to be a promising drug for the control of American foulbrood, an important bacterial disease of honeybee larvae, because of its high antibacterial activity to the pathogen, Paenibacillus larvae, and instability of residue in honey as human food. The low distribution in young larvae, the target of the disease, threw a doubt on the efficacy of ABPC for American foulbrood control.
The fitness of 8 Andalusian horses between 3 and 4 years of age was analysed. The animals were subjected to an exercise test on a sandy track consisting of 2 stages of different intensities. The first stage was of submaximal intensity at 4 speeds which increased progressively (4.17, 5.56, 6.94 and 8.33 m/sec.) covering a distance of 1,000 m in each level. Between each of these speeds, the horses rested for 2 min. The second stage was a maximal speed test over the same distance carried out 2 min after the ending of the maximal phase. Data of heart rate, plasma lactate concentration, velocity, PCV and pH in the blood were obtained. Maximum heart rate, maximum velocity, VLA2, VLA4, peak lactate, minimum pH and maximum PCV were considered functional indexes. A principal component enabled us to segregate horses according to their fitness and in relation to the information provided by the trainers in charge of these horses. The most discriminant variables in order to segregate horses were pHmin, VLA4, HRmax, VLA2 and Vmax. Differences between horses in relation to PCVmax were not found. The influence of each one of these functional indexes on the test exercise tolerance was discussed.
Cardiovascular reflex mechanisms by topical laryngeal instillation of capsaicin (CAPS) or distilled water were evaluated in anesthetized chronic tracheostomized dogs. Both CAPS (10 μg/ml) and water instillation into the isolated upper airway caused a significant decrease in heart rate (P<0.05) and a significant increase in blood pressure (P<0.05) from the values before instillation under both spontaneous and controlled ventilation. The bradycardia was significantly reduced by atropine pretreatment (P<0.05) and the hypertension was significantly decreased by phentolamine and propranolol pretreatments (P<0.01). A higher concentration of CAPS (100 μg/m l) instillation considerably reduced the response to subsequent CAPS (100 μg/ml) instillation, whereas the response to water was sustained, indicating the desensitization of laryngeal CAPS-sensitive endings. All the reflex responses to CAPS and water were eliminated by topical anesthesia with lidocaine. It was concluded that the laryngeal cardiovascular reflex responses were mediated by the afferents such as the laryngeal CAPS-sensitive presumably C-fiber endings or water-responsive receptors and by both the parasympathetic and sympathetic nervous systems as efferents.
Two dogs and a cat with intracranial lesions were evaluated by both computed tomography (CT) and magnetic resonance (MR) imaging. In a dog with vestibular syndrome, better quality images of the medulla oblongata surrounded by thick bones were obtained by MR than by CT, on which the appearance of artifacts impeded the clear image of the area. In a dog with multiple brain metastases of lymphoma, contrast CT delineated lesions more clearly than MR, which was performed one week after CT. During that week dexamethasone which might affect the clarity of MR images of the lesion was administered to reduce brain edema. In a cat with meningeal syndrome of lymphocytic leukemia, only contrast MR imaging identified the width and site of the lesion. These results indicate that it is necessary to select either one of these imaging methods according to the type and site of lesions that are suspected in a particular case.
In the present study, the feasibility of intrathecal indwelling catheters in the preparation of a repeated subarachnoid hemorrhage (SAH) model in dogs, as well as chronic intrathecal administration of therapeutic agents against the ensuing cerebral vasospasm was examined. Briefly, through a small suboccipital incision, two catheters were introduced into the subarachnoid space so that their tips were positioned in the prepontine cistern. One was used to induce SAH by infusing autologous blood, and the other to administer pharmacological agents (saline and/or saline containing a dye in this study) by means of an osmotic pump. The occurrence of cerebral vasospasm was followed by angiography via the catheter placed in the vertebral artery. The obtained results show: i) the injected blood effectively formed a subarachnoid clot in the prepontine cistern, invariably leading to the occurrence of severe cerebral vasospasm of the basilar artery; ii) the fluid injected by the osmotic pump was evenly distributed in the cisterns around the brain stem; iii) on post mortem pathological examination, no injury of the brain or the major arteries ascribable to the placement of catheters was found. Therefore, the present model is considered to be useful for both the investigation of pathophysiology and therapy of cerebral vasospasm following SAH, to be more favorable from the standpoint of animal protection, and more convenient and reliable than those used until now.
Two dogs with pyometra were treated by laparoscopy assisted ovariohysterectomy. Hemostasis of the mesovarium was achieved with an ultrasonic scalpel and hemoclips. Both ovaries and the uterus were exposed via a 10-mm caudal port that was enlarged to 3 cm and the uterine cervix was excised after ligation of the uterine arteries. These cases were the first report on ovariohysterectomy for pyometra by laparoscopy assisted surgery in the veterinary field.
The aim of this study was to establish radioimmunoassay (RIA) for saliva estrone sulfate (E1S), and to elucidate changes in saliva E1S during pregnancy in the sow. Saliva E1S was extracted using a commercially available solid phase column, and the E1S fraction obtained was subjected to RIA. The sensitivity of the RIA was 29.7 pg/tube. The intra-and inter-assay coefficients of variation were 5.5-8.4% and 13.1-19.5%, respectively. Mean recovery for E1S added to saliva samples was as high as 99.9%. A significant positive correlation (r=0.54, n=69, p<0.01) existed between saliva and plasma E1S concentrations. During gestation, the changing patterns of saliva and plasma E1S concentrations were essentially the same, and two peaks of E1S concentrations were observed, one around day 30 and another just before parturition, although E1S concentrations in saliva remained at only 2.4-38.1% (mean 11.4%) of those in plasma E1S. Thus, the present study has made it possible to measure saliva concentrations of E1S and demonstrated a high degree of positive correlation between saliva and plasma E1S concentrations. These results suggest that diagnosis of early pregnancy and of normal or abnormal fetal development could be made by measurements of E1S in saliva.
Objectives of this study were to show postpartum plasma PGF2α metabolite (PGFM) profile, to clarify whether endogenous PGF2α plays a certain role in the uterine involution in cows with dystocia and/or retained placenta, and to examine the effects of fenprostalene, a long-acting PGF2α analog, on the uterine involution and reproductive performance of the cows with abnormal puerperium. A group of 27 cows with dystocia and/or retained placenta showed a massive release of PGF2α after parturition as indicated by a rise of plasma concentrations of PGFM, significantly higher than 33 cows with normal puerperium. The duration of the elevated plasma PGFM concentrations in the cows with abnormal puerperium was shorter than that of the normal cows. In cows with normal puerperium, those showing relatively longer duration of elevated plasma PGFM levels needed a shorter period for postpartum uterine involution than the cows showing a shorter duration of the PGFM elevation (P<0.01), while no such relationship was observed in cows with abnormal puerperium. In field trials, an administration of an exogenous PGF2α, fenprostalene, at 7 to 10 days (78 cows) or 14 to 28 days postpartum (74 cows) was found to be effective in facilitating uterine involution and resumption of ovarian cyclicity, and improved reproductive performance. It may be concluded that a large amount of PGF2α is released for a relatively shorter period in cows after dystocia and/or retained placenta and the elevation of PGFM is not responsible for the uterine involution. The administration of the exogenous PGF2α was shown to be effective at improving the postpartum reproductive performance of cows with abnormal puerperium.
The nucleoprotein genes of Akabane virus S RNA segment from 21 Japanese and two Australian isolates were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) using a primer set containing the initiation and termination codon of the gene. The RT-PCR products were sufficiently produced from the purified virion RNAs of all the isolates, and then analyzed by enzymatic digestion with 11 restriction endonucleases. Digestion with the eight restriction enzymes revealed sequence variation of the isolates. Restriction fragment length polymorphism (RFLP) profiles obtained by digestion revealed the existence of four major groups (genogroups) among the isolates. The two Australian isolates had extremely different RFLP profiles than the Japanese isolates. The data demonstrate the usefulness of analyzing the RFLP patterns to understand the genetic variability of AKA virus isolated in Japan and Australia.
Quantitative measurement of reverse transcriptase-inhibiting (RTI) antibodies in Japanese household cats naturally infected with feline immunodeficiency virus (FIV) was performed by poly A-linked colorimetric reverse transcriptase assay (PAC-RTA). Eight FIV-seropositive plasma samples were diluted twofold from 1:10 to 1:160 and incubated with FIV RT. Fifty percent RTI activity (RTI50) was calculated from a dose response PAC-RTA curve. The plasma of FIV-seropositive cats showed different RTI activities against two Japanese isolates and Petaluma strain. Six of eight plasma samples showed RTI activities against the Japanese isolates (subtype B), but only one showed RTI activity against Petaluma strain (subtype A). It is important to use the appropriate strain as a source of RT for detection of RTI antibodies in cats.