Rapid method utilizing the polymerase chain reaction for detection of canine parvovirus in feces of diarrheic dogs.

By using primers based on the sequence of the VP2 gene of canine parovirus (CPV), we established a rapid and specific assay for identification of the virus from fecal specimens based on the polymerase chain reaction (PCR). By use of a pair of primers, a specific 226-bp sequence was amplified by the PCR. All strains of CPV tested gave a specific amplification product by the PCR, while neither porcine parovirus nor host cell did so. The PCR assay can detect fewer particles of CPV than the conventional methods, being able to detect CPV from fecal specimens in a rapid manner, provided that gel filtration of the samples through a spun column was done to remove inhibitory substances from the fecal specimens. These results suggest that the PCR assay can detect the presence of CPV in dogs early enough to prevent secondary infection by CPV in veterinary hospitals.

Canine parvovirus binds to multiple cellular membrane proteins from both permissive and nonpermissive cell lines.

For identification of canine parvovirus (CPV) binding protein, the SDS-solubilized cell membrane fraction from a permissive cell line. CRPK, was subjected to the virus overlay protein blot assay (VOPBA). Competitive inhibition experiments showed the presence of multiple CPV-binding proteins with molecular masses of 36, 35, 33, 31, 29, 27, 25, and 23 kDa. CPV-binding proteins of same molecular masses were also detected in membrane fractions from nonpermissive, as well as other permissive, cell lines. We confirm that the mechanism of nonpermissiveness to CPV is not operative at the cellular attachment level.

Rapid and efficient method to eliminate substances inhibitory to the polymerase chain reaction from animal fecal samples.

To detect pathogenic viruses in animal fecal specimens by polymerase chain reaction (PCR) assays, it is important to remove or inactivate PCR-inhibitory substances. Recently, it was reported that such inhibitory substances in human feces could be efficiently eliminated by a cationic surfactant, Catrimox-14 (Iowa Biotechnology, Iowa) during extraction of viral RNA. In the present report, Catrimox-14 was successfully applied to detect pathogenic viruses in fecal specimens from a variety of animals. By extraction of viral DNA in the presence of this cationic surfactant, the PCR assay could detect canine parvovirus (CPV) in all fecal specimens prepared from 13 kinds of animals, i.e., cat, chicken, cow, dog, gerbil, goat, golden hamster, horse, mouse, pig, rat, rabbit, or sheep. Pretreatment by gel-filtration or boiling failed to remove or inactivate the PCR-inhibitory substances in fecal specimens from mouse, goat, rat, and sheep.

Influences of alkaline ionized water on milk yield, body weight of offspring and perinatal dam in rats.

The authors previously reported that male offspring of mothers rats given alkaline ionized water (AKW) showed a significantly higher body weight by day 14 after birth than did offspring of mother rats given tap water (TPW); furthermore, marked myocardial necrosis and fibrosis were observed particularly in the former male offspring at the age of 15 weeks. In the present experiment we looked for differences in bioparameters, namely the milk yield of mothers and suckled milk volume of the offspring, between the AKW- and the TPW-treated groups in order to reveal the factors which cause the unusual body weight gain in the offspring. Even though we were able to repeat our previous observation (the body weight of the male offspring of the AKW group increased significantly more by day 14 and 20 after birth and of the female by day 20 after birth than did that of the TPW group (p < 0.05), no significant difference was noted in any of the bioparameters, including those related to milk production and consumption. It is thus suspected that the water-hydrated cation, which was transferred either to the fetus through the placenta or to the offspring through the milk, might be the cause of the unusual body weight increase. Since calcium plays an important role in skeletal formation, it is tentatively concluded that the higher calcium concentration of AKW enriched the mother, serum calcium which was transferred to the fetus through the placenta and to the offspring through the milk.

Histopathological influence of alkaline ionized water on myocardial muscle of mother rats.

We have reported that a marked necrosis and subsequent fibrosis of myocardium occurred among male rats 15 weeks old given alkaline ionized water (AKW) during gestation and suckling periods, and after weaning. In this study, it was examined whether similar lesions would occur in mother rats which were given AKW from day zero of gestation to day 20 of lactation. The myocardial lesion in the mother rats given AKW showed cell infiltration, vacuolation and fibrosis in the papillary muscle of the left ventricle, as were observed in male rats of 15 weeks old. Myocardial degeneration may cause a leakage of potassium into the blood that results in a higher concentration of potassium in the blood in the test group than in that of the control group given tap water.

Influences of alkaline ionized water on milk electrolyte concentrations in maternal rats.

We previously reported that body weight on day 14 after birth in male offspring of rats given alkaline ionized water (AKW) was significantly heavier than that in offspring of rats given tap water (TPW), but no significant difference was noted in milk yield and in suckled milk volume between the two groups. Additionally, the offspring in the AKW group and TPW group were given AKW and TPW, respectively, at weaning, and unexpectedly, the necrotic foci in the cardiac muscle were observed at the 15-week-old age in the AKW group, but not in the TPW group. The present study was designed to clarify the factors which are involved in that unusual increase of body weight and occurrence of cardiac necrosis. Eight dams in each group were given AKW or TPW (control) from day 0 of gestation to day 14 of lactation. The milk samples were collected on day 14 of lactation and analyzed for concentrations of calcium (Ca), sodium (Na), potassium (K), magnesium (Mg) and chloride (Cl). The AKW and TPW were also analyzed. Ca, Na and K levels in milk were significantly higher in the AKW group compared to the TPW group. No significant difference was noted in the Mg and Cl levels between the two groups. These data suggested that the Ca cation of AKW enriched the Ca concentration of the milk and accelerated the postnatal growth of the offspring of rats given AKW.

Effect of heparin on a haemagglutinin of porcine reproductive and respiratory syndrome virus.

Heparin inhibited haemagglutination by porcine reproductive and respiratory syndrome virus (PRRSV) and by Aujesky's disease virus, but failed to inhibit haemagglutination by parainfluenza virus type 3. The minimal inhibitory concentration of heparin required to inhibit 8 HA U of PRRSV haemagglutinin ranged from 0.1 to 1 U ml-1. Mouse erythrocytes failed to combine with the haemagglutination inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by PRRSV. The formation of a haemagglutinin-heparin complex could be observed by sedimenting heparin with the haemagglutinin. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.

Hemagglutination with equine arteritis virus.

Equine arteritis virus (EAV) grown on RK13 cell cultures was tested for hemagglutination (HA) with erythrocytes from a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with erythrocytes from mouse and chicken but not with those of cattle, horse, rabbit, guinea pig, mongolian gerbil, goose or chick embryo. Chickens showed an individual variation in agglutinability of their erythrocytes, requiring selection of birds to obtain erythrocytes for HA. The HA activity was enhanced by treatment of virus materials with Tween 80 followed by treatment with ether. The HA reaction was inhibited by specific antiserum. Higher HA-inhibiting (HI) antibody titers were obtained by the incubation of serum-HA antigen mixture at 4 degrees C for 24 hr. HI antibody titers of individual horse sera showed a significant positive correlation with their neutralizing antibody titers.

Characterization of porcine reproductive and respiratory syndrome virus hemagglutinin.

Porcine reproductive and respiratory syndrome virus hemagglutinin (HAin) was readily adsorbed on mouse erythrocytes at 4, 22, or 37 degrees C, but not on goose erythrocytes. The adsorbed HAin could not be eluted from the cells by resuspending in phosphate buffered saline, by incubating at 37 or 50 degrees C, or by incubating in the presence of neuraminidase. The hemagglutinating activity was not dependent on the pH and NaCl molarity tested. The receptor of mouse erythrocytes for the HAin was relatively stable to trypsin, neuraminidase, sodium deoxycholate (DOC), potassium periodate (KIO4), dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and formalin treatments. The HAin was inactivated by 2-ME and was gradually inactivated by pepsin, formalin and DTT, but not by beta-glucosidase, trypsin, alpha-amylase, papain, phospholipase C, neuraminidase, KIO4, and ethylendiamine tetraacetic acid (EDTA) treatments. The HAin was stable at 37 degrees C or lower temperatures, but not at 56 degrees C or higher. The HAin was relatively resistant to ultraviolet irradiation and sonication. In the equilibrium centrifugation of the HAin preparation on a CsCl density gradient, the HAin activity showed a sharp peak at 1.17 g/cm2. In the SDS-PAGE analysis, the structural polypeptide of HAin in the peak fraction seems to be the nucleocapsid (N) polypeptide with molecular weight of 15 kDa.

Effect of heparin on infection of cells by equine arteritis virus.

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.

Effect of heparin on hemagglutination by equine arteritis virus.

Heparin inhibited hemagglutination (HA) by equine arteritis virus (EAV) as well as did HA by Aujeszky's disease virus (ADV), but failed to inhibit HA by parainfluenza virus type 3 (PIV-3). The minimal concentration of heparin required to inhibit 8 HA U of EAV was 0.1 U/ml. In addition, most EAV hemagglutinin was retained by heparin acrylic beads, as was ADV hemagglutinin, but was not PIV-3 hemagglutinin. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by EAV. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.

Hemagglutination with crane herpesvirus.

A crane herpesvirus (CrHV) grown in chicken embryo fibroblast (CEF) cells was tested for hemagglutination (HA) with erythrocytes from a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with erythrocytes from mouse, ddY and BALB/c strains, but not with those from cattle, sheep and chicken. Mice, ddY strain, showed an individual variation in agglutinability of their erythrocytes and erythrocytes from BALB/c gave a higher HA titer. The HA activity was inhibited by the sera obtained from naturally infected cranes, experimentally infected duck and immunized rabbit with CrHV. HI antibody titers of these sera showed a closely positive correlation with their neutralizing antibody titers.

Adhesion and releasing of Poliovirus to activated sludge of wastewater purifying plants.

Pathogenic enteric viruses are released from infected persons through domestic wastewater to the environment. From that point of view, the knowledge of the viral behavior in wastewater purifying process is important: it is, however, still poorly understood. In this study, we reported the adhesion of Poliovirus to activated sludge samples taken from wastewater purifying plants by using a model system. More than 10(6) particles adhered to one gram (wet) of activated sludge, and the adhered viral particles maintained infectivity for longer period of time and showed higher thermo-resistant than the free viral particles. The adhered viral particles were released by increase of salt concentration or alkaline pH buffer as infectious particles. The data suggest that pathogenic viruses could be enriched and maintain the infectivity in the activated sludge, and released to environments under certain conditions.

[Primary lung cancer with solitary adrenal tumor].

We experienced that two lung cancer patients who had been considered to be potentially resectable were preoperatively pointed out enlarged adrenal glands. Therapeutically, discrimination between benign and malignant adrenal mass lesion is an important problem. Case 1: After a 3 course neoadjuvant chemotherapy, a 50-year-old man had a left adrenalectomy, which revealed non-functional adenoma. One month later, a left lower lobectomy for T3N2 adenocarcinoma was performed. Case 2: A 64-year-old man had a right upper lobectomy for T2N0 adenocarcinoma, firstly. Two months later, a left adrenalectomy was done because a rapid growth and lumbago, which revealed metastatic adenocarcinoma originating from the lung. The patient died of brain and stomach metastases 4 months postoperatively.

[Assisted thoracoscopic resection of huge mediastinal neurogenic tumor with Horner syndrome and superior vena cava syndrome].

A 51-year-old female suffering from right shoulder pain had chest radiography, which showed the huge round shadow (10 x 8.5 x 8.0 cm) on the right upper lung field. Horner syndrome and superior vena cava syndrome were presented. Thoracoscopic appearance showed that the tumor with complete smooth capsulation was neurogenic. After that added ideal thoracotomy (compatible with location and length) was performed, from which tumor was carried out and made a pathologic diagnosis of neurilemoma originated from thoracic sympathetic nerve. Harmonic Scalpel, an ultrasonically activated surgical device was effective in such a case of using near by nerve and small vessels. After operation, superior vena cava syndrome except for Horner syndrome was improved. It is important that we should consider less invasive and safe approach with combination of thoracoscopic and thoracotomic procedures even in case of huge tumor.

[Surgical repair of partial anomalous pulmonary venous connection to the coronary sinus--a case report of PAPVC with mitral and tricuspid valve regurgitation in adult].

We have recently performed a successful repair of a total right anomalous pulmonary venous connection to the coronary sinus associated with mitral and tricuspid regurgitation. A 54-year-old female was inaccurately diagnosed as atrial septal defect associated with mitral and tricuspid regurgitation preoperatively. Operation revealed that all the right pulmonary veins connected to the coronary sinus. Mitral regurgitation which might be caused by distortion of the anterior leaflet was repaired by McGoon's technique. A Carpentire-Edwards ring was employed for the tricuspid annuloplasty. Repair of the atrial septal defect was undertaken with a pericardial septation patch which was sutured in place. The roof of the coronary sinus was widened by cutback, so that blood from the pulmonary veins could drain into the left atrium. The postoperative course was uneventful, except for a supraventricular arrhythmia.