Genogrouping of vaccine breakdown strains (VBS) of feline calicivirus in Japan.

Although prevention of feline calcivirus (FCV) infection by vaccination has been attempted, and isolation of FCV, development of the disease, and a few fatal cases in vaccinated cats have been reported. Fifteen FCV strains isolated from cats that had been vaccinated with commercially available FCV vaccines (F9, FCV-255, and FC-7) were genogrouped. Molecular analysis of viral genomes involved the construction of a phylogenetic tree of capsid genes using the NJ method. Cat anti-F9 serum and rabbit anti-FCV-255 serum were used for virus neutralization tests. Molecular phylogenetic analysis of the amino acid sequences of 15 virus isolates and those of the previously published and GenBank-deposited 9 global and 14 Japanese strains showed that 8 (53%) of the 15 virus isolates as well as the vaccine strains F9 and FCV-255 belonged to genogroup I (G(A)I), and 7 (47%) belonged to genogroup II (G(A)II). Of the 8 G(A)I strains, 2 were isolated from cats that had been vaccinated with an F9 strain live vaccine, 5 from cats vaccinated with an FCV-255-derived vaccine, and 1 from a cat vaccinated with an FC-7-derived vaccine. Of the 7 GAll strains, 5 were isolated from cats that had been vaccinated with the F9 strain live vaccine, 1 from a cat vaccinated with the FCV-255-derived vaccine, and 1 from a cat vaccinated with the FC-7-derived vaccine. These results indicate that more vaccine breakdown strains isolated from the cats vaccinated with the F9 strain-derived vaccine belong to G(A)II than to G(A)I, whereas more vaccine breakdown strains isolated from the cats vaccinated with the FCV-255 strain-derived vaccine belong to G(A)I than to G(A)II, and that when the FC-7 strain-derived vaccine is used, the vaccine breakdown strains belong almost equally to G(A)I and G(A)II. Thus, the genogroups of virus isolates varied with the vaccine strain used (p < 0.05). On the other hand, the neutralizing titres of feline anti-F9 serum and rabbit anti-FCV-255 serum against the 15 isolates were very low, showing no relationships between neutralizing antibody titres and genogroups. The DNA sequence identities between the virus isolates and the vaccine strains were low, at 70.6-82.9%, and no strains were found to have sequences derived from the vaccine strains. Alignment of amino acid sequences showed that the G(A)I or G(A)II virus isolates from the F9-vaccinated cats differed at position 428 of the 5' hypervariable region (HVR) of capsid region of the F9 strain, whereas those from the FCV-255-vaccinated cats differed at positions 438, 453, and 460 of the 5'HVR of capsid region E of the F9 strain. We speculate that these differences influence genogrouping. The amino acid changes within the F9 linear epitopes common to G(A)I and G(A)II were noted at positions 450, 451, 457 of 5'HVR of the capsid region E in the isolates from F9-derived vaccine-treated cats, and 449, 450, and 451 of 5'HVR of capsid region E in the isolates from FCV-255-derived vaccine-treated cats, suggesting that these amino acid changes are involved in escapes. These results suggest that alternate vaccination with the F9 and FCV-255 strains or the use of a polyvalent vaccine containing GAll strains serves to inhibit development.

Transfection of antisense core 2 beta1,6-N-acetylglucosaminyltransferase-1 cDNA suppresses selectin ligand expression and tissue infiltration of B-cell precursor leukemia cells.

B-cell precursor (BCP) leukemia cells infiltrate into peripheral organs and the disease often relapses. Inhibition of tissue infiltration may improve the treatment outcome of BCP-leukemia patients. Selectin ligand has been suggested to play an important role in the infiltration of leukemia cells. However, the regulation mechanisms and involvement in tissue infiltration of selectin ligand expression in BCP-leukemia cells are not fully understood. In this study, we report that BCP-leukemia cells express selectin ligand on O-sialoglycoproteins. Core 2 beta1,6-N-acetylglucosaminyltransferase-1 (C2GnT-1) is mainly expressed in BCP-leukemia cells. Transfection of the antisense C2GnT-1 cDNA resulted in a significant reduction of either selectin ligand expression or selectin-dependent cell adhesion in BCP-leukemia cell line KM3 cells. Migration ability into mouse peripheral organs was reduced significantly in the antisense transfectant. These findings suggest that C2GnT-1 regulates selectin ligand expression. Downregulation of the selectin ligand expression level inhibits tissue infiltration of BCP-leukemia cells. C2GnT-1 may be a candidate of therapeutic target for the inhibition of infiltration of leukemia cells.

Capsid protein genetic analysis and viral spread to the spinal cord in cats experimentally infected with feline calicivirus (FCV).

We investigated primitively the molecular basis of the neural spread of a feline calcivirus isolate (FCV-S) from the spinal cord of a cat that died after manifesting excitation. Experimental infections of cats with three clones from parent virus isolate FCV-S, isolated based on plaque size, were performed, and virus recovery from the spinal cord and the nucleotide and predicted amino acid sequences of the viral capsid protein region (ORF2) were compared. In the experimental infection with the one-time cloned virus (C1L1) isolated from a large plaque, the C1L1 was recovered from the spinal cord. In contrast, seven-times cloned C6L7 (from large plaque) and five-times cloned C5S2 (isolated from small plaque) were not recovered from the spinal cord. Genetic analysis of the capsid protein gene of the three viral clones revealed that four bases were different and two amino acids were different at positions 34 (Val in C6L7 and Ala in C1L1 and C5S2) and 46 (Leu in C6L7 and Pro in C1L1 and C5S2) between C6L7 (with large plaque) and C5S2 (with small plaque). The amino acid at position 434 of C1L1 was different from those of C6L7 and C5S2 (Gly in C1L1, D (Asp) in C6L7 and C5S2). From these results, the plaque size seemed not to be related to the spread of virus to the spinal cord. Clone C1L1, which spread to the spinal cord, had a difference of one amino acid from the other two clones, which may be related to the ability to spread to the spinal cord.

Purification and properties of beta-galactosidase from Aspergillus oryzae.

Beta-Galactosidase [EC 3.2.1.23] has been purified from a culture of Aspergillus oryzae by 2-propanol fractionation, column chromatography on DEAE-Sephadex A-50 and Sephadex G-200. The preparation was homogeneous on ultracentrifugation and disc electrophoresis. The enzyme showed pH optima of 4.5 with ONPG-1 as a substrate and 4.8 with lactose as a substrate. The stable pH range was from 4.0 to 9.0 and the optimum temperature was 46 degrees. The Michaelis constants were 7.2 X 10-minus 4 M with ONPG and 1.8 X 10-minus 2 M with lactose. Hg-2+, Cu-2+, N-bromosuccinimide, and sodium laurylsulfate caused marked inhibition. The apparent molecular weight was calculated to be about 105,000 by Sephadex gel filtration and sucrose density gradient centrifugation.

Muscle layer- and region-dependent distributions of oxytocin receptors in the porcine myometrium.

The aim of the present study was to clarify smooth muscle- and region-dependent distributions of the oxytocin receptor that mediates oxytocin-induced contraction in the nonpregnant porcine myometrium by means of mechanical and radioligand ([3H]-oxytocin) binding studies. In Krebs solution, oxytocin (0.1-300 nM) caused concentration-dependent contractions of the cornual myometrium, and the longitudinal muscle was more sensitive than the circular muscle. [Arg8]-vasopressin and [deamino-Cys1, D-Arg8]-vasopressin also contracted the myometrium, and the order of the potency was oxytocin > [Arg8]-vasopressin > [deamino-Cys(1), D-Arg(8)]-vasopressin. Treatment with a high concentration of oxytocin selectively inhibited the contraction of oxytocin and [Arg8]-vasopressin without affecting the responses of acetylcholine and high-K+. Selective cross inhibition was also observed in the presence of a high concentration of [Arg(8)]-vasopressin. The oxytocin-induced contraction was resistant to tetrodotoxin and atropine, but was reduced by verapamil or by the removal of external Ca2+, indicating that oxytocin has a direct action on smooth muscle cells and that extracellular Ca2+ plays an important role for the contraction. In Kumagai solution, oxytocin caused contraction of the cornual longitudinal muscle (-logEC50 = 8.5) but not the circular muscle. Longitudinal muscles of other regions (corpus and cervix) were also responsive to oxytocin, but the -logEC50 value differed from region to region (cornua > corpus = cervix). On the other hand, oxytocin failed to cause contraction of the corpus and cervical circular muscles. 3H-Oxytocin bound to crude membrane preparations of the myometrium in a concentration-dependent (0.084-2.7 nM) saturable manner. Scatchard analysis of equilibrium binding data revealed the presence of a single class of binding site with an apparent dissociation constant (Kd, 1.1-1.5 nM), but receptor density (Bmax) differed in the two muscle layer types (longitudinal muscle: circular muscle = 5:1) and tended to decrease from the cornua to the cervix. In conclusion, the receptor specific for oxytocin is present in the porcine myometrium and mediates the contractile responses of both oxytocin and [Arg8]-vasopressin. The distribution of the oxytocin receptors differs according to the type of muscle layer (longitudinal muscle > circular muscle) and the region of the uterus.

Detection of feline herpesvirus 1 DNA by the nested polymerase chain reaction.

The thymidine kinase region of feline herpesvirus 1 (FHV 1) genome in ocular/nasal swabs from cats with clinical manifestations of upper respiratory disease was amplified by nested polymerase chain reaction (nested PCR). Two primer pairs were prepared for nested PCR. FHV 1 DNA in ocular/nasal swabs was extracted using instaGene-DNA purification matrix. Nested PCR for the FHV 1 culture supernatants was ten times as sensitive as single PCR. On comparing viral isolation with single PCR and nested PCR for the detection of FHV 1 in ocular/nasal secretions, of 5 samples that yielded infectious virus in cell culture, 3 (60%) were positive in single PCR and 5 (100%) were positive in nested PCR. When 22 ocular/nasal swabs that did not yield FHV 1 were assayed, 3 were negative in both single PCR and nested PCR, 2 were positive in both single and nested PCR and 17 were positive in only nested PCR. Thus, FHV 1 was detected in 19/22 (86.4%) by the nested PCR and in 2/22 (9%) by single PCR. These results show that nested PCR is 4.8 (24 positive samples/5 positive samples) times as sensitive as single PCR.

Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA.

PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V. parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.

An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody.

To detect antibody against feline herpesvirus 1 (FHV-1) in the sera of cats, the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using nuclear antigen was investigated. The standardized optical density readings (ODs) of the ELISA obtained by the 1-step serum dilution (1:80) method were compared with the serum neutralization test (SNT) results, with a correlation of 0.993, and with the hemagglutination inhibition (HI) test results, with a correlation of 0.851. The ODs for the ELISA titers were obtained using the serial serum dilution method and were compared with the SNT results, with a correlation of 0.933, and with the HI test results, with a correlation of 0.987. In the experimental infection of 4 specific-pathogen-free cats, the results of different serologic tests (SNT and HI) and the ELISA using the serial serum dilution method revealed rapid production of antibodies after inoculation, whereas the ELISA using the one-step serum dilution method indicated that titers increased more slowly. These results indicate that with the present ELISA using nuclear antigen, there are fewer demands on time and labor, making the method convenient for monitoring FHV-1 infection.

Phylogenetic analysis of the Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum based upon 16S rRNA.

The nucleotide sequences of 16S rRNA genes of Erysipelothrix rhusiopathine and Erysipelothrix tonsillarum were determined. The sequences are almost similar (99.8%) with only three nucleotides mismatched.

Chromosome-sized DNA of Malassezia pachydermatis by pulsed-field gel electrophoresis.

The genome of Malassezia pachydermatis isolates from dogs was resolved into six chromosomes by using pulsed-field gel electrophoresis and their molecular sizes were calculated as 820, 1,100, 1,400, 1,470, 1,660 and 1,820 Kb, respectively. Comparison of electrophoretic patterns suggested that the chromosomes of M. pachydermatis were homozygous.

Properties of a calicivirus isolated from cats dying in an agitated state.

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCV-SAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3.0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70.4 per cent nucleotide and 77.3 per cent amino acid homology and FCV-F9 had a 68.6 per cent nucleotide and 73.9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.

[Two cases of canine histoplasmosis in Japan].

Histoplasmosis is distributed in tropical, subtropical and temperate zones of the world. The disease is one of the imported mycoses in Japan. To date, although more than 30 human and one canine case of histoplasmosis have been reported in Japan, some including that of the canine might have been infected domestically, since the patients have no history of going abroad. The pathogen of histoplasmosis is thus believed to be present in our country. We examined skin biopsies from two dogs in Tokyo and Kumamoto, and found fungal elements 1-2 or 2-4 microEm in diameter in the macrophages. The homology of DNA sequences for the ITS rRNA gene were correspondent to Ajellomyces capsulatus at a rate of more than 97.4%. Therefore, the two dogs were diagnosed as having been infected with Histoplasma capsulatum which is the anamorph of A. capsulatus. Since the dogs had no history of having been outside Japan and had not been brought from an endemic area, they might have been infected domestically. Further epidemiological surveys on canine histoplasmosis may be able to estimate autochthonous human cases in Japan.

Drug resistant plasmid of Actinobacillus pleuropneumoniae isolated from swine pleuropneumonia in Thailand.

Actinobacillus pleuropneumoniae, isolated from the pneumonic lung tissue of pigs, was resistant to streptomycin, sulfadiazine and tetracycline. This isolate, TA5, possessed 2 plasmids (pTA51 = 3.7 Kb, pTA52 = 6.2 Kb); one of the plasmids (pTA52) was coded for resistance to SM and SA. Another isolate of A. pleuropneumoniae, TA8, also possessed 2 plasmids (pTA81 = 3.7 Kb, pTA82 = 4.4 Kb), with a plasmid (pTA82) coded for resistance to SM and SA. All 4 plasmids were not transmissible and small.

[Drug sensitivity, conjugative R plasmids and plasmid profiles of Salmonella isolated from humans with infectious enteritis].

Using 92 Salmonella strains isolated from patients suspected of having infectious diseases of the intestinal tract who visited 13 hospitals in Japan during the six years between 1991 and 1996, we investigated the drug susceptibility, prevalence of conjugative R plasmid, and the plasmid profiles. 1) Of the bacterial isolates tested, 52.2% showed drug-resistance. Regarding the drug-resistance patterns, 70.8% of the isolates were resistant to a single drug, while 29.2% were multi drug-resistant. 2) Dividing the resistance patterns by the serotypes, among Salmonella Enteritidis isolates, single-drug resistance to SM was the most frequent, being detected in 27 isolates. Single-drug resistance to NA and two-drug resistance to SM/TC were the second-most frequent, each being detected in isolates. Among Salmonella Hadar isolates, four isolates showed two-drug resistance to SM/TC, and one isolate showed single-drug resistance to TC. Among Salmonella Typhimurium isolates, one isolate each showed three-drug resistance to ABPC/CER/KM and KM/TC/CP. Among Salmonella Agona isolates, one isolate each showed two-drug resistance to SM/TC and single-drug resistance to SM. Among Salmonella Derby isolates, two isolates showed single-drug resistance to SM. 3) The prevalence of conjugative R plasmid was investigated in 48 drug-resistant isolates, and six isolates (12.5%) contained the plasmid. 4) The prevalence of the plasmid was investigated in 29 drug-resistant S. Enteritidis isolates, and 22 isolates (75.9%) contained the plasmid. These isolated were classified by the plasmid profiles into types H1 to H7. 5) Regarding the plasmid profiles of the S. Enteritidis isolates, a position corresponding to 60 Kbp was the most frequently detected in 90.5%.

Phylogenetic analysis of field isolates of feline calcivirus (FCV) in Japan by sequencing part of its capsid gene.

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B-F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.00%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH2)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.

[P300 latency in patients with respiratory insufficiency due to sequelae of pulmonary tuberculosis].

We studied latent (mild) pulmonary encephalopathy in 14 patients with mild chronic respiratory insufficiency due to the sequelae of pulmonary tuberculosis. All of the patients were between 49 and 62 (mean age: 57.9). None of them had any impairment of daily activities and apparently had a clear consciousness. First, the P300 component evoked by auditory stimuli was examined. Immediately after that, the PO2, PCO2, pH were measured. Then the Hasegawa's dementia scale, the mini-mental state, the "Kanahiroi" test, Zung's depression score, digit span test were also assessed in the 14 patients. P300 components in 7 age-matched normal volunteers were also examined and compared with those in the 14 patients. The mean P300 latency in the patients were significantly prolonged compared with that in the normal volunteers (p less than 0.01). The P300 latency was well correlated with the PCO2, PO2, pH. The results of the "Kanahiroi" test also correlated with these parameters. We suggest that patients with mild respiratory insufficiency due to the sequelae of pulmonary tuberculosis often have latent (mild) pulmonary encephalopathy, and that P300 latency and the "Kanahiroi" test are very useful to detect and evaluate such latent pulmonary encephalopathy.

[Judicial judgements on anesthesia malpractice in Japan].

We reviewed 75 judicial precedents on anesthetic malpractice during surgical procedure which had appeared in legal journals in the period between 1963 and 1997. Anesthetic techniques employed were: general anesthesia (35 cases), spinal anesthesia (19 cases), local anesthesia (12 cases), and others (9 cases). Anesthesiologists were involved in 16 lawsuits, of which anesthesiologists lost 6 suits between 1986 and 1995. There were 8 cases classified as to be caused by respiratory problems including 2 cases of wrong gas supply. The defendants lost all the 8 cases. On the other hand, the plaintiff lost all the cases of malignant hyperthermia (n = 7). There is a tendency of increase in law suit with general anesthesia. Recent judgments suggested the importance of anesthetic managements, correct recording and appropriate monitoring by anesthesiologist during and immediately after surgery. Spinal anesthesia should be performed by anesthesiologist, and the frequency of anesthetic accident should be decreased. Japan is still in short of anesthesiologists and efforts should be paid to increase the number of anesthesia specialists.

[Significance of product documents (tenpubunsho) cited in judicial precedents in the field of anesthesiology].

The significance of the product documents (Tempubunsho) was mentioned in 21 out of 94 medical malpractice cases published in several laws reports 1963-97. The number of such cases has been increasing in recent years. Among the items described in the product documents, the precautions for use were most frequently mentioned (12 cases) followed by the instructions for administration and dosage (3 cases). In most cases, the judgment was against the medical institution involved due to the violation of legal obligations with respect to the contents of the product documents. Product documents will be taken much more seriously in future lawsuits. Attention should be focused on the contents of the product documents, especially when revised. When the administration of a drug deviates from the contents of the product document, medical evidence is required to support this deviation.

[Construction of a scale for independent and interdependent construal of the self and its reliability and validity].

A hypothetical model about person's independent and interdependent construal of the self was proposed, based on the theories proposed by Markus and Kitayama (1991), Triandis (1989) and Yamaguchi (1994). The purpose of this study was to construct a scale for measuring individual difference on the independent and interdependent construal of the self and to examine its reliability and validity. Four hundred and ninety-four subjects (180 males and 314 females) responded to a questionnaire. Sixteen items were selected for the scale through item analysis. This scale showed one-factor structure and a high reliability. As for the construct validity, the scale showed significant correlations with Collectivism scale, Need for Uniqueness scale and Public Self-Consciousness scale. These results supported the hypothetical model about independent and interdependent construal of the self.

[Independent and interdependent construal of the self: the cultural influence and the relations to personality traits].

The purpose of the present study was to investigate the independent and interdependent construal of the self in relation to the cultural difference and personality traits. A scale developed by Kiuchi (1995) to measure the person on the dimension of independent versus interdependent, the SII was administered to three groups of students. They were: (1) 81 college students who once lived in Europe or America, (2) 99 students of a college of music, and (3) 296 undergraduates of other majors and backgrounds. Mean SII scores clearly showed that the first group gave the highest priority to independent construal of the self, the second music college group follows them, and the last group of regular undergraduates gave the highest priority to interdependent construal of the self. In addition, SII scores were shown to have significant correlations with social anxiety, self-monitoring, sex identity, and self-esteem. Implication of these results are discussed.