Two cases of atretic cephalocele, and histological evaluation of skin appendages in the surrounding skin.

Atretic cephalocele is a small skin-covered lesion, usually located at or near the mid-line of the scalp. Histologically, it is composed of syncytial cells expressing neurone-specific enolase and epithelial membrane antigen. The syncytial cells form capillary-like structures *(pseudovascular areas) and collagenic fibrosis with densely packed collagen bundles (fibrous areas). Such findings suggest that the atretic cephalocele is a mild form of cephalocele, with its pathogenesis lying in the spectrum of neural tube closure abnormalities. However, few descriptions of abnormalities of the skin overlying and surrounding atretic cephalocele are available. We report two cases of atretic cephalocele that showed hamartomatous change in the surrounding cutaneous appendages. These findings suggest that atretic cephalocele is associated with abnormalities not only of the neural tube, but also of the surrounding skin.

Adaptive Capon beamforming for lensless electron cyclotron emission imaging with high spatial resolution.

Electron cyclotron emission (ECE) imaging diagnostics incorporating a lensless approach have been developed for measurements involving active spatial selectivity and direction-of-arrival estimation. The Capon method for adaptive-array analysis was proposed to improve the spatial resolution of the two-dimensional ECE imaging technique. Broadband noise source emissions were used to simulate the ECE to verify the practical effectiveness of the Capon method in the ECE imaging. Multiple noise source emission positions were properly estimated with a high spatial resolution using the Capon method.

A report on small team clinical ethics consultation programmes in Japan.

Clinical ethics support, including ethics consultation, has become established in the field of medical practice throughout the world. This practice has been regarded as useful, most notably in the UK and the USA, in solving ethical problems encountered by both medical practitioners and those who receive medical treatment. In Japan, however, few services are available to respond to everyday clinical ethical issues, although a variety of difficult ethical problems arise daily in the medical field: termination of life support, euthanasia and questions about patient autonomy. In light of these conditions, a group of 17 volunteer educators and researchers from the area of biomedical ethics, including the authors, have formed the Clinical Ethics Support and Education Project, and began providing Japan's first small team clinical ethics consultation service in October, 2006. Members include scholars of biomedical ethics, scholars of philosophy and ethics, legal professionals and legal scholars, nurses and doctors, consisting of five women and 12 men. Consultation teams, made up of a small number of members, were organised each time a request for consultation was received. Over approximately 15 months (October 2006-December 2007), the programme received 22 consultation requests from medical practitioners and medical institutions, and three from the families of patients. In this paper, we will discuss the status of our consultation service and examples of consultation cases we have handled. In addition, we will examine the process of evaluating small team clinical ethics consultation services, as well as the strengths and weakness of such programmes.

Identification of thermo-acidophilic bacteria isolated from the soil of several Japanese fruit orchards.

AIMS: To investigate the occurrence and distribution of thermo-acidophilic bacteria (TAB) associated with various commercial fruit crop soils in Japan and to assess their ability to produce the odorous phenolic compound, guaiacol. METHODS AND RESULTS: Phylogenetic analysis based on the 5' end of the 16S rRNA gene (approximately 500 bp), was performed on 62 TAB isolated from the soil of several Japanese fruit orchards. The results suggested that 60 of the bacterial strains analysed belonged to the genus Alicyclobacillus, while the remaining two belonged to the genus Bacillus. The majority of strains (58%) were identified as Alicyclobacillus acidoterrestris. This group partitioned into three phylogenetically distinct subgroups (A-C). Isolates identified as A. acidiphilus (two strains), A. acidoterrestris (36 strains), and A. hesperidum subsp. aigle (one strain), produced guaiacol from vanillic acid. Levels of guaiacol production varied significantly among strains. The guaiacol producing phenotype was conserved among certain species, however no correlation was observed between levels of guaiacol production and 16S rRNA gene-based phylogenetic relatedness. CONCLUSIONS: Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans were widely distributed among various fruit orchards in Japan. Guaiacol production was common at the species/subspecies level; however the amount of guaiacol produced by each strain varied significantly. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a comprehensive phylogenetic survey of Alicyclobacillus species in Japanese fruit orchards. Quality control standards for guaiacol producing Alicyclobacillus have also been described.

Low shear stress can initiate von Willebrand factor-dependent platelet aggregation in patients with type IIB and platelet-type von Willebrand disease.

Platelets exposed to shear stress aggregate in the absence of exogenously added agonists, utilizing distinct platelet membrane receptors and ligands depending upon the level of shear stress applied. Using a modified cone and plate type viscometer, we previously demonstrated that, under low shear stress (18 dyn/cm2), aggregation is mediated by platelet membrane glycoprotein (GP) IIb-IIIa and fibrinogen, whereas aggregation induced by high shear stress (108 dyn/cm2) requires the binding of von Willebrand factor (vWF) to both GPIb-IX and GPIIb-IIIa (Ikeda, Y., M. Handa, K. Kawano, T. Kamata, M. Murata, Y. Araki, H. Anbo, Y. Kawai, K. Watanabe, I. Itagaki, et al. 1991. J. Clin. Invest. 87:1234-1240). Here we report that vWF-dependent aggregation occurs under low shear stress in citrated platelet-rich plasma (PRP) from two types of congenital bleeding disorders, platelet-type von Willebrand disease (vWD) and type IIB vWD, in both of which ristocetin-induced aggregation is known to be heightened. Aggregation induced by low shear stress was enhanced in both types of disorders compared to normal controls, and the enhancement was completely abolished by anti-vWF monoclonal antibody NMC-4, which blocks the GPIb-binding site on vWF. Under high shear stress, the extent of maximal aggregation was not different between controls and the patient groups although maximal aggregation was reached much more quickly in the latter. When citrated PRP was exposed to a gradient of shear stress (6 to 108 dyn/cm2 over a 5-min period), vWF-dependent aggregation, as judged from the inhibitory effect of NMC-4, first occurred at 14 dyn/cm2 in platelet-type vWD and at 10-12 dyn/cm2 in type IIB vWD, as compared with more than 81 +/- 20.1 dyn/cm2 in control platelets. These results suggest that an abnormality in either vWF or GPIb-IX triggers the aggregation-inducing interaction of the two molecules under low shear stress, which might explain the intravascular platelet clumping, that presumably underlies the thrombocytopenia observed in these bleeding disorders.

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.

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.

Transmembrane calcium influx associated with von Willebrand factor binding to GP Ib in the initiation of shear-induced platelet aggregation.

We found that the binding of multimeric vWF to GP Ib under a shear force of 108 dynes/cm2 resulted in the transmembrane flux of Ca2+ ions with a two- to three-fold increase in their intracellular concentration ([Ca2+]i). The blockage of this event, obtained by inhibiting the vWF-GP Ib interaction, suppressed aggregation. In contrast, the blockage of vWF binding to GP IIb-IIIa, as well as the prevention of activation caused by increased intracellular cAMP levels, inhibited aggregation but had no significant effect on [Ca2+]i increase. A monomeric recombinant fragment of vWF containing the GP Ib-binding domain of the molecule (residues 445-733) prevented all effects mediated by multimeric vWF but, by itself, failed to support the increase in [Ca2+]i and aggregation. These results suggest that the binding of multimeric vWF to GP Ib initiates platelets aggregation induced by high shear stress by mediating a transmembrane flux of Ca2+ ions, perhaps through a receptor-dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of GP IIb-IIIa; the latter receptor then binds vWF and mediates irreversible aggregation.

An oxygraphic method for the determination of cholesterol and measurement of the slow oxygen-consuming reactions of hepatic microsomes.

Oxygen-consuming reactions of cholesterol oxidase [EC 1.1.3.6] and microsomes were measured with a galvanic oxygen electrode which was attached to an offset amplifier for sensitive measurement of the reaction processes. The sensitivity of this oxygraphic method for detection of oxygen consumption was ten times greater than that of the usual method. The minimum rate of slow oxygen-consuming reactions which could be estimated was about 5 nmoles of oxygen per min, and the minimum amount of oxygen consumption which could be determined was also about 5 nmoles. An oxygraphic method for direct and rapid determination of cholesterol was demonstrated using one-twentieth the amount of cholesterol oxidase which is used for the colorimetric method. The processes of cyanide-suppressed beta-NADH-dependent oxygen consumption and cyanide-insensitive alpha-NADH-dependent oxygen consumption, which were difficult to follow by the usual method, were followed using a small amount of microsomes (less than 1mg protein/ml). Furthermore, the temporary cessation of alpha-NADH-dependent oxygen consumption caused by ferricyanide and the corresponding oxidation-reduction of reduced cytochrome b5 were followed in the presence of ADP. ADP did not inhibit the oxygen consumption. The results indicate that the oxygen consumption with alpha-NADH is due to electron transfer from alpha-NADH via NADH-cytochrome b5 reductase and cytochrome b5, in which the rate-determining step lies at some reaction after the reduction of cytochrome b5.

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

Analysis of the reactivity of microsomal cytochrome P-450 toward drugs and exogenous ligands.

Three forms of cytochrome P-450 in rat liver microsomes (Comai, K. and Gaylor, J.L. (1973) J. Biol. Chem. 284, 4947-4955) were characterized as two types of P-450 in the membrane-bound state. One was reactive not only toward cyanide but also aniline, aminopyrine, and hexobarbital, and the other was less reactive toward cyanide and rather unreactive toward the drugs. Cyanide titrations of microsomes in the presence and absence of the drugs were performed spectrophotometrically for analysis of the interactions. The affinity of cyanide for the less reactive P-450 was about twenty times less than that for the reactive P-450. About 40% of P-450 in the microsomal membrane was reactive and the rest was the less reactive form. The reactive P-450 involved two forms having different affinities for cyanide. The ratio of their amounts was 1:2. Triton WR-1339 interfered with cyanide binding to the reactive P-450 mainly. The relative amount of each form of P-450 as well as the dissociation constant of cyanide could be estimated by the present method, and the modes of interaction of the membrane-bound P-450 with the drugs and exogenous ligands were deduced.

Identification and properties of the prosthetic group of choline oxidase from Alcaligenes sp.

Choline oxidase from Alcaligenes sp. catalyzed the oxidation of choline and betaine aldehyde to betaine with concomitant consumption of oxygen and production of hydrogen peroxide. The values of Km for choline and betaine aldehyde were 0.87 and 6.2 mM, respectively. The molecular weight of the enzyme was estimated to be 66,000 by SDS-gel electrophoresis and 72,000 by gel-filtration using a high performance liquid chromatograph. The prosthetic group of the enzyme was identified as 8 alpha-[N(3)-histidyl]-FAD from the electrophoretic mobility at pH 6.25 of the hydrolysate of the methylated histidylflavin. The visible absorption spectrum of the enzyme showed peaks at 358 and 453 nm and a shoulder at about 480 nm. The covalently bound FAD was reduced on addition of either choline or betaine aldehyde under anaerobic conditions and was reoxidized by aeration. The enzyme was found to contain 1 mol of FAD per mol enzyme. Amino acid analysis of a purified flavin peptide gave the following molar ratios of amino acids to flavin: pro(1), Asp + Asn(3), Ser(1), His(1), and Arg(1). Aspartic acid was the N-terminal amino acid. The partial sequence of amino acids in the flavin peptide was as follows: Formula (See Text).

Circular dichroism studies on flavoproteins containing covalently bound coenzymes.

Absorption and circular dichroism spectra of cholesterol oxidase from Schizophyllum commune and choline oxidase from Alcaligenes sp. were measured and compared. The prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD (1, 2), while that of choline oxidase is 8 alpha-[N(3)-histidyl]-FAD (3). In the CD spectra of the two enzymes in either the oxidized or reduced state, the corresponding bands in the visible region are of approximately the same intensity and shape but of opposite sign. A notable feature in the CD spectra of the two enzymes after light irradiation is the appearance of a CD band in the longer wavelength region (550-650 nm) and the opposite signs of the CD band in this region in the two enzymes. The similarity of the shape and intensity of the CD spectra of the two enzymes suggests that the environments surrounding the flavin moieties are very similar, and the sign reversal of the CD bands suggests that the mutual orientations between the transition moment of flavin and that of its environment differ in the two enzymes.

Continuous measurement of shear-induced platelet aggregation.

A mechanical device for continuous recording of shear-induced platelet aggregation (SIPA) has been developed using a turbidometric technique. The device consists of three components: light source, a thermostated cone-plate streaming chamber and optical detection unit. There was a good correlation between platelet count and transmitted light intensity under the conditions in which no activation of platelets occurred. When platelets were exposed to shear stress at 37 degrees C, transmitted light intensity increased with time. Scanning electronmicroscopic examinations revealed that more prominent platelet aggregation was demonstrated when light intensity increased. Reproducible recording of SIPA was obtained when experiments were repeated using the same sample. No change in light intensity was observed when formalin-fixed platelets were sheared. Both PGE1, and anti-GPIIb/IIIa mouse monoclonal antibody, AP2, showed complete inhibition of SIPA. It is indicated that our device for continuous measurement of SIPA is an important new tool to study one aspect of platelet functions.

Radial keratotomy to treat myopic refractive error after cataract surgery.

PURPOSE: To assess the predictability and effectiveness of radial keratotomy in patients with myopic refractive error and unacceptable anisometropia after cataract surgery. SETTING: A prospective multicenter study. METHODS: This study comprised 40 eyes of 40 Japanese patients who had had cataract surgery. Radial keratotomy was performed, and the 6 month postoperative data were analyzed. RESULTS: Mean patient age was 71.0 years +/- 7.4 (SD) (range 51 to 84 years) and mean preoperative anisometropia -3.41 +/- 1.69 D (range -1.25 to -7.75 D). The surgery decreased mean anisometropia to -1.01 +/- 0.94 D (P < .000001, Wilcoxon signed-rank test), a mean reduction of 2.22 +/- 1.23 D (range 0.75 to 5.88 D). Postoperative anisometropia ranged from 0.81 to -3.13 D. The surgical effects were overestimated by the nomograms developed for the correction of naturally occurring myopia in the eyes of white patients. Multiple regression analysis revealed that optical zone size and number of incisions were significantly correlated with the amount of myopic correction, and the regression equation (R2 = 0.77) was expressed as follows: Effects = (-1.45 x optical zone size) + (0.24 x incision number) + 7.60. A new nomogram was derived based on this equation. CONCLUSIONS: Radial keratotomy was a safe and efficient procedure to treat myopic refractive error in pseudophakic eyes. Separate nomograms are necessary for white and Asian populations.

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.

Biologically active components against Drosophila melanogaster from Podophyllum hexandrum.

In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, a dichloromethane extract of Podophyllum hexandrum was found to give an insecticidal activity against larvae of Drosophila melanogaster Meigen. From the extract, an insecticidal compound was isolated by bioassay-guided fractionation. The compound was identified as podophyllotoxin (1) by comparison of its spectroscopic characteristics with literature data. In bioassays for insecticidal activity, 1 showed a LC(50) value of 0.24 micromol/mL diet against larvae of D. melanogaster and a LD(50) value of 22 microg/adult against adults. Acetylpodophyllotoxin (1A), however showed slight insecticidal activity in both assays, indicating that the 4-hydroxyl group was an important function for enhanced activity of 1.

Antimutagenic activity of polymethoxyflavonoids from Citrus aurantium.

The methanol extract from Citrus aurantium showed a suppressive effect on umu gene expression of SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract from C. aurantium was successively re-extracted with hexane, dichloromethane, butanol, and water. A dichloromethane fraction showed a suppressive effect. The suppressive compounds in the dichloromethane fraction were isolated by SiO(2) column chromatography and identified as tetra-O-methylscutellarein (1), sinensetin (2), and nobiletin (3) by EI-MS and (1)H- and (13)C NMR spectroscopy. These compounds suppressed the furylfuramide-induced SOS response in the umu test. Gene expression was suppressed 67%, 45%, and 25% at a concentration of 0.6 micromol/mL, respectively. The ID(50) value (50% inhibition dose) of compound 1 was 0. 19 micromol/mL. These compounds were assayed with other mutagens, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver metabolizing enzymes, activated Trp-P-1, and UV irradiation. These compounds showed of all mutagen-induced SOS response in the umu test. In addition, compounds 1-3 exhibited antimutagenic activity in the S. typhimurium TA100 Ames test.

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.

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.