Serological responses of Australian horses using a commercial duplex indirect ELISA following vaccination against strangles.

OBJECTIVE: To determine the nature of serological responses in Australian horses using a commercial duplex indirect ELISA (iELISA) following vaccination against strangles. DESIGN: A group (n = 19) of client-owned horses from five properties were recruited to receive a primary course of a Streptococcus equi subsp. equi (S. equi) extract vaccine. Serological responses were determined by duplex iELISA incorporating S. equi-specific fragments of two cell wall proteins, SEQ2190 and SeM (antigens (Ag) A and C, respectively). METHODS: The horses were administered a primary strangles vaccination course. Blood was collected immediately prior to each of the three vaccinations at 2-week intervals and additionally at 28 and 56 days following the 3rd vaccination (V3). RESULTS: Significant increases in mean antibody levels of horses following vaccination were limited only to AgC, which was significantly increased at T2/V3, 14 days following V2 (ratio of geometric means = 3.7; 95% confidence interval (CI): 1.6, 8.4; P = 0.003). There was no increase in mean antibody to Ag A (ratio of geometric means = 1.4; 95% CI: 0.6, 3.2; P = 0.39). Four horses (22%) exceeded the test cut-off for AgC following vaccination. CONCLUSION: Vaccination of Australian horses is unlikely to interfere greatly with detection of strangles using the duplex iELISA. No responses would be anticipated to AgA following vaccination with Equivac© S/Equivac© 2in1 and only a minority are likely to respond to AgC. We conclude that the results of this study validate the usefulness of the duplex iELISA to assist control measures for strangles outbreaks in Australian horse populations.

The potential impact of a single amino-acid substitution on the efficacy of equine influenza vaccines.

REASONS FOR PERFORMING STUDY: The protection induced by an equine influenza (EI) vaccine strain depends on its antigenic relatedness to the challenge virus. Although the World Organisation for Animal Health (OIE) recommend that both Florida sublineage clade 1 (Fc1) and clade 2 (Fc2) viruses should be included in EI vaccines, Japanese EI vaccines have not, thus far, been updated to include a Fc2 virus. OBJECTIVES: To evaluate the efficacy of antibodies raised against Japanese EI vaccine strains in the neutralisation of recent Fc2 viruses. STUDY DESIGN: Antigenic analysis. METHODS: Virus neutralisation tests were performed using antisera from experimentally infected horses and from horses that had received a primary course of the currently available vaccines. RESULTS: Antiserum raised against the Japanese EI vaccine strain, A/equine/La Plata/1993, exhibited poor cross-neutralising activity against the Fc2 viruses isolated recently in Ireland and the UK, which have the substitution of alanine to valine at position 144 in antigenic site A of the haemagglutinin gene. In contrast, the antiserum exhibited good cross-neutralising activity against the Fc2 viruses without the substitution. This finding was supported in experiments with antisera collected from vaccinated horses. CONCLUSIONS: This suggests that the efficacy of the Japanese EI vaccine for some of the recent Fc2 viruses is suboptimal and that vaccines should be updated in accordance with the OIE recommendations.

Intrasplicing--analysis of long intron sequences.

We propose a new model for the splicing of long introns, which we call intrasplicing. The basic idea of this model is that the splicing of long introns may be facilitated by the splicing of inner parts of the intron prior to the splicing of the long intron itself. Since long introns have up to about 100,000 bases, this model seems to be a likely explanation of their splicing. To investigate the possibility of this model, we develop a new computational method for the analysis of DNA sequences with respect to splicing. We analyze the genomic sequence of four species with our method and derive several results indicating that intrasplicing may be an appropriate model for the splicing of at least part of the long intron sequences.

Yeast [PSI+] "prions" that are crosstransmissible and susceptible beyond a species barrier through a quasi-prion state.

The yeast [PSI(+)] element represents an aggregated form of release factor Sup35p and is inherited by a prion mechanism. A "species barrier" prevents crosstransmission of the [PSI(+)] state between heterotypic Sup35p "prions." Kluyveromyces lactis and Yarrowia lipolytica Sup35 proteins, however, show interspecies [PSI(+)] transmissibility and susceptibility and a high spontaneous propagation rate. Cross-seeding was visualized by coaggregation of differential fluorescence probes fused to heterotypic Sup35 proteins. This coaggregation state, referred to as a "quasi-prion" state, can be stably maintained as a heritable [PSI(+)] element composed of heterologous Sup35 proteins. K. lactis Sup35p was capable of forming [PSI(+)] elements not only in S. cerevisiae but in K. lactis. These two Sup35 proteins contain unique multiple imperfect oligopeptide repeats responsible for crosstransmission and high spontaneous propagation of novel [PSI(+)] elements.

Calcium regulation of microtubule sliding in reactivated sea urchin sperm flagella.

The changes in the bending pattern of flagella induced by an increased intracellular Ca(2+) concentration are caused by changes in the pattern and velocity of microtubule sliding. However, the mechanism by which Ca(2+) regulates microtubule sliding in flagella has been unclear. To elucidate it, we studied the effects of Ca(2+) on microtubule sliding in reactivated sea urchin sperm flagella that were beating under imposed head vibration. We found that the maximum microtubule sliding velocity obtainable by imposed vibration, which was about 170-180 rad/second in the presence of 250 microM MgATP and <10(-9) M Ca(2+), was decreased by 10(-6)-10(-5) M Ca(2+) by about 15-20%. Similar decrease of the sliding velocity was observed at 54 and 27 microM MgATP. The Ca(2+)-induced decrease of the sliding velocity was due mainly to a decrease in the reverse bend angle. When the plane of beat was artificially rotated by rotating the plane of vibration of the pipette that held the sperm head, the asymmetric bending pattern also rotated at 10(-5) M Ca(2+) as well as at <10(-9) M Ca(2+). The rotation of the bending pattern was observed at MgATP higher than 54 microM ( approximately 100 microM ATP). These results indicate that the Ca(2+)-induced decrease of the sliding velocity is mediated by a rotatable component or components (probably the central pair) at high MgATP, but is not due to specific dynein arms on particular doublets. We further investigated the effects of a mild trypsin treatment and of trifluoperazine on the Ca(2+)-induced decrease in sliding velocity. Axonemes treated for 3 minutes with a low concentration (0.1 microgram/ml) of trypsin beat with a more symmetrical waveform than before the treatment. Also, their microtubule sliding velocity and reverse bend angle were not affected by high Ca(2+) concentrations. Trifluoperazine (25-50 microM) had no effect on the decrease of the sliding velocity in beating flagella at 10(-5) M Ca(2+). However, the flagella that had been 'quiescent' at 10(-4) M Ca(2+) resumed asymmetrical beating following an application of 10-50 microM trifluoperazine. In such beating flagella, both the sliding velocity and the reverse bend angle were close to their respective values at 10(-5) M Ca(2+). Trypsin treatment induced a similar recovery of beating in quiescent flagella at 10(-)(4) M Ca(2+), albeit with a more symmetrical waveform. These results provide first evidence that, at least at ATP concentrations higher than approximately 100 microM, 10(-6)-10(-5) M Ca(2+) decreases the maximum sliding velocity of microtubules in beating flagella through a trypsin-sensitive regulatory mechanism which possibly involves the central pair apparatus. They also suggest that calmodulin may be associated with the mechanism underlying flagellar quiescence induced by 10(-4) M Ca(2+).

[Comparison of the morphology and the reactivity of Chlamydia pneumoniae isolated in north-Kanto Area, Ibaraki Prefecture, Japan].

To compare the morphology among Chlamydia pneumoniae (C. pneumoniae), strain TW183 and strains which were isolated in the area of Kasumigaura, Ibaraki from 1992 to 1995. C. pneumoniae were infected on HL cell monolayers and cultured in 5% CO2 at 35.5 degrees C for about 60 hrs. The cells were harvested and fixed with 2.5% glutaraldehyde, and then the regular procedure for observation of Chlamydia in inclusion by transmission electron microscope was performed. Immunoblot assay was carried out by using highly and partially purified C. pneumoniae TW183 and 4 isolates with partial purification as antigens. The results were as follows: the shape of TW183 and the isolates included pear and round shapes, respectively. Immunoblotting profiles were the same in terms of band-formation patterns with the serum from a patient infected with C. pneumoniae. These results may indicate that the round shape of C. pneumoniae elementary body (EB) is predominantly pandemic in Japan, although pear-shaped EBs of C. pneumoniae were found in the neighboring prefecture of Chiba.

A reverse-sandwich enzyme-linked immunosorbent assay for verocytotoxin 1 and 2 antibodies in human and bovine sera.

A reverse-sandwich enzyme-linked immunosorbent assay (ELISA), in which an antibody is sandwiched by antigens, was established for the titration of antibodies to verocytotoxins (VT) in human and animal sera. This assay has two advantages over a conventional indirect ELISA: (i) higher specificity and sensitivity and (ii) the ability to comparably titrate antibodies from different species. The VT1 (Shiga-like toxin 1) antibody-positive rates were 5% in 202 normal adult humans and 99% in 93 normal cattle at a dairy farm. This ELISA is most suitable for seroepidemiologic studies of infections with VT-producing Escherichia coli in humans and various animal species.

[Detection of anti-Chlamydia trachomatis antibody by means of enzyme immunoassay using synthetic peptide antigen].

Newly developed diagnostic kits for the detection of Anti-Chlamydia trachomatis, Peptide-Chlamvdia (LOY: Meiji Milk Products Co., Ltd., Tokyo; for IgG and IgA), were evaluated using the microimmunofluorescence assay (MIF) as the gold standard. These results were also compared to results of testing by Sero-IPALISA and immunoblot (I-B). Detection by LOY in based on enzyme immunoassay with synthetic peptides as the antigen. Thirty serum samples from pediatric patients and 130 serum samples from gynecology patients were used. All 26 pediatric samples that were positive for Chlamydia pneumoniae IgG antibody tested negative with LOY, indicating that the presence of the antibody against C. pneumoniae did not affect the assay by LOY. For 90 gynecological samples, the total, the positive and the negative agreement rates for IgG were quite high; i.e. 87.8%, 90.0% and 70.0% (LOY vs MIF), 85.6%, 85.0% and 90.0% (Sero-IPALISA vs MIF), and 92.0%, 94.9% and 70.0% (I-B vs MIF), respectively. On the other hand, many cases of MIF (-) and LOY (+) discrepancy were seen in IgA detection. In order to better understand the basis for such disagreement. 34 serum samples were collected from patients whose cervical samples were negative for the Chlamydia group antigen based on the assay with IDEIA-Chlamydia. They were then assayed by MIF and LOY. The total, the positive and the negative agreement rates for IgG were 91.2%, 100% and 90.9%, while the total and the negative agreement rates for IgA were 88.2% and 88.2% (there were no IgA positive cases). Furthermore, 6 serum samples (1 case of MIF (+) LOY (+) and 5 cases of MIF (-) LOY (+)) were provided to determine whether LOY detects C. trachomatis specific IgA antibody. Increasing amounts of C. trachomatis serovar L2 were added to the serum samples resulting in a progressive decrease in their reactivity in the LOY assay. These results lead us to speculate that LOY can reveal even low levels of C. trachomatis specific IgA antibody. In conclusion, LOY can be used as an useful kit for detecting C. trachomatis antibody.

Towards Automatic Construction of Cell-Lineage of C. elegans from Nomarski DIC Microscope Images.

This study aims at automatic construction of a cell lineage from 4D (multi-focal, time-lapse) images, which are taken using a Nomarski DIC (differential-interference contrast) microscope. A system with such abilities would be a powerful tool for studying embryo genesis and gene function based on mutants, whose cell lineage may differ from that of wild types. We have designed and implemented a system for this purpose, and examined its ability through computational experiments. The procedure of our system consists of two parts: (1) Image processing which detect the positions of the nuclei from each 2D microscope image, and (2) Constructing a hypothetical cell lineage based on the information obtained in (1). We have also developed a tool which allows a human expert to easily filter out erroneous nuclei candidates generated in (1). We present computational results and also discuss other ideas which may improve the performance of our system.

Spectrum of cytokine gene expression in intestinal mucosal lesions of Crohn's disease and ulcerative colitis.

BACKGROUND/AIMS: We investigated the mRNA expression of spectrum of cytokines in the colonic mucosa in inflammatory bowel disease (IBD). METHODS: The expression of cytokine gene was evaluated by using the reverse transcription-polymerase chain reaction and the radioactivity of amplified cDNA standardized by coamplified beta-actin cDNA. RESULTS: Crohn's disease and ulcerative colitis showed significantly increased expression of IL-1beta, IL-6, IL-8 and TNF-alpha mRNA as compared with controls (p < 0.05). CONCLUSION: Proinflammatory cytokines such as IL-1beta, IL-6, IL-8 and TNF-alpha are closely involved in the immune abnormalities of inflammatory mucosal lesions in IBD.

[Detection of chlamydial antibodies by immunoblotting technique--in comparison of three commercially available test kits and micro-IF].

We reexamined the assay conditions of immunoblotting (W-B) technique to detect antibodies (IgG and IgA) to C. trachomatis serovar L2. Partially purified Chlamydia (35% urografin) was used as the antigen. The marker bands of W-B for positive and/or negative are major outer membrane protein (MOMP) band and either one or more bands staining in 40-62 KDa area. We then compared the sensitivity and specificity of three commercially available test kits and micro-IF by using W-B as a standard. The kits compared were sero-IPALISA-IgG-IgA, IP-Azyme-IgG-IgA and HITAZYME-IgG-IgA, and micro-IF. Serum samples were collected from the outpatient departments of gynecology, urology, and internal medicine and pediatrics. The results of agreement between W-B and test kits in IgG detection were as follows: in sero-IPALISA, total agreement was 85.7%, positive agreement 83.3%, negative agreement 90.9% and in IPAzyme: 85.7%, 83.3%, 90.9%, respectively and in HITAZYME: 82.9%, 75%, 100%, respectively. These results are almost the same as micro-IF: 85.7%, 79.2%, 100%, respectively. These kits may have relatively high sensitivity and specificity in IgG antibody detection. In IgA detection, the total agreement between W-B and sero-IPALISA was 82.9%, positive agreement 100%, negative agreement 66.7%, in IPAzyme: 77.1%, 58.8%, 94.4%, respectively and in HITAZYME: 65.7%, 64.7%, 66.7%, respectively and in micro-IF: 82.9%, 70.6%, 94.9%, respectively. Although these agreements are not so high in IgA detection, the W-B technique gives fairly consistent results as well as those kits. This indicates that W-B technique with MOMP and other protein bands (40-62 KDa) as marker for positive reaction is a useful method for detection of chlamydial antibodies.

Study of cytokines in ulcerative colitis.

We investigated the lymphocyte-activation antigens and the expression of cytokine genes in the mucosa of ulcerative colitis (UC). Fresh colonic mucosal biopsy specimens from patients with UC and controls were fixed for the immunohistochemical study of CD4, HLA-DR, and CD25, and other specimens were prepared for the RNA analysis of cytokines. Gene expression was evaluated by the reverse transcription-polymerase chain reaction, and the radioactivity of dot-blotted amplified cDNA was standardized by co-amplified beta-actin cDNA. The inflamed mucosa of active UC showed increased CD4+DR+ and CD25+ cells in comparison with control subjects. Active UC showed significantly increased mRNA expression of IL-1 beta, IL-2R alpha, IL-6, IL-8, and TNF alpha compared with the controls. We found no significant difference in the mRNA expression for IL-2, IL-4, IL-10, and IFN-gamma between active UC and controls. Increased CD4+DR+ and CD25+ cells in active UC mucosa indicate mucosal CD4(+) T cell activation in the lamina propria, but we did not clarify Th1 or Th2 specific T cell activation from our study of cytokine mRNA expression. The increased mRNA expression for IL-1 beta, IL-6, and TNF alpha in the mucosal lesions of UC indicates that these inflammatory cytokines may play important roles in the pathogenesis of UC.

Activated CD4+ and CD8+ cells in the colonic mucosa of ulcerative colitis patients: their relationship to HLA-DR antigen expression on the colonic epithelium and serum soluble CD25 levels.

This study was performed to clarify the relationship between activated (HLA-DR-expressing) CD4+ and CD8+ cells in the colonic lamina propria of ulcerative colitis and other immunological factors, i.e., epithelial DR expression, serum soluble CD25 levels, and colonic mucosal CD25+ cells. The frequency of epithelial DR expression was positively correlated with the numbers of CD4+ and CD8+ cells. The percentages activated CD4+/CD4+ cells were higher in mucosae with DR- epithelium than in mucosae with DR+ epithelium. The serum soluble CD25 levels were increased in ulcerative colitis, and there was an inverse correlation between these levels and the relative number of activated CD4+ cells in untreated active disease. These results suggest that interactions among mucosal CD4+ cells, colonic epithelium, and serum soluble CD25 might play an important role in the pathogenesis of ulcerative colitis.

Quercetin potentiates TNF-induced antiviral activity.

Tumor necrosis factor (TNF) produces a dose-dependent inhibition of vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), and herpes simplex virus type 1 (HSV-1) replication in WISH cells. The antiviral activity of TNF against VSV and EMCV is greatly enhanced by combination with quercetin. Induction of 2',5'-oligo-adenylate (2-5A) synthetase by TNF is also enhanced by quercetin. Addition of polyclonal antibodies to human interferon (IFN)-beta completely blocked both enhancement of antiviral activity and 2-5A synthetase induction.

[Effect of storage conditions on the infectivity of Chlamydia pneumonia].

In order to facilitate the isolation of C. pneumoniae, strain similar TWAR, with high frequency, we investigated the effect of various factors on the infectivity of Chlamydia using two laboratory strains, C. pneumoniae TWAR and C. trachomatis serovar D. The factors tested were the effects of different temperatures for storage conditions, saliva from healthy person, storage media for Chlamydia, and the frequency of freezing and thawing. Chlamydial suspension was prepared in the two media, SPG (sucrose-phosphate-glutamate buffer pH 7.5), and CT-GM (culture medium for Chlamydia which contains 1 micrograms/ml cycloheximide and 0.04% glucose). Chlamydial suspension was allowed to stand in each of four different thermal conditions: 37 degrees C, room temperature (25 degrees C), 4 degrees C, 0 degrees C and -75 degrees C for 1, 3, 6, 24, 48, 72, 96, 120 and 144 hours. For storage at -75 degrees C, one of three groups of glass vial tubes containing Chlamydia was covered with an "airmat" to prevent the rapid freezing of Chlamydia. The effect of various factors on the infectivity was assayed by inoculation of the suspension on HeLa 229 cell monolayers. Results showed that the infectivity rapidly decreased at 37 degrees C and room temperature, while at 4 degrees C, 0 degrees C and -75 degrees C, relatively high infectivity was maintained and contained until days 4 to 6. This decreasing pattern was similar to among the media used. We were not able to find any differences in the infectivity among the samples with or without the "airmat".(ABSTRACT TRUNCATED AT 250 WORDS)

[Isolation and identification of Chlamydia spp. from effusion of otitis media].

Three chlamydial strains isolated from patients of otitis media with effusion were studied by comparing reactivity to monoclonal antibody (MAb) and polyclonal antibody (PAb) produced against one clinical isolate (named Mk), which was first isolated by Dr Mukai (Mukai Microbiological Research Laboratory, Yamato-shi, Kanagawa prefecture). Commercially supplied antibody (Microtrak (Syva), Culture-set (Ortho diagnostic system)) was also used. To isolate the Chlamydia spp, the yolk sacs of eggs were immediately inoculated with sample effusions (0.2 to 0.4 ml per sac) as soon as the samples were received. The eggs were observed every day for a period of 12 days thereafter for signs of life or death. One to two blind passages were first done in the eggs and then in HeLa 229 cells. The reactivity was examined by both micro-IF tests, among various strains of Chlamydia (C. trachomatis: L2. C. pneumoniae, C. psittaci: Budgerigar, Izawa, Meningopneumonitis (MP)) and by immunoblot analysis. Chlamydia spp were isolated in two of the twenty-nine sample effusions (6.9%). These isolates were then tested for reactivity to MAb and PAb. It was found that MAb reacted with MP and Mk, but not with Budgerigar, Izawa and C. pneumoniae. The antibody of Culture-set reacted with C. trachomatis C. pneumoniae and C. psittaci. No reactivity was observed in Mk by MicroTrak. Immunoblot analysis revealed that MAb reacted with about 95 KDa protein of Mk, the two clinically isolated Chlamydia spp and MP. By using PAb from rabbits, similar blotting patterns were observed in Mk, the clinical isolates and MP.(ABSTRACT TRUNCATED AT 250 WORDS)