Evaluation of immunochromatographic test of Shiga toxin 2e in enrichment cultures of swine edema disease clinical samples.

To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1-196.2 ng/ml and 0-12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.

Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli.

Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable "sandwich" assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1-64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.

Luteolin suppresses 5-hydroxytryptamine elevation in stimulated RBL-2H3 cells and experimental colitis mice.

Increased 5-hydroxytryptamine may be associated with the development and progression of inflammatory bowel disease. In this study, we examined the suppressive effect of flavonoids on the increased intra- and extracellular 5-hydroxytryptamine levels in rat mast RBL-2H3 cells, known to produce 5-hydroxytryptamine by the phorbol 12-myristate 13-acetate stimulation. Among the flavonoids examined, luteolin and quercetin significantly reduced the cellular 5-hydroxytryptamine concentration. Gene and protein expression analyses revealed that luteolin significantly suppressed cellular tryptophan hydroxylase 1 expression induced by phorbol 12-myristate 13-acetate stimulation. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling was also suppressed by luteolin, suggesting that this pathway is one of targets of 5-hydroxytryptamine modulation by luteolin. An in vivo experimental colitis model was prepared by administering 2.5% dextran sodium sulfate in drinking water to C57BL/6 mice for seven days. The ingestion of 0.1% dietary luteolin suppressed the increasing 5-hydroxytryptamine in the colorectal mucosa. In conclusion, luteolin possesses a suppressive effect on extensive 5-hydroxytryptamine formation in both experimental RBL-2H3 cells and colitis models.

Elevation of the serotonin-derived quinone, tryptamine-4,5-dione, in the intestine of ICR mice with dextran sulfate-induced colitis.

Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic inflammatory disorders associated with oxidative stress. The intestines produce 5-hydroxytryptamine that may negatively affect disease state under inflammatory conditions when overproduced. 5-Hydroxytryptamine is a substrate for myeloperoxidase and is converted into reactive tryptamine-4,5-dione. Here, an experimental colitis model was established through oral administration of 5% dextran sulfate sodium to ICR mice for 7 days. Furthermore, the formation of tryptamine-4,5-dione in the colorectal mucosa/submucosa and colorectal tissue was analyzed by chemical and immunochemical methodologies. First, free tryptamine-4,5-dione in the homogenate was chemically trapped by o-phenylenediamine and analyzed as the stable phenazine derivative. Tryptamine-4,5-dione localization as adducted proteins in the colorectal tissue was immunohistochemically confirmed, and as demonstrated by both methods, this resulted in the significant increase of tryptamine-4,5-dione in dextran sulfate sodium-challenged mice compared with control mice. Immunohistochemical staining confirmed tryptamine-4,5-dione-positive staining at the myeloperoxidase accumulation site in dextran sulfate sodium-challenged mice colorectal tissue. The tryptamine-4,5-dione locus in the mice was partly matched with that of a specific marker for myeloperoxidase, halogenated tyrosine. Overall, the results possibly indicate that tryptamine-4,5-dione is generated by neutrophil myeloperoxidase in inflammatory tissue and may contribute to the development of inflammatory bowel disease.

Immunochromatographic detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli with cross-detection of cholera toxin.

Here, we report the development of an immunochromatographic test strip that can detect heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli. Five types of monoclonal antibody (mAb)-producing hybridomas were isolated: three mAbs were A subunit specific and two were B subunit specific. Four mAbs also cross-reacted with both LT proteins derived from swine and human E. coli strains, but only one mAb 57B9 additionally cross-reacted with cholera toxin. Thus, mAb 57B9 was used to form a gold colloid-conjugated antibody for the immunochromatographic test by combination with polyclonal anti-LT rabbit IgG. This test strip detected not only LT in the culture supernatant of LT gene-positive strains, but also cholera toxin in the culture supernatant of Vibrio cholerae. These results indicate that this test strip is suitable for the diagnosis of both enterotoxigenic E. coli and V. cholerae infection.

Development of a simple and rapid diagnosis method for swine edema disease to specifically detect Stx2e protein by immunochromatographic test.

Edema disease in piglets is caused by Shiga toxin 2e (Stx2e)-producing Escherichia coli. However, there is currently no available Stx2e-specific immunochromatographic test strip to differentiate Stx2e from other types of Shiga toxin 2. In the present study, to develop an Stx2e-specific immunochromatographic test strip, we isolated nine different monoclonal antibody-producing hybridoma clones from Stx2e toxoid-immunized mice and confirmed that six antibodies were A subunit-specific whereas three antibodies were B subunit-specific. Only one A subunit-specific monoclonal antibody (45B2) was cross-reactive with prototype Stx2 (Stx2a) at the same sensitivity, but the remaining eight monoclonal antibodies were not. In immunochromatographic tests using the highly sensitive antibodies, test strips using some combinations of gold colloid-conjugated monoclonal antibody with the B subunit-specific monoclonal antibody on the membrane detected Stx2e, but not other types of Shiga toxin 2. These test strips had the ability to detect Stx2e in the culture supernatant of clinically isolated Stx2e gene-positive strains, but not in those of Stx2e gene-negative strains. These results indicate that our test strip is practical for the specific detection of Stx2e to diagnose swine edema disease.

Isolation of B subunit-specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2.

Shiga toxin 2 (Stx2)-specific mAb-producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)-specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat-labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)-specific whereas five were Stx2B-specific antibody-producing clones. The in vitro neutralization activity of Stx2B-specific mAbs against Stx2 was greater than that of Stx2A-specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B-specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B-specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the VH and VL regions of clones 45 and 75D9 were determined. Our Stx2B-specific mAbs may be new candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease.

Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to ß-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing ß-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.