Detection of cholera toxin (ctxA and ctxAB) genes in Vibrio cholerae isolated from clinical and environmental samples in Limbang Sarawak by multiplex polymerase chain reaction (PCR)

Aims@#Cholera epidemics have been occurred in Malaysia since 1991 till 2003 which can be proved from the records by the Infectious Diseases Division of the Ministry of Health. Moreover, there were also course of cholera epidemics from the year 1994 to 2003 which had been happened in Sarawak. Cholera outbreaks in Malaysia mostly caused by the El Tor O1 Vibrio cholerae serogroup. The aims of this study were to detect the presence of V. cholerae in clinical and environmental samples (n=28) from Limbang, Sarawak by collaboration with Sarawak Government Hospital and to detect the toxin genes from the isolates.@*Methodology and results@#All the isolates were sub-cultured in alkaline peptone water (APW). The boiled-cell method was used for DNA extraction. The total DNA extracted was amplified by polymerase chain reaction (PCR). Two types of PCR were used in this study which are 16S rRNA PCR and multiplex PCR. The results obtained from the study found out that 16 out of 28 (57.14%) samples were confirmed to be V. cholerae species. Four primers specific for V. cholerae were used in multiplex PCR (O1 type, O139 type, ctxA and ctxAB) to confirm the species type and the toxin genes. All samples shown positive for V. cholerae O1 serotype and 100% positive to all genes for the identification of ctxA and ctxAB genes. @*Conclusion, significance and impact of study@#From this study, it showed that multiplex PCR can be used for research purposes in molecular genetics field involving cholera outbreak.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine / 生物医学与环境科学（英文）

OBJECTIVE@#To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine.@*METHODS@#Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 μg/mouse) alone, or CTA1-DD (5 μg/mouse) alone, or H3N2 split vaccine (3 μg/mouse) plus CTA1-DD (5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured.@*RESULTS@#H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus.@*CONCLUSION@#CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

A Group of Descending Glutamatergic Neurons Activated by Stress in Corticolimbic Regions Project to the Nucleus Accumbens

The nucleus accumbens (NAc) is the major component of the ventral striatum that regulates stress-induced depression. The NAc receives dopaminergic inputs from the ventral tegmental area (VTA), and the role of VTA-NAc neurons in stress response has been recently characterized. The NAc also receives glutamatergic inputs from various forebrain structures including the prelimbic cortex (PL), basolateral amygdala (BLA), and ventral hippocampus (vHIP), whereas the role of those glutamatergic afferents in stress response remains underscored. In the present study, we investigated the extent to which descending glutamatergic neurons activated by stress in the PL, BLA, and vHIP project to the NAc. To specifically label the input neurons into the NAc, fluorescent-tagged cholera toxin subunit B (CTB), which can be used as a retrograde neuronal tracer, was injected into the NAc. After two weeks, the mice were placed under restraint for 1 h. Subsequent histological analyses indicated that CTB-positive cells were detected in 170~680 cells/mm² in the PL, BLA, and vHIP, and those CTB-positive cells were mostly glutamatergic. In the PL, BLA, and vHIP regions analyzed, stress-induced c-Fos expression was found in 20~100 cells/mm². Among the CTB-positive cells, 2.6% in the PL, 4.2% in the BLA, and 1.1% in the vHIP were co-labeled by c-Fos, whereas among c-Fos-positive cells, 7.7% in the PL, 19.8% in the BLA, and 8.5% in the vHIP were co-labeled with CTB. These results suggest that the NAc receives a significant but differing proportion of glutamatergic inputs from the PL, BLA, and vHIP in stress response.

Epidemiology, management, and prevention of cholera

Cholera is an acute secretory form of diarrhea caused by a potent enterotoxin (cholera toxin) after ingestion of toxigenic Vibrio cholerae of the O1 or O139 serogroups. Although cholera is very common in Africa and Asia as a whole, the incidence of cholera has been very low in recent years in Korea. Dehydration and electrolyte abnormalities due to massive watery diarrhea can lead to death, and the mortality rates in untreated patients with severe cholera can exceed 70%. Effective rehydration therapy is the cornerstone of the management of patients with cholera and can reduce the mortality rate to less than 0.2%. Antibiotics reduce the volume and duration of diarrhea, but are recommended for patients with severe disease because of the rapid emergence and spread of multidrug-resistant V. cholerae across the globe. Two oral cholera vaccines are available, and the World Health Organization recommends that these oral vaccines be considered in integrated prevention programs in endemic countries at risk for outbreaks.

Antibody Production, Anaphylactic Signs, and T-Cell Responses Induced by Oral Sensitization With Ovalbumin in BALB/c and C3H/HeOuJ Mice

PURPOSE: Two mouse strains, BALB/c and C3H/HeOuJ, broadly used in the field of food allergy, were compared for the evaluation of the allergenic potential of ovalbumin (OVA). METHODS: Sensitization was made by administering 2 different OVA doses (1 and 5 mg), with cholera toxin as Th2-polarizing adjuvant. Antibody levels, severity of anaphylaxis, and Th1 and Th2 responses induced by the allergen were assessed. In addition, because the mice selected had functional toll-like receptor 4, the influence of contamination with lipopolysaccharide (LPS) on the immunostimulating capacity of OVA on spleen cells was also evaluated. RESULTS: Both strains exhibited similar susceptibility to OVA sensitization. The 2 protein doses generated similar OVA-specific IgE and IgG1 levels in both strains, whereas C3H/HeOuJ mice produced significantly more IgG2a. Oral challenge provoked more severe manifestations in C3H/HeOuJ mice as indicated by the drop in body temperature and the severity of the anaphylactic scores. Stimulation of splenocytes with OVA led to significantly higher levels of Th2 and Th1 cytokines in BALB/c, and these were less affected by protein contamination with LPS. CONCLUSIONS: The antibody and cytokine levels induced by OVA in BALB/c mice and the observation that BALB/c spleen cell cultures were more resistant than those of C3H/HeOuJ mice to the stimulus of LPS make this strain prone to exhibit Th2-mediated food allergic reactions and very adequate for the study of the features of OVA that make it allergenic.

Eosinophils are Required for Immune Responses Induced by Oral Immunization

Eosinophils are multifunctional leukocytes that reside in several tissues, most abundantly in the small intestinal lamina propria under the steady state. To date, the phenotypic and functional characteristics of small intestinal eosinophils have remained poorly understood. In this study, we found that proliferation of ovalbumin (OVA)-specific CD4+ T cells isolated from the mesenteric lymph nodes of eosinophil-deficient DeltadblGATA mice were decreased relative to wild-type mice after oral immunization with OVA and cholera toxin (CT), the typical mucosal adjuvant that induces CD4+ T cell-dependent responses. DeltadblGATA mice showed reduced mucosal secretion of OVA-specific IgA and IgG1 while maintaining a systemic level of anti-OVA IgG1 upon oral immunization with OVA and CT. These findings suggest that eosinophils might have a role in the modulation of T cell-mediated immune responses including mucosal antibody responses in the gastrointestinal tract following oral immunization.

Curcumin utilizes the anti-inflammatory response pathway to protect the intestine against bacterial invasion

BACKGROUND/OBJECTIVES: Curcumin, a major component of the Curcuma species, contains antioxidant and anti-inflammatory properties. Although it was found to induce apoptosis in cancer cells, the functional role of curcumin as well as its molecular mechanism in anti-inflammatory response, particularly in intestinal cells, has been less investigated. The intestine epithelial barrier is the first barrier and the most important location for the substrate coming from the lumen of the gut. SUBJECTS/METHODS: We administered curcumin treatment in the human intestinal epithelial cell lines, T84 and Caco-2. We examined endoplasmic reticulum (ER) stress response by thapsigargin, qPCR of XBP1 and BiP, electrophysiology by wild-type cholera toxin in the cells. RESULTS: In this study, we showed that curcumin treatment reduces ER stress and thereby decreases inflammatory response in human intestinal epithelial cells. In addition, curcumin confers protection without damaging the membrane tight junction or actin skeleton change in intestine epithelial cells. Therefore, curcumin treatment protects the gut from bacterial invasion via reduction of ER stress and anti-inflammatory response in intestinal epithelial cells. CONCLUSIONS: Taken together, our data demonstrate the important role of curcumin in protecting the intestine by modulating ER stress and inflammatory response post intoxication.

Development of oral cholera vaccine and its vaccination / 中华预防医学杂志

The application of the cholera vaccine is one of the cholera prevention and control strategies. Cholera vaccines stimulate mucosal immune to play the role of antibacteria and antitoxin. When the cholera toxin B subunit is added in the cholera vaccine, it could also defend against some diarrhea associated pathogens by cross-protection. Oral inactivated cholera vaccines are commercially available now. The oral live vaccine candidates are under development. The development of cholera vaccine is not only on the technical aspect, based on the situations of epidemic areas and population, cost, storage and transportation condition should also be considered. Though the argument on the use of cholera vaccine in epidemic areas and population in high risk existed previously, its vaccination has reached agreement now based on the clinical trials and evaluations during epidemic period.

Antibiotic resistance and molecular characterization of Vibrio cholera strains isolated from an outbreak of cholera epidemic in Jiangsu province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To assess the antibiotic resistance and molecular characterization of cholera strains and to provide basis for clinical treatment and prevention of cholera.</p><p><b>METHODS</b>4 stains isolated from an outbreak of cholera epidemic in Huai'an City in Jiangsu province in September 2010 were characterized using antibiotic susceptibility, biotype analysis, virluence genes detection, ctxB gene sequencing, and PFGE analysis.</p><p><b>RESULTS</b>The 4 strains were all resistant to sulphamethoxazole/trimethoprim, erythromycin, streptomycin. High drug susceptibility of the samples was found to 6 kinds of antibiotics such as amikacin, norfloxacin, ciprofloxacin, gentamicin, chloramphenicol, ampicillin. The isolates expressed phenotypic traits of both serogroup O1 ogawa and El Tor and carried 9 kinds of virulence genes, ctxA, ace, zot, toxR, tcpI, ompU, rtxC, tcpA, and hlyA gene. They were also identified as harboring the classical ctxB genotype based on amino acid residue substitutions. The PFGE profiles of NotI showed a single banding pattern, while SfiI's was 2 banding patterns.</p><p><b>CONCLUSION</b>The bacterium type of Vibrio cholerae causing the epidemic outbreak of cholera belonged to the atypical EL Tor variant which was also identified as toxicogenic strain. The mapping of the strains prompted that there should be the common contamination source. Drug sensitivity test can guide the clinical drug use, in order to reduce the emergence of resistant strains.</p>

High-efficiency expression of a receptor-binding domain of SARS-CoV spike protein in tobacco chloroplasts / 生物工程学报

Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.

Molecular characteristics and antibiotic resistance of Vibrio cholerae O139 in Shandong province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the molecular epidemiological characteristics and antibiotic resistance profiles of Vibrio cholerae O139 in Shandong province.</p><p><b>METHODS</b>A total of 13 strains of V. cholerae O139 (9 clinical strains and 4 environmental strains) isolated from cholera epidemics in Shandong province since 1997 were recovered and confirmed with serum agglutination and biochemical reaction. Pulsed-field gel electrophoresis (PFGE) was carried out for molecular subtyping. Virulence genes and drug resistance related genes were detected by PCR. Antibiotic susceptibility tests were performed using micro-broth dilution method.</p><p><b>RESULTS</b>Thirteen strains of V. cholerae O139 were differentiated into seven pulsetypes. One clinical strain and two environmental strains isolated from Jining in 2013 were clustered into the pulsetype namely KZGN11O139. CN0077, and an identical PFGE pattern of KZGN11O139. CN0002 was found among three clinical strains from Jinan in 2005, Jining in 2005 and Heze in 2009. Other pulsotypes were unique in China and found only in Shandong province. Because of deletion of ctxAB and tcpI, the PFGE patterns of two strains isolated from Yantai in 2000 and 2004 were different from other 11 strains which harbored ctxAB, tcpA, tcpI, rtxA, hlyA and toxR. All strains contained one or more drug resistance related genes such as intI 1, intI 4 and sxt, and were resistant to two kinds of antibiotics at least. Among the 12 kinds of antibiotics, the resistant ratioes to kamamycin, trimethoprim-sulfamethoxazole, ampicillin and gentamicin were 11/13, 9/13, 7/13 and 7/13, respectively.</p><p><b>CONCLUSION</b>Molecular subtyping indicates possible epidemiological links among V.cholerae O139 in Shandong province, and almost all strains were toxigenic and drug resistant.</p>

Establishment of a triplex real-time PCR for the detection of cholera toxin gene ctx and heat labile enterotoxin gene elt / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish a triplex TaqMan real-time PCR system containing internal amplification control (IAC) to detect cholera toxin gene ctxA and enterotoxigenic Escherichia coli (ETEC)heat-labile enterotoxin gene elt.</p><p><b>METHODS</b>Primers and probes were designed based on the sequences of ctxA, elt and IAC. Both sensitivity and specificity were analyzed and interactions between different reactions were evaluated.</p><p><b>RESULTS</b>This system showed that the sensitivity of ctxA was 94 copies/reaction while the elt 79 copies/reaction and the amplification efficiency were 94.7% and 98.1%, respectively. Under the ratio of copy numbers on gene ctxA to elt as between 1 : 1-1 : 10, when both targets were detected, with impact was less on each other. However, when the amount of elt or ctxA was 100 times of IAC, the amplification of IAC was significantly inhibited.</p><p><b>CONCLUSION</b>This system showed both satisfactory sensitivity and specificity, thus could be used to detect pathogenic bacteria in diarrhea stools. The detection of IAC could prompt the presence of PCR inhibitors in samples being tested.</p>

Combination of salermide and cholera toxin b induce apoptosis in MCF-7 but not in MRC-5 cell lines

Sirtuin1 is an enzyme that deacetylates histones and several non-histone proteins including P53 during the stress. P300 is a member of the histone acetyl transferase family and enzyme that acetylates histones. Hereby, this study describes the potency combination of Salermide as a Sirtuin1 inhibitor and cholera toxin B [CTB] as a P300 activator to induce apoptosis Michigan Cancer Foundation-7 [MCF-7] and MRC-5. Cells were cultured and treated with a combination of Salermide and CTB respectively at concentrations of 80.56 and 85.43 micro mol/L based on inhibitory concentration 50 indexes at different times. The percentage of apoptotic cells were measured by flow cytometry. Real-time polymerase chain reaction was performed to estimate the messenger ribonucleic acid expression of Sirtuin1 and P300 in cells. Enzyme linked immunosorbent assay and Bradford protein techniques were used to detect the endogenous levels of total and acetylated P53 protein generated in both cell lines. Our findings indicated that the combination of two drugs could effectively induced apoptosis in MCF-7 significantly higher than MRC-5. We showed that expression of Sirtuin1 and P300 was dramatically down-regulated with increasing time by the combination of Salermide and CTB treatment in MCF-7, but not MRC-5. The acetylated and total P53 protein levels were increased more in MCF-7 than MRC-5 with incubated combination of drugs at different times. Combination of CTB and Salermide in 72 h through decreasing expression of Sirtuin1 and P300 genes induced acetylation of P53 protein and consequently showed the most apoptosis in MCF-7 cells, but it could be well-tolerated in MRC-5. Therefore, combination of drugs could be used as an anticancer agent

Effect of Cholera Toxin Administered Supraspinally or Spinally on the Blood Glucose Level in Pain and D-Glucose Fed Animal Models

In the present study, the effect of intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration with cholera toxin (CTX) on the blood glucose level was examined in ICR mice. The i.t. treatment with CTX alone for 24 h dose-dependently increased the blood glucose level. However, i.c.v. treatment with CTX for 24 h did not affect the blood glucose level. When mice were orally fed with D-glucose (2 g/kg), the blood glucose level reached to a maximum level at 30 min and almost returned to the control level at 120 min after D-glucose feeding. I.c.v. pretreatment with CTX increased the blood glucose level in a potentiative manner, whereas i.t. pretreatment with CTX increased the blood glucose level in an additive manner in a D-glucose fed group. In addition, the blood glucose level was increased in formalin-induced pain animal model. I.c.v. pretreatment with CTX enhanced the blood glucose level in a potentiative manner in formalin-induced pain animal model. On the other hand, i.t. pretreatment with CTX increased the blood glucose level in an additive manner in formalin-induced pain animal model. Our results suggest that CTX administered supraspinally or spinally differentially modulates the regulation of the blood glucose level in D-glucose fed model as well as in formalin-induced pain model.

Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance.

Purpose: The aim of this study was to understand the epidemiological linkage of clinical and environmental isolates of Vibrio cholerae and to determine their genotypes and virulence genes content. Materials and Methods: A total of 60 V. cholerae strains obtained from clinical specimens (n = 40) and surface waters (n = 20) were subjected to genotyping using PFGE and determination of their virulence-associated gene clusters. Result: PCR analysis showed the presence of chromosomally located hly and RTX genetic elements in 100% and 90% of the environmental isolates, respectively. The phage-mediated genetic elements such as CTX, TLC and VPI were detected in 5% of the environmental isolates suggesting that the environmental isolates cannot acquire certain mobile gene clusters. A total of 4 and 18 pulsotypes were obtained among the clinical and environmental V. cholerae isolates, respectively. Non-pathogenic environmentally isolated V. cholerae constituted a distinct cluster with one single non-O1, non-O139 strain (EP6) carrying the virulence genes similar to the epidemic strains. This may suggest the possible potential of conversion of non-pathogenic to a pathogenic environmental strain. Conclusions: The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.

Caspase-11, the Main Executioner in Non-canonical Inflammasome

Murine caspase-11 is orthologus to human caspase-4 and caspase-5, and is required for the response to cholera toxin B and infection with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Caspase-11 rather than caspase-1 is the main offender involved in sepsis. Hence, caspase-11 plays an essential pro-inflammatory role in innate immune response to bacterial infections.

Immunogenicity and Protective Efficacy of a Dual Subunit Vaccine Against Respiratory Syncytial Virus and Influenza Virus

Respiratory syncytial virus (RSV) and influenza virus are the most significant pathogens causing respiratory tract diseases. Composite vaccines are useful in reducing the number of vaccination and confer protection against multiple infectious agents. In this study, we generated fusion of RSV G protein core fragment (amino acid residues 131 to 230) and influenza HA1 globular head domain (amino acid residues 62 to 284) as a dual vaccine candidate. This fusion protein, Gcf-HA1, was bacterially expressed, purified by metal resin affinity chromatography, and refolded in PBS. BALB/c mice were intranasally immunized with Gcf-HA1 in combination with a mucosal adjuvant, cholera toxin (CT). Both serum IgG and mucosal IgA responses specific to Gcf and HA1 were significantly increased in Gcf-HA1/CT-vaccinated mice. To determine the protective efficacy of Gcf-HA1/CT vaccine, immunized mice were challenged with RSV (A2 strain) or influenza virus (A/PR/8/34). Neither detectable viral replication nor pathology was observed in the lungs of the immune mice. These results demonstrate that immunity induced by intranasal Gcf-HA1/CT immunization confers complete protection against both RSV and homologous influenza virus infection, suggesting our Gcf-HA1 vaccine candidate could be further developed as a dual subunit vaccine against RSV and influenza virus.

Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

Background & objectives: El Tor Vibrio cholerae O1 carrying ctxBC trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. Methods: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpAC, tcpAE, hlyAC and hlyAE were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. Results: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. Interpretation & conclusions: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxBC).

Cross-reactivity of outer membrane proteins of Campylobacter species with cholera toxin.

Campylobacter jejuni is a foodborne pathogen and a leading cause of diarrhoea worldwide. It is believed that a cholera toxin-like toxin (CTLT) produced by C. jejuni may mediate watery diarrhoea. However, the production of a CTLT by C. jejuni is controversial. A cholera toxin gene (ctx) homologue has not been identified in Campylobacter species. We investigated the identity of the CT cross-reactive antigen from Campylobacter species previously and the results are reviewed here. Filtrates of C. jejuni grown in four different liquid media, reported to promote CTLT production, were tested by Chinese hamster ovary (CHO) cell elongation assay for functional toxin and for reactivity with CT antibody using GM1 ganglioside ELISA (GM1 ELISA) and immunoblotting. Protein sequence of the CT antibody-reactive band was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Non-jejuni species (C. coli, C. lari, C. foetus, C. hyointestinalis and C. upsaliensis) were investigated by CHO cell assay and immunoblotting. Filtrates from seven C. jejuni reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay. However, filtrates from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains irrespective of GM1 ELISA reactivity, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein (PorA) of C. jejuni. CT antibody reacted with a C. jejuni recombinant PorA on immunoblotting. All non-C. jejuni strains were negative by CHO cell assay, but the common 53-kDa proteins reacted with CT antibody on immunoblots. The cross-reactivity of PorAs of Campylobacter species with CT may lead to the erroneous conclusion that Campylobacter species produce a functional CTLT.

Molecular mechanism of acquisition of the cholera toxin genes.

One of the major pathogenic determinants of Vibrio cholerae, the cholera toxin, is encoded in the genome of a filamentous phage, CTX. CTX makes use of the chromosome dimer resolution system of V. cholerae to integrate its single stranded genome into one, the other, or both V. cholerae chromosomes. Here, we review current knowledge about this smart integration process.