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.
Vibrio cholerae--genetics ; Cholera Toxin

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.
Adjuvants, Immunologic ; Administration, Intranasal ; Animals ; Cholera Toxin ; Female ; Immunity, Humoral ; Influenza A Virus, H3N2 Subtype ; immunology ; Influenza Vaccines ; Mice, Inbred BALB C ; Nasal Mucosa ; immunology ; Random Allocation ; Recombinant Fusion Proteins

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.
Animals ; Basolateral Nuclear Complex ; Cholera Toxin ; Depression ; Hippocampus ; Mice ; Neurons* ; Nucleus Accumbens* ; Prosencephalon ; Ventral Striatum ; Ventral Tegmental Area

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.
Africa ; Anti-Bacterial Agents ; Asia ; Cholera Toxin ; Cholera Vaccines ; Cholera* ; Dehydration ; Diarrhea ; Disease Outbreaks ; Eating ; Enterotoxins ; Epidemiology* ; Fluid Therapy ; Humans ; Incidence ; Korea ; Mortality ; Serogroup ; Vaccines ; Vibrio cholerae ; World Health Organization

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.
Anaphylaxis ; Animals ; Antibody Formation* ; Body Temperature ; Cell Culture Techniques ; Cholera Toxin ; Cytokines ; Food Hypersensitivity ; Hypersensitivity ; Immunoglobulin E ; Immunoglobulin G ; Mice* ; Ovalbumin* ; Ovum ; Spleen ; T-Lymphocytes* ; Toll-Like Receptor 4

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.
Animals ; Antibody Formation ; Cholera Toxin ; Eosinophils* ; Gastrointestinal Tract ; Immunity, Mucosal ; Immunization* ; Immunoglobulin A ; Immunoglobulin G ; Intestine, Small ; Leukocytes ; Lymph Nodes ; Mice ; Mucous Membrane ; Ovalbumin ; Ovum ; T-Lymphocytes

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.
Actins ; Apoptosis ; Bacteria ; Cholera Toxin ; Curcuma ; Curcumin* ; Electrophysiology ; Endoplasmic Reticulum ; Epithelial Cells ; Humans ; Intestines* ; Membranes ; NF-kappa B ; Skeleton ; Thapsigargin ; Tight Junctions

OBJECTIVETo assess the antibiotic resistance and molecular characterization of cholera strains and to provide basis for clinical treatment and prevention of cholera.

METHODS4 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Anti-Bacterial Agents ; Cholera ; Cholera Toxin ; Disease Outbreaks ; Drug Resistance, Bacterial ; Drug Resistance, Microbial ; Epidemics ; Genotype ; Humans ; Vibrio cholerae ; Vibrio cholerae O1 ; Virulence

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.
Administration, Oral ; Cholera ; Cholera Toxin ; Cholera Vaccines ; Cross Protection ; Diarrhea ; Humans ; Vaccination ; Vaccines, Attenuated ; Vaccines, Inactivated

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.
Chloroplasts ; metabolism ; Cholera Toxin ; Protein Interaction Domains and Motifs ; Recombinant Fusion Proteins ; biosynthesis ; SARS Virus ; Spike Glycoprotein, Coronavirus ; biosynthesis ; Tobacco ; metabolism ; Vaccines, Subunit ; biosynthesis