Diagnostic techniques for rapid detection of Vibrio cholerae O1/O139.

Cholera caused by the toxigenic Vibrio cholerae is still a major public health problem in many countries. This disease is mainly due to poor sanitation, hygiene and consumption of unsafe water. Several recent epidemics of cholera showed its increasing intensity, duration and severity of the illness. This indicates an urgent need for effective management and preventive measures in controlling the outbreaks and epidemics. In preventing and spread of epidemic cholera, rapid diagnostic tests (RDTs) are useful in screening suspected stool specimens, water/food samples. Several RDTs developed recently are considered as investigative tools in confirming cholera cases, as the culture techniques are difficult to establish and/or maintain. The usefulness of RDTs will be more at the point-of-care facilities as it helps to make appropriate decisions in the management of outbreaks or epidemiological surveillance by the public health authorities. Apart from RDTs, several other tests are available for the direct detection of either V. cholerae or its cholera toxin. Viable but non-culturable (VBNC) state of V. cholerae poses a great challenge in developing RDTs. The aim of this article is to provide an overview of current knowledge about RDT and other techniques with reference to their status and future potentials in detecting cholera/V. cholerae.

Single-Molecule Detection with Lightguiding Nanowires: Determination of Protein Concentration and Diffusivity in Supported Lipid Bilayers.

Determining the surface concentration and diffusivity of cell-membrane-bound molecules is central to the understanding of numerous important biochemical processes taking place at cell membranes. Here we use the high aspect ratio and lightguiding properties of semiconductor nanowires (NWs) to detect the presence of single freely diffusing proteins bound to a lipid bilayer covering the NW surface. Simultaneous observation of light-emission dynamics of hundreds of individual NWs occurring on the time scale of only a few seconds is interpreted using analytical models and employed to determine both surface concentration and diffusivity of cholera toxin subunit B (CTxB) bound to GM1 gangliosides in supported lipid bilayer (SLB) at surface concentrations down to below one CTxB per µm2. In particular, a decrease in diffusivity was observed with increasing GM1 content in the SLB, suggesting increasing multivalent binding of CTxB to GM1. The lightguiding capability of the NWs makes the method compatible with conventional epifluorescence microscopy, and it is shown to work well for both photostable and photosensitive dyes. These features make the concept an interesting complement to existing techniques for studying the diffusivity of low-abundance cell-membrane-bound molecules, expanding the rapidly growing use of semiconductor NWs in various bioanalytical sensor applications and live cell studies.

A binding motif for Hsp90 in the A chains of ADP-ribosylating toxins that move from the endoplasmic reticulum to the cytosol.

Cholera toxin (Ctx) is an AB-type protein toxin that acts as an adenosine diphosphate (ADP)-ribosyltransferase to disrupt intracellular signalling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER-associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognises two peptide sequences from CtxA1: an N-terminal RPPDEI sequence (residues 11-16) and an LDIAPA sequence in the C-terminal region (residues 153-158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full-length CtxA1. Both sequences were necessary for the ER-to-cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI-like motif at the N- or C-termini of the A chains from four other ER-translocating toxins that act as ADP-ribosyltransferases: pertussis toxin, Escherichia coli heat-labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP-ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER-to-cytosol export of CtxA1 and possibly other toxin A chains as well.

Real-Time Detection of Markers in Blood.

The real-time selective detection of disease-related markers in blood using biosensors has great potential for use in the early diagnosis of diseases and infections. However, this potential has not been realized thus far due to difficulties in interfacing the sensor with blood and achieving transparent circuits that are essential for detecting of target markers (e.g., protein, ions, etc.) in a complex blood environment. Herein, we demonstrate the real-time detection of a specific protein and ion in blood without a skin incision. Complementary metal-oxide-semiconductor technology was used to fabricate silicon micropillar array (SiMPA) electrodes with a height greater than 600 µm, and the surface of the SiMPA electrodes was functionalized with a self-assembling artificial peptide (SAP) as a receptor for target markers in blood, i.e., cholera toxin (CTX) and mercury(II) ions (Hg). The detection of CTX was investigated in both in vitro (phosphate-buffered saline and human blood serum, HBO model) and in vivo (mouse model) modes via impedance analysis. In the in vivo mode, the SiMPA pierces the skin, comes into contact with the blood system, and creates comprehensive circuits that include all the elements such as electrodes, blood, and receptors. The SiMPA achieves electrically transparent circuits and, thus, can selectively detect CTX in the blood in real time with a high sensitivity of 50 pM and 5 nM in the in vitro and in vivo modes, respectively. Mercury(II) ions can also be detected in both the in vitro and the in vivo modes by changing the SAP. The results illustrate that a robust sensor that can detect a variety of molecular species in the blood system in real time that will be helpful for the early diagnosis of disease and infections.

Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54% ±â€¯0.43 of the total soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37 °C, and approximately half of the binding affinity remained at 25 °C. Our results suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.

Cholera Outbreak due to Raw Seafood Consumption in South Korea, 2016.

Three cases of cholera occurred in South Korea during a period of three weeks in August 2016. All the cases were associated with the consumption of raw seafood in southern coastal area of South Korea. Epidemiologic investigations were performed to track the spread of cholera, including persons in contact with the cholera patients, seafood, and seawater from the fish tank and marine environments. A microbiological investigation demonstrated that cholera isolated from the three patients and a seawater sample at the Korea Strait showed identical serotype (O1 Ogawa), biotype (El tor), and toxin (ctx-positive). Pulsed-field gel electrophoresis analysis showed that the three clinical strains are identical (100%) and shared 97% identity with the seawater sample.

Selection of affinity peptides for interference-free detection of cholera toxin.

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB5.

CTA1: Purified and display onto gram-positive enhancer matrix (GEM) particles as mucosal adjuvant.

The A1 subunit of cholera toxin (CTA1) retains the adjuvant function of CT, without its toxic side effects, making the molecule a promising mucosal adjuvant. However, the methods required to obtain a pure product are both complicated and expensive, constricting its potential commercial applicability. Here, we fused the peptidoglycan binding domain (PA) to the C-terminus of CTA1, which enabled the fusion protein to be expressed by Bacillus subtilis, and secreted into the culture medium. CTA1 was then purified and displayed on GEM particles using a one step process, which resulted in the formation of CTA1-GEM complexes. Next, the CTA1-GEM complexes were used as an adjuvant to enhance the immune responses of mice to the influenza subunit vaccine. It was observed that the CTA1-GEM complexes enhanced specific systemic (IgG) and mucosal (IgA) immune responses against antigen, and induced cellular immune responses as well. The data presented here suggests that CTA1-GEM complexes can serve as a viable mucosal adjuvant.

[The application of MALDI-ToF mini-sequencing for detecting genetically altered versions of cholera agent].

The genetically altered modifications of V.cholerae eltor are characterized by occurrence of single-nucleotide polymorphisms in gene ctxB. To detect these modifications the technique is proposed based on mini-sequencing with MALDI-ToF by of products of reaction with selected probes adjacent to 115 and 203 positions of gene mentioned previously. The mass-spectrometry analysis of the results of reaction of mini-sequencing of strains of V.cholerae eltor isolated during epidemic complications at the territory of the Siberia and the Far East revealed mass-specters corresponding to values of molecular masses of probes (ctxB115, ctxB203) and those complementary completed to points of corresponding replacements (T/C) of didesoxinucleotides (ddTTP, ddCTP). For analyzed strains of V.cholerae eltor isolated in the 1970s, elongation is establishedfor both probes by didesoxinucleotide that testifies presence in their genome ctxB3 allele with thymine in 115 and 203 positions, distinctive for typical representatives of V.cholerae eltor. For V.cholerae eltor, isolated in 1990s, hybridization to points of replacement of didesoxicytosine and presence of ctxB1 allele with cytosine at analyzed positions, distinctive to vibrio of classic biovars. This allele is detected in genome of one of modifications of atypical genetically altered clones ofV.cholerae eltor. This technique, by its sensitivity and specificity, matches direct sequencing of gene ctxB of strains of V.cholerae eltor and proves promising for analysis of other valuable single-nucleotide polymorphisms.

Blood Group O-Dependent Cellular Responses to Cholera Toxin: Parallel Clinical and Epidemiological Links to Severe Cholera.

Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A(-/-)) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A(-/-) cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations.

Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli.

Herein, we report an Escherichia coli-based expression and purification method of recombinant cholera toxin B subunit (CTB). The CTB gene (E. coli codon optimized) is cloned into commercial pET-22b(+) vector using standard molecular biology techniques and the resulting vector is transformed into BL21(DE3) electrocompetent cells. The bacterial cells are grown and induction with isopropyl ß-D-1-thiogalactopyranoside (IPTG) results in accumulation of CTB in the culture medium. CTB is purified from the culture medium using a simple two-step chromatography process: immobilized metal affinity chromatography (IMAC) followed by ceramic hydroxyapatite (CHT). CTB is purified to >95 % homogeneity with a yield of over 10 mg per liter of culture. Depending on the application, endotoxin is removed using a commercially available endotoxin removal resin to <1 EU/mg.

Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously.

Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

Recognition of human milk oligosaccharides by bacterial exotoxins.

The affinities of the most abundant oligosaccharides found in human milk for four bacterial exotoxins (from Vibrio cholerae and pathogenic Escherichia coli) were quantified for the first time. Association constants (Ka) for a library of 20 human milk oligosaccharides (HMOs) binding to Shiga toxin type 2 holotoxin (Stx2) and the B subunit homopentamers of cholera toxin, heat-labile toxin and Shiga toxin type 1 (CTB5, HLTB5 and Stx1B5) were measured at 25°C and pH 7 using the direct electrospray ionization mass spectrometry assay. Notably, all four bacterial toxins bind to a majority of the HMOs tested and five of the HMOs (2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II and lacto-N-fucopentaose III) are ligands for all four toxins. These five HMOs are also reported to bind to other bacterial toxins (e.g. toxin A and toxin B of Clostridium difficile). In all cases, the HMO affinities (apparent Ka) are relatively modest (≤15,000 M(-1)). However, at the high concentrations of HMOs typically ingested by infants, a significant fraction of these toxins, if present, is expected to be bound to HMOs. Binding measurements carried out with 2'-fucosyllactose or lacto-N-fucopentaose I, together with a high-affinity ligand based on the native carbohydrate receptor, revealed that all four toxins possess HMO-binding sites that are distinct from those of the native receptors, although evidence of competitive binding was found for lacto-N-fucopentaose I with Stx2 and 2'-fucosyllactose and lacto-N-fucopentaose I with HLTB5. Taken together, the results of this study suggest that, while HMOs are expected to bind extensively to these bacterial toxins, it is unlikely that HMO binding will effectively inhibit their interactions with their cellular receptors.

[Improvement of laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera Vibrio biovar El Tor].

AIM: Improvement of laboratory diagnostics of cholera taking into the account appearance of hybrid variants of cholera vibrio El Tor biovar in the 1990s. MATERIALS AND METHODS: Phenotypic and molecular-genetic properties of typical toxigenic (151 strains) and hybrid (102 strains) variants of El Tor biovar cholera vibrios, isolated in the Caucuses in 1970-1990 and 1993-1998, respectively, were studied. Toxigenicity gene DNA fragments, inherent to El Tor biovars or classic, were detected by using a reagent kit "Genes of Vibrio cholerae variant ctxB-rstR-rstC, REF" developed by us. RESULTS: Reagent kit "Genes of V. cholerae variant ctxB-rstR-rstC, REF" is proposed to be used for laboratory diagnostics of cholera during study of material from humans or environmental objects and for identification of V. cholerae 01 on genome level in PCR-analysis as a necessary addition to the classic scheme of bacteriological analysis. CONCLUSION: Laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera vibrio El Tor biovar is based on a complex study of material from humans and environmental objects by routine bacteriologic and PCR-analysis methods with the aim of detection of gene DNA fragments in the studied material, that determine biovar (classic or El Tor), identification of V. cholerae O1 strains with differentiation of El Tor vibrios into typical and altered, as well as determination of enterotoxin, produced by the specific cholera vibrio strain (by the presence ctxB(El) or ctxB(Cl) gene DNA fragment, coding biosynthesis of CT-2 or CT-1, respectively).

Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample.

Pathogenic Vibrio cholerae produces a cholera toxin which is the cause of a severe diarrheal disease called "Cholera". Available detection methods, including standard bacteriological test and immuno-based detection, are specific to the suspected pathogenic V. cholerae O1 and O139, but they are not specific to the cholera toxin producible strain. This work combined the polymerase chain reaction (PCR) of cholera toxin gene, ctxA gene, and microcantilever-based DNA sensor to improve the sensitivity and specificity of detection. Gold coated microcantilever, 250 µm long and 50 µm wide, with an embedded polysilicon wire acting as a piezoresistive material was modified by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) for immobilization of specific DNA probe via avidin layer on the surface. The avidin and 5' biotinylated single-stranded DNA (ssDNA) probe concentrations were optimized for the immobilization at 50 µg/mL and 1 µM, respectively. The hybridization between ssDNA probe on this DNA sensor and target DNA creates nanomechanical bending and resistance change of piezoresistive material inside the beam. This microcantilever-based DNA sensor offers a detection sensitivity of 3.25 pg or 14 nM of DNA template for ctxA gene detection. The lowest number of V. cholerae O1 in food sample with and without the enrichment process that the polymerase chain reaction (PCR) for ctxA gene combined with this DNA sensor can detect is 0.835 and 835 cells/g, respectively. This detection sensitivity is 10 times higher than that of the conventional PCR method.

Twin outbreak of cholera in rural North Karnataka, India.

BACKGROUND & OBJECTIVES: Successive outbreaks of acute watery diarrhoea occurred in Talikoti and Harnal, located in Bijapur District of the southern Indian s0 tate of Karnataka, in July and August 2012, respectively. These outbreaks were investigated to identify the aetiology and epidemiology. METHODS: Information was collected from the local population and health centres. Stool and water samples were collected from the admitted patients and their drinking water sources. Standard microbiological and PCR techniques were employed for isolation and characterization of the pathogen. RESULTS: While 101 people (0.38%) were affected in Talikoti, 200 (20.94%) were affected in Harnal which is a small remote village. All age groups were affected but no death occurred. While the outbreak was smaller, longer and apparently spread by person to person contact in Talikoti, it occurred as a single source flash outbreak at Harnal. A single clone of toxigenic Vibrio cholerae O1 Ogawa biotype El Tor was isolated from the two stool samples obtained from Talikoti and subsequently from three of five stool samples obtained from Harnal indicating village to village spread of the aetiological agent. Striking similarity in antibiotic resistance profiles of these isolates with a particular strain isolated from the city of Belgaum, 250 km away, in 2010, prompted tracking the lineage of the V. cholerae isolates by DNA fingerprinting. Random amplified polymorphic DNA (RAPD) fingerprinting assay helped confirm the origin of the incriminating strain to Belgaum. INTERPRETATION & CONCLUSIONS: Our study reported the first twin outbreak of cholera in two remote areas of Bijapur district, Karnataka, south India. It also indicated the need for immediate preparedness to deal with such emergencies.

Crystallization of the HigBA2 toxin-antitoxin complex from Vibrio cholerae.

The genome of Vibrio cholerae encodes two higBA toxin-antitoxin (TA) modules that are activated by amino-acid starvation. Here, the TA complex of the second module, higBA2, as well as the C-terminal domain of the corresponding HigA2 antitoxin, have been purified and crystallized. The HigBA2 complex crystallized in two crystal forms. Crystals of form I belonged to space group P2(1)2(1)2, with unit-cell parameters a = 129.0, b = 119.8, c = 33.4 Å, and diffracted to 3.0 Šresolution. The asymmetric unit is likely to contain a single complex consisting of two toxin monomers and one antitoxin dimer. The second crystal form crystallized in space group P3(2)21, with unit-cell parameters a = 134.5, c = 55.4 Å. These crystals diffracted to 2.2 Šresolution and probably contain a complex with a different stoichiometry. Crystals of the C-terminal domain of HigA2 belonged to space group C2, with unit-cell parameters a = 115.4, b = 61.2, c = 73.8 Å, ß = 106.7°, and diffracted to 1.8 Šresolution.

Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks.

INTRODUCTION: Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants. METHODOLOGY/PRINCIPAL FINDINGS: In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and easily scalable system may enable the implementation of pCTB to mass vaccination against outbreaks, thereby providing better protection of high-risk populations in developing countries.