Seroepidemiology of cholera in Gulf coastal Texas

Single serum samples from 559 volunteers from a Texas Gulf Coast area were examined for vibriocidal antibody to Vibrio cholerae O1 (biotype El Tor, serotype Inaba) by a microtiter method. Elevated levels of vibriocidal antibody were present in 14% of the subjects. Also, 6.8% of the subjects had elevated levels of antibody to the enterotoxin of V. cholerae O1 by the immunoglobulin G enzyme-linked immunosorbent assay. Recent infection, defined on the basis of elevations in both vibriocidal and antitoxin antibodies, had occurred in 1.3% of the subjects. When subjects who reported Brucella infection, travel to a cholera-endemic area, and/or cholera vaccination within a year of the study were removed from the analysis, a prevalence of recent infection of 0.89% was obtained. Significantly higher titers of vibriocidal antibody were found in those with exposure to seawater (fishermen, shrimpers, merchant marines, and dock workers) than in those without such exposure (P less than 0.005). Furthermore, titers of antitoxin antibody were significantly higher in those who consumed shellfish than in nonconsumers. Finally, titers of vibriocidal antibody were significantly higher in Vietnamese subjects than in non-Vietnamese subjects. The results of this study indicate that an endemic focus of infection with V. cholerae occurs in this area.     

小鼠血清中杀O139霍乱弧菌抗体检测方法的初步建立

目的研究探索O139霍乱弧菌杀弧菌抗体的检测方法。方法用微孔板培养和琼脂平板克隆计数相结合的杀弧菌抗体检测方法,对实验菌株及稀释度、补体浓度等关键参数进行筛选;对50份小鼠免疫血清进行杀弧菌抗体滴度检测,并与O139群霍乱弧菌LPS Ig G抗体滴度进行相关分析;对该方法的特异性、线性和精密性进行了验证。结果筛选出最佳菌株为20100603菌株,最佳稀释度倍数为2 000倍,补体最佳稀释倍数为16倍。O139群霍乱弧菌小鼠免疫血清检测到较高的杀弧菌抗体滴度而PBS小鼠免疫血清未检测到杀弧菌抗体滴度。小鼠免疫血清杀弧菌抗体滴度与O139群霍乱弧菌LPS Ig G抗体滴度之间存在正相关关系。验证结果显示,在抑制剂浓度达到1.0~2.0 A600时,抑制率100%;线性回归方程为y=-1.093x+5.058,其相关系数为-0.999,P0.05;方法批内CV值为15.72%,批间CV值为23.47%。结论初步建立了O139霍乱弧菌杀弧菌抗体的检测方法,该方法具有较高的特异性、线性和精密度。     

Immunogenicity of synthetic saccharide fragments of Vibrio cholerae O1 (Ogawa and Inaba) bound to Exotoxin A

Recombinant exotoxin A (rEPA) from Pseudomonas aeruginosa conjugated to Vibrio cholerae O1 serotype-specific polysaccharides (mono-, di- and hexasaccharide) were immunogenic in mice. Monosaccharide conjugates boosted the humoral responses to the hexasaccharide conjugates. Prior exposure to purified Ogawa lipopolysaccharide (LPS) enabled contra-serotype hexasaccharide conjugates to boost the vibriocidal response, but Inaba LPS did not prime for an enhanced vibriocidal response by a contra-serotype conjugate. Prior exposure to the carrier, and priming B cells with the LPS of either serotype, resulted in enhanced vibriocidal titers if the Ogawa hexasaccharides were used, but a diminished response to the Inaba LPS. These studies demonstrate that the 'functional' B cell epitopes on the LPS differ from those of the neoglycoconjugates and that the order of immunization and the serotype of the boosting conjugate can influence the epitope specificity and function of the antisera.     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.     

Immunogenicity of a Killed Bivalent (O1 and O139) Whole Cell Oral Cholera Vaccine,Shanchol, in Haiti

Background

Studies of the immunogenicity of the killed bivalent whole cell oral cholera vaccine, Shanchol, have been performed in historically cholera-endemic areas of Asia. There is a need to assess the immunogenicity of the vaccine in Haiti and other populations without historical exposure to Vibrio cholerae.

Methodology/Principal Findings

We measured immune responses after administration of Shanchol, in 25 adults, 51 older children (6–17 years), and 47 younger children (1–5 years) in Haiti, where cholera was introduced in 2010. A≥4-fold increase in vibriocidal antibody titer against V. cholerae O1 Ogawa was observed in 91% of adults, 74% of older children, and 73% of younger children after two doses of Shanchol; similar responses were observed against the Inaba serotype. A≥2-fold increase in serum O-antigen specific polysaccharide IgA antibody levels against V. cholerae O1 Ogawa was observed in 59% of adults, 45% of older children, and 61% of younger children; similar responses were observed against the Inaba serotype. We compared immune responses in Haitian individuals with age- and blood group-matched individuals from Bangladesh, a historically cholera-endemic area. The geometric mean vibriocidal titers after the first dose of vaccine were lower in Haitian than in Bangladeshi vaccinees. However, the mean vibriocidal titers did not differ between the two groups after the second dose of the vaccine.

Conclusions/Significance

A killed bivalent whole cell oral cholera vaccine, Shanchol, is highly immunogenic in Haitian adults and children. A two-dose regimen may be important in Haiti, and other populations lacking previous repeated exposures to V. cholerae.     

Colonization of professional divers by toxigenic Vibrio cholerae O1 and V. cholerae non-O1 at dive sites in the United States, Ukraine and Russia

Abstract Vibrio cholerae , recognized as the causative agent of epidemic cholera, was isolated from healthy professional divers and from water samples collected at dive sites in the United States, Ukraine and Russia. Swabs of nose, ear and throat of divers and their tank regulators, i.e. the divers and their diving gear, were taken before and after routine dives. Blood samples were collected before and 30–60 days after each dive to measure IgG and IgA titers against the whole cell antigen of V. cholerae O1. Nine strains of V. cholerae O1 and nine strains of V. cholerae non-O1 were isolated during this study. These isolates were identified by conventional biochemical tests and indirect fluorescent antibody staining methods, using fluorescein isothiocyanate-labeled monoclonal antibody, COLTA, prepared against the 'A' antigenic factor of the lipopolysaccharide of V. cholerae O1, and serotyped by slide agglutination. Seven of the nine strains of V. cholerae O1 isolated and successfully cultured during the studies, were toxigenic by enzyme-linked immunosorbent assay and polymerase chain reaction. Analyses of IgG and IgA antibodies of the divers showed that most of the divers had prior exposure to V. cholerae O1. V. cholerae serotype non-O1 strains isolated during the study were found to be non-toxigenic.     

Cholera toxin modulates the systemic immune responses against Vibrio cholerae surface antigens after repeated inoculations

The immunomodulating properties of a low cholera toxin (CT) dose over the systemic antibody response against Vibrio cholerae antigens after a comparatively extensive period of time were evaluated. Groups of 10 mice were injected intraperitoneally three times at 0, 30 and 86 days with 500 microl of buffer or 10(8) viable recombinant V. cholerae bacteria (lacking cholera toxin A subunit) with or without 100 ng of CT. Sera were obtained from inoculated mice at 0, 14, 28, 37, 58, 80, 93, 114, 236 and 356 days after the first injection. Vibriocidal activity and IgM and IgG anti-lipopolysaccharide (LPS) or outer membrane protein (OMP) antibodies levels were estimated by ELISA in sera of inoculated mice. Anti-LPS IgG subclasses were measured 2 weeks after each immunization by ELISA. Treatment of mice with CT markedly influenced the immune response to LPS but not against OMP of V. cholerae. Simultaneous intraperitoneal administration of CT with V. cholerae resulted in marked enhancement of both IgM anti-LPS and vibriocidal titers which subsisted for a relatively extensive period of time after repeated antigen administration. No differences were observed in IgM and IgG anti-OMP titers after extended periods of time between CT and control treatments. A similar pattern of IgG anti-LPS subclasses was observed in the serum samples analyzed. These results suggest that long term CT administration modulates the IgM anti-V. cholerae LPS response and the serum vibriocidal activity.     

Oral immunization in adult mice to live and heat-killed Vibrio cholerae

Effects of systemic and intestinal local immune responses in mice fed ad libitum and forcedly with live or heat-killed Vibrio cholerae on the elimination of vibrios from the intestine were investigated. In mice fed with live vibrios, ad libitum feeding could induce potential delayed-type hypersensitivity and rapid production of vibriocidal antibody in the serum whereas forcedly feeding suppressed the delayed-type hypersensitivity and retarded the antibody production. In contrast, when killed microorganisms were used as antigens, significant delayed-type hypersensitivity and rapid response of vibriocidal antibody were induced in forcedly fed mice although ad libitum feeding suppressed the induction of the delayed-type hypersensitivity and retarded the production of vibriocidal antibody. The elimination of vibrios from the intestine of mice was promoted in both mice groups fed ad libitum and forcedly with live vibrios but not with killed microorganisms. Total IgA in the intestinal contents of mice fed with live vibrios both ad libitum and forcedly were higher than those of mice fed with killed antigens. In addition, when the extracts of intestinal contents were absorbed by live antigens, IgA contents in mice fed with live vibrios were reduced more markedly than those in mice immunized orally by the feeding with killed antigens. These findings suggested that the elimination of vibrios from the organ was closely related to local IgA antibody response to heat-labile substance of live Vibrio cholerae.     

A semi-automated vibriocidal assay for improved measurement of cholera vaccine-induced immune responses

Vibriocidal antibody assay has been a surrogate standard assay in the evaluation of cholera vaccine efficacy because it has a good correlation with protection. Although the optical density-based vibriocidal assay in a 96-well microtiter-plate format is widely used in clinical trials, it has limitations as vibriocidal titers are altered by incubation time and samples with the same end-point titers could have potentially different vibriocidal kinetics. In the present study, we developed an improved agar-plate assay coupled with an automated colony counting system. Through testing 30 pairs of human sera from vaccinees administered with a cholera vaccine or placebo, these two assays showed good correlations for the vibriocidal titers and fold increases in titers between pre- and post-vaccinated sera as determined by the Pearson correlation coefficient and the Regression coefficient. Notably, the newly-developed semi-automated assay demonstrated that serum samples with the same end-point titers turned out to have distinct vibriocidal kinetics that were not distinguishable by the microtiter-plate assay. The semi-automated assay responded specifically to Vibrio cholerae but not to irrelevant bacteria such as Salmonella typhi and Escherichia coli. These results demonstrate that the semi-automated assay provides better sensitivity, accuracy, and stability of the assay results with minimized efforts than conventional microtiter-plate assay and could provide a useful tool as an in vitro surrogate assay for the evaluation of cholera vaccine efficacy.     

Vibriocidal Antibody Responses to a Bivalent Killed Whole-Cell Oral Cholera Vaccine in a Phase III Trial in Kolkata,India

Background

During the development of a vaccine, identification of the correlates of protection is of paramount importance for establishing an objective criterion for the protective performance of the vaccine. However, the ascertainment of correlates of immunity conferred by any vaccine is a difficult task.

Methods

While conducting a phase three double-blind, cluster-randomized, placebo-controlled trial of a bivalent killed whole-cell oral cholera vaccine in Kolkata, we evaluated the immunogenicity of the vaccine in a subset of participants. Randomly chosen participants (recipients of vaccine or placebo) were invited to provide blood samples at baseline, 14 days after the second dose and one year after the first dose. At these time points, serum geometric mean titers (GMT) of vibriocidal antibodies and seroconversion rates for vaccine and placebo arms were calculated and compared across the age strata (1 to 5 years, 5 to 15 years and more than 15 years) as well as for all age groups.

Results

Out of 137 subjects included in analysis, 69 were vaccinees and 68 received placebo. There were 5•7 and 5•8 geometric mean fold (GMF) rises in titers to Vibrio cholerae Inaba and Ogawa, respectively at 14 days after the second dose, with 57% and 61% of vaccinees showing a four-fold or greater titer rise, respectively. After one year, the titers to Inaba and Ogawa remained 1•7 and 2•8 fold higher, respectively, compared to baseline. Serum vibriocidal antibody response to V. cholerae O139 was much lower than that to Inaba or Ogawa. No significant differences in the GMF-rises were observed among the age groups.

Conclusions

The reformulated oral cholera vaccine induced a statistically significant anti-O1 Inaba and O1 Ogawa vibriocidal antibody response 14 days after vaccination, which although declined after one year remained significantly higher than baseline. Despite this decline, the vaccine remained protective five years after vaccination.     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

Dietary l-proline supplementation confers immunostimulatory effects on inactivated Pasteurella multocida vaccine immunized mice

This study was conducted to determine the immunostimulatory effect of l-proline on inactivated vaccine immunized mice. Ninety-five female KM mice were randomly divided into five groups: (1) mice received dietary supplementation with 0.4 % l-proline and immunized with inactivated vaccine (V–P group); (2) mice received dietary supplementation with 0.3 % l-alanine (isonitrogenous control) and immunized with inactivated vaccine (V–A group, negative control); (3) mice were immunized with inactivated vaccine with oil adjuvant (V–O group, positive control); (4) mice were immunized with inactivated vaccine with aluminum hydroxide adjuvant (V–H group, positive control); (5) mice immunized with phosphate-buffered saline (control group). All mice were dead in the control group between 36 and 48 h post infection. Mice in the V–P group showed 100 % protection after challenge with P. multocida serotype A (CQ2) at dose of 4.4 × 10⁵ CFU (2LD50). Meanwhile, serum antibody titers in the V–P group were higher than those in the V–A group before infection and those in the V–A and V–O groups at 36 h post infection. Moreover, serum IL-1β levels in the V–P group were lower than those in V–O group. Furthermore, serum GSH-PX levels in the V–P group were higher than those in the V–A and V–O groups. Collectively, dietary proline supplementation confers beneficial immunostimulatory effects in inactivated P. multocida vaccine immunized mice.     

O139霍乱灭活全菌体菌苗免疫后人群抗体水平测定

Ｏ１３９霍乱弧菌经甲醛灭活制备而成的全菌体菌苗在广西田阳县进行临床考核。每组５０名中学生经肌肉途径接种４５亿菌和９０亿菌两种剂量,一个月后８０％接种者血清中杀弧菌抗体升高,最高可达１∶１６０,免疫后３个月开始缓慢下降,但仍有６８－７２％接种者抗体效价在１∶２０以上。高剂量组半数接种者的杀弧菌抗体可维持存在６个月。血清中凝集抗体升高明显,但３个月后下降也明显。与对照组Ｏ１菌苗相比,两种抗体水平升高与下降趋势一致。采用ＥＬＩＳＡ法测定抗毒抗体,Ｏ１３９菌苗组与Ｏ１菌苗组结果相似,均有一定上升。上述结果提示Ｏ１３９霍乱灭活菌苗可作为短效应急菌苗使用     

Immunochemical Studies on Lipopolysaccharide from Agglutinable and Non-Agglutinable Vibrios

Lipopolysaccharide (LPS) prepared from Vibrio cholerae and a non-agglutinable (NAG; not agglutinable with O-group I serum according to Gardner and Venkatraman [13]) vibrio strain, isolated from a patient with cholera-like clinical symptoms, have been compared with respect to their chemical composition and immunological behavior. In addition to a significant difference in the chemical composition between the two lipopolysaccharides, the LPS from V. cholerae, unlike that from the NAG vibrio, requires prior treatment with alkali for it to be an effective antigen in the indirect hemagglutination test with sheep cells. It has been suggested that the alkali acts by removing excess O-acetyl group from LPS of agglutinable vibrios. LPS from the NAG vibrio consistently showed a lower antibody response in rabbits in terms of agglutinin and vibriocidal titer. Also, the class of agglutinin antibody elicited by LPS of the NAG vibrio was predominantly immunoglobin M, and that from V. cholerae was immunoglobulin G under comparable conditions.     

Intranasal immunization with recombinant toxin-coregulated pilus and cholera toxin B subunit protects rabbits against Vibrio cholerae O1 challenge

Intranasal immunization, a noninvasive method of vaccination, has been found to be effective in inducing systemic and mucosal immune responses. The present study was aimed at investigating the efficacy of intranasal immunization in inducing mucosal immunity in experimental cholera by subunit recombinant protein vaccines from Vibrio cholerae O1. The structural genes encoding toxin-coregulated pilus A (TcpA) and B subunit of cholera toxin (CtxB) from V. cholerae O1 were cloned and expressed in Escherichia coli . Rabbits were immunized intranasally with purified TcpA and CtxB alone or a mixture of TcpA and CtxB. Immunization with TcpA and CtxB alone conferred, respectively, 41.1% and 70.5% protection against V. cholerae challenge, whereas immunization with a mixture of both antigens conferred complete (100%) protection, as assayed in the rabbit ileal loop model. Serum titers of immunoglobulin G (IgG) antibodies to TcpA and CtxB, and anti-TcpA- and anti-CtxB-specific sIgA in intestinal lavage of vaccinated animals were found to be significantly elevated compared with unimmunized controls. Vibriocidal antibodies were detected at remarkable levels in rabbits receiving TcpA antigen and their titers correlated with protection. Thus, mucosal codelivery of pertinent cholera toxoids provides enhanced protection against experimental cholera.     

Biofilm acts as a microenvironment for plankton-associated Vibrio cholerae in the aquatic environment of Bangladesh

The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait. A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.     

The suppressive effect of circulating specific antibody on the response to oral immunisation with Vibrio cholerae

Species IgG antibody given intravenously 3-4 hours prior to oral immunisation with Vibrio cholerae led to a specific depression of both the systemic and loca limmune response. One vibriocidal unit of IgG antibody, which itself would given undetectable levels of circulating specific antibody, was significantly immunosuppressive. The suppression is considered to be due to central repression of the antigen-reactive lymphocyte, rather than to antigen exclusion at the gut mucosal surface. The repression appeared less pronounced in some immunoglobulin classes than in others.     

Difference of Phenotype and Genotype Between Human and Environmental: Isolated Vibrio cholerae in Surabaya,Indonesia

Cholera due to Vibrio cholerae has been spreading worldwide, although the reports focusing on Indonesian V. cholerae are few. In this study, in order to investigate how V. cholerae transmitted to human from environment. We extended an epidemiological report that had investigated the genotype of V. cholerae isolated from human pediatric samples and environmental samples. We examined 44 strains of V. cholerae isolated from pediatric diarrhea patients and the environment such as shrimps or oysters collected in three adjacent towns in Surabaya, Indonesia. Susceptibilities were examined for 11 antibiotics. Serotype O1 or O139 genes and pathogenic genes including cholera toxin were detected. Multi-locus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC)-PCR were also performed to determine genetic diversity of those isolates. Serotype O1 was seen in 17 strains (38.6%) with all pathogenic genes among 44 isolates. Other isolates were non-O1/non-O139 V. cholerae. Regarding antibiotic susceptibilities, those isolates from environmental samples showed resistance to ampicillin (11.4%), streptomycin (9.1%) and nalidixic acid (2.3%) but those isolates from pediatric stools showed no resistance to those 3 kinds of antibiotics. MLST revealed sequence type (ST) 69 in 17 strains (38.6%), ST198 in 3 strains (6.8%) and non-types in 24 strains (54.5%). All the ST69 strains were classified to O1 type with more than 95% similarity by ERIC-PCR, including all 6 (13.6%) isolates from environmental samples with resistance to streptomycin. In conclusion, V. cholerae O1 ST69 strains has been clonally spreading in Surabaya, exhibiting pathogenic factors and antibiotic resistance to streptomycin, especially in the isolates from environment.     

Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

The antimicrobial action of the xanthine oxidase-xanthine system on the causative agent of cholera

As revealed in experiments on V. cholerae, the enzymatic link xanthine oxidase-xanthine produces a vibriostatic effect at the concentration of xanthine oxidase equal to 0.0125 g/l and a vibriocidal effect at the concentration of xanthine oxidase equal to 0.025 g/l in a medium with pH 7.5-7.6. In the presence of protein the antivibrionic activity of the xanthine oxidase link is decreased. The introduction of bivalent iron into the enzymatic link xanthine oxidase-xanthine enhances its vibriocidal action on V. cholerae.