Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

Development of an improved synthetic medium for a better production of the new cholera toxin and its immunological relationship with the toxin produced by Vibrio cholerae O139 strains

Abstract An improved synthetic medium (M4) comprising syncase medium supplemented with sodium chloride (1%) and sucrose (0.5%) pH adjusted to 7.4 was developed for a better production of the new cholera toxin (NCT). The culture filtrates prepared in the M4 medium caused significantly ( P < 0.05) more fluid accumulation than that in syncase medium. Crude toxin, prepared in the M4 medium with V. cholerae O1 strains (X-392 and 2740-80) caused a reaction similar to that of the same amount of NCT (32 μg) prepared in the syncase medium. The neutralization of the optimal loop reacting dose of the NCT prepared in the M4 medium by anti-NCT raised against syncase prepared toxin indicates the release of the same kind of toxin in both media. These observations indicate that the modified M4 medium may be used for NCT preparation and further characterization. All the strains of Vibro cholerae O139 used in this study produced a toxin antigenically similar to NCT.     

烟草生产霍乱毒素B亚单位疫苗的免疫分析

用纯化的ＣＴＢ后腿肌肉注射免疫小鼠,每次１２．５μｇ,每隔１４天加强一次,共注射三次,末次免疫后两周收集血清,免疫小鼠诱导产生了特异性抗ＣＴＢ抗体。在ＣＨＯ细胞中,免疫小鼠血清能够中和ＣＴ的细胞病变效应;小鼠肠结扎实验表明,抗ＣＴＢ血清能够抑制ＣＴ结合细胞表面受体ＧＭ１神经节苷脂。这表明转基因烟草产ＣＴＢ在小鼠中能够产生抗细菌毒素的保护免疫力。     

大肠杆菌热稳定肠毒素与霍乱毒素B亚单位融合蛋白免疫原性的评价

构建了霍乱毒素B亚单位(CTB)和大肠杆菌热稳定肠毒素(ST）的重组表达载体pMCST1、pMCST2。二的不同是,前为单拷贝ST,后为双拷贝ST。在大肠杆菌DH50α中融合蛋白高效表达。用这两种重组蛋白分别免疫小鼠,都诱导产生了高滴度的抗CTB和抗ST的血清。表明这两组融合蛋白具有良好的CTB和ST的免疫原性,为进一步构建抗CTB和抗ST的产毒性细菌腹泻疫苗打下了基础。     

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1

BT23, a Vibrio cholerae O1 El Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.     

Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139

Abstract The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V. cholerae O1 of the E1 Tor biotype. Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V. cholerae tested carried cholera toxin genes and other gene associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the E1 Tor biotype of V. cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.     

霍乱毒素无毒B亚单位（CTB）黏膜免疫佐剂的研究进展

霍乱毒素（CT）具有很强的黏膜免疫佐剂活性,是当今研究热点之一,但CT的黏膜免疫佐剂效应机理尚未完全弄清。本文主要就霍乱毒素无毒B亚单位（CTB）的黏膜免疫佐剂作用进行综述。     

Distribution of the zot (zonula occludens toxin) gene among strains of Vibrio cholerae 01 and non-01

Abstract The distribution of the zot gene that encodes the zonula occludens toxin, a newly described toxin of Vibrio cholerae , among clinical, environmental and food isolates of V. cholerae 01 and non-01 was investigated. Both the zot gene and the ctx gene that encode cholera toxin were found in 247 of 257 clinical strains and 62 of 415 environmental or food isolates of V. cholerae 01. The zot gene, but not the ctx gene was found in 37 strains (one clinical strain and 36 environmental or food isolates). In addition, two of 31 clinical strains and six of 98 environmental or food isolates of V. cholerae non-01 possessed both the zot gene and the ctx gene. These results demonstrated the predominantly concurrent occurrence of the zot gene and ctx genes among strains of V. cholerae 01 which suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea produced by V. cholerae 01.     

转基因烟草生产霍乱毒素B亚单位的纯化

提取转基因烟草叶片总蛋白,用经溴化氰活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联有抗ＣＴ ＩｇＧ色谱柱,得到了表达产物ＣＴＢ蛋白质。经ＰＡＧＥ、Ｗｅｓｔｅｒｎ ｂｌｏｔ、琼脂糖免疫扩散和免疫电泳等方法鉴定表明该蛋白与天然ＣＴＢ相同。     

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production

Abstract The implication in cholera toxin (CT) production of the newly identified gene, lypA , that encodes the lysophospholipase L₂ of Vibrio cholerae , was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L₂ activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of El Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae .     

转基因烟草表达霍乱毒素B亚单位的研究

将霍乱毒素Ｂ亚单位（ＣＴＢ）基因克隆到质粒ｐＢｉｎ４３８中,分别构建植物表达载体ｐＢＩ－ＣＴＢ、ｐＢＩ－ＳＰＣＴＢ和ｐＢＩ－ＣＴＢＥＲ。采用叶盘法分别转化烟草Ｋ３２６,各表达载体得到了一批较基因植株。转基因烟草的ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分析表明ＣＴＢ基因整合到了烟草基因组中。转基因植株的ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ ｂｌｏｔ分析表明ｐＢＩ－ＳＰＣＴＢ和ｐＢＩ－ＣＴＢＥＲ的转基因植株能有效表     

Length of the linker and the interval between immunizations influences the efficacy of Vibrio cholerae O1, Ogawa hexasaccharide neoglycoconjugates

Ogawa hexasaccharide neoglycoconjugates induce protective antibodies in mice. Similar Ogawa conjugates but with a longer linker that connects the carrier to shorter saccharides are immunogenic, but generally ineffective at inducing vibriocidal or protective antibodies. The efficacy of Ogawa hexasaccharide neoglycoconjugates of different linker lengths were tested. The majority of mice given immunizations separated by a 14-day gap did not produce vibriocidal or protective antibodies. Mice immunized 28 days apart with immunogens containing the shortest or medium length linker, but not the longest, produced vibriocidal and protective antibodies. A nonprotective, priming dose of purified Ogawa LPS followed 5 days later with a booster of the Ogawa neoglycoconjugates (di-, tetra-, or hexasaccharide) resulted in vibriocidal antibodies at day 10.     

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139

Abstract The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae , in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene ( ctx ) positive and ctx⁻ strains of V. mimicus detected the presence of zot gene. An Acc -I- Eco RV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 to 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx⁻ toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

Incidence and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

Toxigenic Vibrio cholerae from environmental sources associated with the cholera outbreak after ‘AILA’ cyclone in West Bengal,India

Aims: West Bengal experienced a devastating storm named ‘AILA’ in its coastal and southern districts. We attempted to understand the transmission dynamics emphasizing on potable water to detect the presence of toxigenic strains of Vibrio cholerae, followed by the natural devastation. Methods and Results: A total of 33 water samples (from tap, tube well and ponds) were analysed. From them, 11 (33·3%) samples were found to be contaminated with V. cholerae, among which 5 (45%) isolates were V. cholerae O1 biotype Ogawa. Antibiogram profile reveals that most of the V. cholerae O1 isolates were highly sensitive against fluroquinolone group of antibiotics, but less sensitive against cefuroxime (50%), cefotaxime (40·9%), ceftriaxone (38·63%), trimethoprim (37·3%), streptomycin (29·2%) and furazolidon (4·54%). Three (36%) V. cholerae isolates were found to be ctxB positive (2 ctxB classical). Conclusions: Potable water plays a crucial role in cholera transmission. Natural disasters like the reported one aided with feacal–oral contamination enhance the possibilities of drinking water contamination. Significance and Impact of the Study: The application of the modified technique, making use of the enrichment subsequently followed by culture and PCR, will help us to detect the presence of toxigenic V. cholerae contamination in different aquatic environment. Moreover, natural extremes have a direct role in increase of salinity level, followed by higher predominance of V. cholerae along with their toxicity development in terms of genetic modification.     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。     

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.