O139霍乱弧菌的研究进展

Ｏ１３９霍乱弧菌是一种新发现的可引起霍乱流行的病原体，该病的流行已引起各国的关注。Ｏ１３９霍乱弧菌的研究对菌苗研制和控制疾病有重要意义。本文综述了最近国外Ｏ１３９霍乱弧菌的生物学特性、免疫反应及其毒素共调菌毛（ｔｃｐ）的研究进展。     

云南省O139霍乱弧菌引起的一次食源性霍乱爆发

霍乱是由O1霍乱弧菌(Vibrio cholerae O1)或O139霍乱弧菌(Vibrio cholerae O139)引起的一种肠道传染病,是发展中国家的一个主要公共卫生问题,2000年以来世界卫生组织每年报告霍乱病例数范围是101383～184311例,其中非洲报告病例数占86.8%～96.9%,亚洲占3.1%～8.2%,拉丁美洲占0.0%～2.3%,非洲、亚洲和拉丁美洲报告病死率范围分别是1.7%～3.3%、0.3%～0.6%和0.0%[1].     

O139霍乱弧菌检测方法的研究─诊断血清的制备

本文以Ｏ１３９死菌免疫健康家兔,经吸收去除非特异性凝集素制成的特异性诊断血清,专供诊断霍乱弧菌Ｏ１３９之用。采用玻片凝集试验对５株Ｏ１３９菌株及１２６株肠道菌进行验证,在敏感性和特异性方面均获得满意的结果。     

O139霍乱弧菌质粒基因组文库的建立及O抗原基因的筛选

合成O抗原的基因是串联在一起的一个基因簇,提取O139霍乱弧菌基因组DNA,限制性内切酶EcoRⅠ酶切,电泳回收4～20kb的DNA片段,构建质粒基因组文库。随机筛选重组克隆,获得一株可与O.139霍乱弧菌抗血清发生凝集反应的重组克隆,命名为大肠杆菌DH5α(pMG320)。经鉴定分析重组克隆所表达的O抗原具有良好的免疫原性及反应原性。酶切分析质粒pMG320,推知其O抗原基因大小约4.6kb。这为今后O.139霍乱疫苗的研制及O.139霍乱弧菌O抗原基因的结构和功能研究提供了条件。     

霍乱弧菌O139多糖抗原的提取方法及优化

目的:研究霍乱弧菌O139多糖抗原的提取方法,以获得高纯度的多糖抗原。方法:采用热酚水法提取霍乱弧菌O139的脂多糖(LPS),并增加了DNaseⅠ、RNase消化步骤,以去除DNA及RNA的污染;进一步采用酸水解法获得脱毒的O特异性多糖(O-SP)。结果:经生化方法检测,证实提纯的LPS和O-SP纯度较高,能满足进行霍乱免疫研究的要求。结论:该方法简便可行,值得推广。     

Production and cross-reactivity patterns of a panel of high affinity monoclonal antibodies to Vibrio cholerae O139 Bengal

Abstract A series of monoclonal antibodies of different isotypes specific for Vibrio cholerae O139, the new pandemic strain of cholera, was produced. These mAbs reacted only with the reference strain (MO45) representing serovar O139 but did not react with any of the other reference strains representing serovars O1 to O140. Significantly, the mAbs did not agglutinate the R-cultures of V. cholerae (CA385, 20–93) which demonstrated the exceptional specificity of these mAbs and indicated that the mAbs recognized antigenic determinants unique for the O139 serovar. There was heterogeneity in the intensity of reactivity of the mAbs with strains of V. cholerae O139 isolated from diverse sources. Apart from 4H6, the other mAbs agglutinated all the O139 strains examined. 2D12 and 2F8 were the best mAbs based on the intensity of agglutination with all the O139 strains. Evaluation of 3A10 in comparison with a polyclonal anti-O139 antibody raised in rabbit using the slide agglutination format revealed that 3A10 fared as well as the polyclonal antibody for the laboratory identification of the O139 serovar. The acquisition of these mAbs provide reagents which would be very useful in the development of simple immunodiagnostic assays for the diagnosis of V. cholerae O139 infections.     

Vibrio cholerae O22 might be a putative source of exogenous DNA resulting in the emergence of the new strain of Vibrio cholerae O139

The new epidemic strain O139 of Vibrio cholerae, the etiologic agent of cholera, has probably emerged from the pandemic strain O1 El Tor through a genetic rearrangement involving the horizontal transfer of exogenous O-antigen- and capsule-encoding genes of unknown origin. In V. cholerae O139, these genes are associated with an insertion sequence designated IS1358_O139. In this work, we studied the distribution of seven genes flanking the IS1358_O139 element in 13 serovars of V. cholerae strains. All these O139 genes and an IS1358 element designated IS1358_O22-1 were only found in V. cholerae O22 with a similar genetic organization. Sequence analysis of a 4.5-kb fragment containing IS1358_022-1 and the adjacent genes revealed that these genes are highly homologous to those of V. cholerae O139. These results suggest that strains of V. cholerae O22 from the environment might have been the source of the exogenous DNA resulting in the emergence of the new epidemic strain O139.     

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.     

Characterization of an Enterotoxin Produced by Vibrio cholerae O139

A cholera-like enterotoxin was purified from Vibrio cholerae O139 strain AI-1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al-1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI-1841 were identical to those of the CTs. Three toxins equally reacted against anti-569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti-B subunit antiserum by double gel immunodiffusion. Anti-569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.     

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.     

Purification and characterization of Vibrio cholerae O139 fimbriae

Abstract A Vibrio cholerae O139 (strain Al-1841) isolated from a patient with a cholera-like disease in Bangladesh predominantly produced new curved, wavy fimbriae (Al-1841 fimbriae) and small numbers of previously reported V. cholerae non-O1 S7-like pili. The former was purified and characterized. The molecular mass of the Al-1841 fimbrial subunit was less than 2.5 kDa, and it was immunologically different from that of V. cholerae non-O1 S7 pili. This novel fimbrial antigen was detected in all 182 Gram-negative strains from five genera tested but was absent from the Gram-positive bacteria tested. The purified Al-1841 fimbriae did not agglutinate human or rabbit erythrocytes.     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

O139霍乱弧菌LPS基因在大肠杆菌中的克隆和表达

利用粘粒载体pCOS5构建了国内分离的O139霍乱弧菌的基因组文库,并从文库中筛选获得可以表达O139霍乱弧菌脂多糖的重组克隆株E.coliJM109(pMG310)。重组粘粒pMG310经酶切分析,所克隆的外源DNA片段大小为37kb。实验证明：重组克隆株E.coliJM109(pMG310)所表达的脂多糖具有良好的免疫原性及反应原性。