vi—PHA试剂的研制及其在检测伤寒带者中的应用

Purified S. typhi Vi antigen is sensitized with equal volume of tannic acid treated formalational sheep erythrocytes (SRBC) at a final concentration of 1 microgram/ml. The Vi-passive hemagglutination assay (Vi-PHA) diagnostic reagent is developed to detect Vi antibodies to S. typhi for the detection of chronic carriers after typhoid fever and the screening S. typhi healthy carriers from food-handlers, which is characterized with high sensitivity, strong specificity and good stability. This Vi-PHA reagent is able to detect 1.16 micrograms/ml of Vi antibodies and doesn't make any cross reaction with healthy sera. For the sera of other diseases, the cross rate is only 0.84%. Using this reagent, 19 positive sera (6.93%) are detected from 274 convalescent sera from typhoid fever, 14 convalescents of which are stool-culture S. typhi positive, that persists a positive rate of 73.68%; 3 positive sera are detected from 106 foodhandlers, one of which is stool-culture S. typhi positive. Therefore, the reagent is simple, convenient, rapid and easy to be applicated in basic unit.     

Salmonella Typhi sense host neuroendocrine stress hormones and release the toxin haemolysin E

Salmonella enterica serovar Typhi (S. typhi) causes typhoid fever. We show that exposure of S. typhi to neuroendocrine stress hormones results in haemolysis, which is associated with the release of haemolysin E in membrane vesicles. This effect is attributed to increased expression of the small RNA micA and RNA chaperone Hfq, with concomitant downregulation of outer membrane protein A. Deletion of micA or the two-component signal-transduction system, CpxAR, abolishes the phenotype. The hormone response is inhibited by the β-blocker propranolol. We provide mechanistic insights into the basis of neuroendocrine hormone-mediated haemolysis by S. typhi, increasing our understanding of inter-kingdom signalling.     

Surveillance for typhoid fever in Matsuyama city during 1974-1981 and detection of Salmonella typhi in sewage and river waters

Eighty-two cases of typhoid fever were found in Matsuyama city in the period from 1974 to 1981. Seventy-six cases were found to be infected with Salmonella typhi other three with Salmonella paratyphi A, and the remaining three were diagnosed only clinically. The strains of S. typhi isolated from these patients showed such a variety of Vi-phage types as D1, D2, E1, M1, 53 and degraded Vi-positive strain (DVS). The concurrent survey of the city sewage and river waters for typhoid bacilli was conducted with total 578 samples taken therefrom. S. typhi was isolated from 120 of those samples. The Vi-phage types of the isolates were closely related with those of the isolates from the patients. The periodical examinations of the city sewage and the draining river may serve as a useful means for the controlling typhoid fever epidemics.     

Molecular cloning of a Salmonella typhi LT-like enterotoxin gene

Diarrhoea is a common event during typhoid fever; nevertheless, the possible participation of a diarrhoea-inducing enterotoxin has not been described (Roy et al., 1985). Recombinant bacteriophage lambda FDC1 was isolated from a genomic library of Salmonella typhi, the causal agent of typhoid fever, by screening with a probe for the B subunit gene of the heat-labile, cholera-like, Escherichia coli enterotoxin (LT). Lambda FDC1 codes for an enterotoxin that causes secretion in rat ileal loops, that elongates Chinese hamster ovary (CHO) cells, that is recognized by antibodies against LT, and does not bind in vitro to ganglioside GM1. These results should allow further studies towards elucidating a possible role for the S. typhi enterotoxin in the pathogenesis of typhoid fever.     

The detection of antigenemia in typhoid patients at different periods in the course of the infection

The enzyme immunoassay of serum samples, obtained from 37 typhoid patients and previously subjected to bacteriological study with negative results, permitted the detection of S. typhi specific antigens and, as a consequence, the rapid diagnosis of the disease. The prevalence of various antigenic components of S. typhi was observed at different stages of infection. The presence of surface O and Vi antigens was characteristic of acute stages of S. typhi infection. At the same time L-form antigens, in rare cases in combination with surface O and Vi antigens, were characteristic of patients at the period following the acute stage of the disease, as well as chronic carriers. This is indicative of the necessity of introducing specific antibodies to the antigenic determinants of S. typhi L forms into the assortment of diagnostic immune preparations.     

Leukocyte migration inhibition by a specific antigen in human typhoid fever.

Cellular reactivity to heat-killed Salmonella typhi antigen was investigated by the leukocyte migration inhibition (LMI) method in 33 S. typhi infected patients and in 32 control persons. In the typhoid group a statistically significant LMI was observed as compared to the members of the control group. A correlation was found between the level of the cellular sensitivity and the time elapsed between onset of the disease and performance of the test. Previous typhoid vaccination had no influence on the LMI. No correlation was found between the agglutinin titres and the sensitivity demonstrated by the LMI test. The value of the method in studies of cellular immunity in typhoid fever is discussed.     

Cutting edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection

Infection of mice with Salmonella typhimurium gives rise to a disease similar to human typhoid fever caused by S. typhi. Since S. typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against S. typhimurium is a longstanding matter of debate. By infecting mice on a susceptible background and deficient in B cells (Igmu-/- mice) with different strains of S. typhimurium, we could for the first time formally clarify the role of B cells in the response against S. typhimurium. Compared with Igmu+/+ mice, LD50 values in Igmu-/- mice were reduced during primary, and particularly secondary, oral infection with virulent S. typhimurium. After systemic infection, Igmu-/- mice cleared attenuated aroA- S. typhimurium, but vaccine-induced protection against systemic infection with virulent S. typhimurium involved both B cell-dependent and -independent effector mechanisms. Thus, B cell-mediated immunity plays a distinct role in control of S. typhimurium in susceptible mice.     

Demonstration of an antigenic protein specific for Salmonella typhi

Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 ? resolution by X-ray crystallography shows a 16-stranded β-barrel with three β-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics.     

Characterisation and distribution of a cryptic Salmonella typhi plasmid pHCM2

pHCM2 is a 106 kbp cryptic plasmid harboured by Salmonella typhi CT18, originally isolated from a typhoid patient in Vietnam. The genome of S. typhi CT18, including pHCM2, has recently been completely sequenced and annotated. Bioinformatic analysis revealed that 57% of the coding sequences (CDSs) encoded on pHCM2 display over 97% DNA sequence identity to the virulence-associated plasmid of Yersinia pestis, pFra. pHCM2 encodes no obvious virulence-associated determinants or antibiotic resistance genes but does encode a wide array of putative genes directly related to DNA metabolism and replication. PCR analysis of a series of S. typhi isolates from Vietnam detected pHCM2-related DNA sequences in some S. typhi isolated before, but not after, 1994. Similar pHCM2-related sequences were also detected in S. typhi isolated from other regions of South East Asia and Pakistan but not elsewhere in the world.     

伤寒沙门菌动物模型研究进展

目前伤寒沙门菌引起的人类伤寒仍是一种严重危害人类健康的疾病,且近年来多重耐药伤寒沙门菌株频频出现,使伤寒的治疗更加棘手.由于该菌具有严格的宿主特异性,又缺乏理想的动物模型,其致病机制的研究、疫苗及药物的研发受制约.新近研究发现,免疫系统人源化小鼠模型和诱导型一氧化氮合酶基因敲除小鼠模型可用于伤寒沙门菌的体内实验.本文就其应用现状及缺憾作一综述.     

Immunofluorescent Identification of Salmonella typhi During a Typhoid Outbreak

Immunofluorescence and conventional bacteriological methods were compared for their ability to detect Salmonella typhi in 134 fecal specimens from 105 individuals associated with an outbreak of typhoid fever. Smears prepared from untreated fecal material (direct method) and after a preliminary incubation in selenite F broth (delayed method) were tested with an anti-Vi serum conjugated with fluorescein isothiocyanate. The delayed method was more sensitive than the direct method in detecting S. typhi. The delayed method was positive in 40 of 41 patients positive by culture methods, but gave positive or questionable reactions in 11 presumably uninfected individuals. The fluorescent-antibody test employing a Vi conjugate is a satisfactory screening procedure for detecting S. typhi, but all positives must be confirmed bacteriologically.     

The effect of immunostimulants on the resistance of white mice to the causative agent of typhoid fever

The influence of prodigiosan, salmosan, polyribonate and levamisole on the body nonspecific and specific resistance to S. typhi strain 4446 has been studied. Prodigiosan and salmosan have proved to be the most effective. The injection of these compounds simultaneously with typhoid vaccine (both chemical adsorbed vaccine and alcohol-treated vaccine, enriched with Vi-antigen) significantly increases the survival rate of immunized animals (by 35-45%), elevates the resistance index (1.5- to 2.3-fold) and the effectiveness index of the vaccine (17- to 32-fold) in comparison with the controls. Besides, prodigiosan and salmosan alone are capable of increasing nonspecific resistance to S. typhi strain 4446, which is manifested by an increase of the survival rate of stimulated animals by 61.87%. Proceeding from the results thus obtained, the possibility of good prospects for prodigiosan and salmosan in the prophylaxis of typhoid fever in humans may be inferred.     

Prevalence of multi-drug resistant Salmonella typhi among clinically diagnosed typhoid fever patients in Lagos, Nigeria

A total of 635 clinically diagnosed typhoid fever patients were bled from three different health institutions in the metropolis of Lagos, Nigeria over a period of 15 months, May 1997 to July 1998. Out of the total blood cultured, 101 (15.9%) isolates of Salmonella species were isolated of which 68 (67.3%) were S. typhi, 17 (16.8%) and 16 (15.8%) were S. paratyphi A. and S. arizonae respectively. The overall isolation rate of S. typhi among patients is 10.7%, with most isolates 45.9% found among the severely-ill young adults, age group 16-30 years. All isolates were subjected to anti-microbial susceptibility testing using 12 different antibiotics: chloramphenicol, ampicillin, cotrimoxazole, gentamicin, colistin sulfate, nalidixic acid, nitrofurantoin, cefotaxime, tetracycline, streptomycin, ofloxacin and ciprofloxacin. All the S. typhi and S. paratyphi A isolates showed resistance to two or more of the 10 of 12 antibiotics tested particularly the 3-first-line antibiotics commonly used (chloramphenicol, ampicillin and cotrimoxazole) in the treatment of typhoid fever in Nigeria. No isolate showed resistance to ofloxacin and ciprofloxacin, however, nalidixic acid and gentamicin showed a moderate and appreciable inhibition to most of our isolates.     

The function of Salmonella typhi at different stages in the pathogenesis of typhoid infection

The complex study of the adhesive, colicinogenic and antigenic properties of S. typhi of different origin has revealed that adhesive properties can be observed more frequently in the strains isolated from the blood and bile and are completely absent in the strains isolated from feces. S. typhi strains of various origin do not essentially differ in their sensitivity to colicins and in the capacity for their production. Among the strains isolated from feces and bile, agents in the W-form occur more frequently than among the strains isolated from the blood. Escherichia coli, isolated from typhoid patients and carriers at the moment of the persistence of S. typhi in the body, are characterized by faintly pronounced antagonistic properties, enhanced sensitivity to colicins and rather pronounced hemagglutinating activity.     

Role of Genomic Rearrangements in Producing New Ribotypes of Salmonella typhi

Salmonella typhi is the only species of Salmonella which grows exclusively in humans, in whom it causes enteric typhoid fever. Strains of S. typhi show very little variation in electrophoretic types, restriction fragment length polymorphisms, cell envelope proteins, and intervening sequences, but the same strains are very heterogeneous for ribotypes which are detected with the restriction endonuclease PstI. In addition, the genome of S. typhi has been proven to undergo genomic rearrangement due to homologous recombination between the seven copies of rrn genes. The relationship between ribotype heterogeneity and genomic rearrangement was investigated. Strains of S. typhi which belong to 23 different genome types were analyzed by ribotyping. A limited number of ribotypes were found within the same genome type group; e. g., most strains of genome type 3 belonged to only two different ribotypes, which result from recombination between rrnH and rrnG operons. Different genome type groups normally have different ribotypes. The size and identity of the PstI fragment containing each of the seven different rrn operons from S. typhi Ty2 were determined, and from these data, one can infer how genomic rearrangement forms new ribotypes. It is postulated that genomic rearrangement, rather than mutation, is largely responsible for producing the ribotype heterogeneity in S. typhi.     

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.     

The Vi antigen of Salmonella typhi: molecular analysis of the viaB locus.

Strains of Salmonella typhi isolated from the blood of patients with typhoid fever invariably express a capsular polysaccharide, termed the Vi antigen. Vi antigen expression is controlled by two separate chromosomal loci, viaA and viaB. The viaA locus is commonly found in enteric bacteria. In contrast, the viaB locus appears to be specific to Vi-expressing strains of Salmonella and Citrobacter. Here the cloning, expression and analysis of viaB determinants from S. typhi Ty2 is described. Whole-cell DNA from strain Ty2 was size-fractionated and cloned into the pLA2917 cosmid vector. A recombinant cosmid, pVT1, conferring a Vi-positive phenotype upon Escherichia coli and upon the Vi-non-expressing strain Ty21a of S. typhi, was characterized and used for further studies. Transposon Tn5 insertion mutagenesis demonstrated that the Vi-antigen-encoding region on pVT1 consisted of a 15 kb fragment. A subclone, designated pVT3, which contained an 18 kb insert, was sufficient to confer Vi antigen expression upon E. coli and S. typhi Ty21a. Results of recombination experiments indicated that this DNA sequence was the viaB locus of S. typhi Ty2. In E. coli SE5000 maxicells, the viaB determinants encoded at least eight polypeptides, with molecular masses of 80, 65, 59, 48, 44, 39, 35 and 28 kDa. Functional characterization of viaB mutations in S. typhi Ty2 suggested that the 80 and 65 kDa proteins were required for cell-surface localization of the Vi antigen.