Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

Efficient transformation of a Yersinia enterocolitica strain (serotype O:3) by pBR322 DNA

Abstract The Yersinia enterocolitica strain MS201 (serotype O:3) was transformed by pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from the transformed Y. enterocolitica is able to transform plasmid-free MS201 at a significantly higher frequency, suggesting the existence of a restriction-modification system in MS201.     

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.     

Molecular heterogeneity in Yersinia enterocolitica and 'Y. enterocolitica-like' species--Implications for epidemiology, typing and taxonomy

Yersinia enterocolitica is an extremely heterogeneous species. Serotyping and biotyping have been used extensively, in the past, to study its heterogeneity and epidemiology. Application of methods like ribotyping, pulsed-field gel electrophoresis and a host of other genomic techniques have further revealed molecular heterogeneity in this species. Furthermore, these methods may be used effectively to supplement serotyping and biotyping schema for studying epidemiology of Y. enterocolitica. This is evident from the ability of some of these methods to subtype strains belonging to serogroups O:3, O:9 and O:8 - which are most commonly encountered in human Yersiniosis. Multilocus enzyme electrophoresis and nucleotide sequencing have reiterated the taxonomic relationships of this organism. However there is paucity of information about the molecular heterogeneity of 'Y. enterocolitica-like' species, which need to be addressed in the future. Also, newer techniques such as amplified fragment length polymorphism, VNTR-based typing and multilocus sequence typing should be applied to further understand epidemiology, population structure and evolutionary genetics of Y. enterocolitica and 'Y. enterocolitica-like' species.     

正交实验法在PCR反应条件优化中的应用

本研究以小肠结肠炎耶尔森氏菌为研究材料,将纯理论数学方法一正交设计实验法。应用于分子生物学技术PCR体系的优化之中．首先采用搜索性正交实验,在此实验结果基础上。进一步设计细调性正交实验来寻求PCR反应的最适条件．所得优化扩增体系稳定,重复性好,表明正交实验设计方法高效快速,科学性强,是PCR反应条件优化中值得推广使用的有效方法。     

Identification of Murine T Cells Reactive with the Bacterial Superantigen Yersinia pseudotuberculosis-Derived Mitogen (YPM) and Factors Involved in YPM-Induced Toxicity in Mice

We previously reported that Yersinia pseudotuberculosis-derived mitogen (YPM) acts as a superantigen to human T cells. In this study, we assessed the superantigenicity and toxicity of YPM using murine experimental models. YPM activated T cells to produce interleukin-2 in a major histocompatibility complex class II molecule-dependent manner. The T-cell blasts induced by YPM expressed T-cell receptor (TCR) β-chain variable region (Vβ)7, VβS.1, Vβ8.2 and Vβ8.3. The injection of YPM into mice pre-sensitized with D-galactosamine induced lethal shock. This shock was blocked by the injection of monoclonal antibodies (mAbs) to CD4, TCR Vβ7 plus Vβ8, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), but not by injection to CD8 or unrelated Vβ. These results indicate that YPM-induced shock requires the presence of CD4⁺ T cells bearing TCR Vβ7 and Vβ8, and that endogenous TNF-a and IFN-γ mediate the lethal effects.     

Multilocus variable number tandem repeat analysis as a tool to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A

Aims:  To identify variable number tandem repeat (VNTR)-containing loci, and to use multilocus VNTR (MLVA) to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A isolated from diverse sources.
Methods and Results:  The whole genome sequence of Y. enterocolitica 8081 was analysed and eight VNTR loci with repeat sizes between 4 and 9 bp, and each containing more than four repeat copies were selected for MLVA typing of 88 strains of Y. enterocolitica . Of these, four loci were polymorphic and generated 26 MLVA genotypes among 81 strains of Y. enterocolitica biovar 1A. MLVA was found to be quite discriminatory (DI = 0·87). Cluster analysis and population modelling using minimum spanning tree (MST) clearly clustered Y. enterocolitica biovar 1A into two major groups.
Conclusions:  The MLVA is easy to perform and can be used to discern clonal relationship among strains of Y. enterocolitica . Also the phylogenetic relationships obtained with MLVA genotypes were in good agreement with those established by other typing methods.
Significance and Impact of the Study:  The MLVA method reported is relatively more discriminatory than the other genotyping methods and has the potential to be used as an epidemiological tool for the study of strains of Y. enterocolitica biovar 1A.     

Whole cell protein profiling reiterate phylogenetic relationships among strains of Yersinia enterocolitica biovar 1A as discerned earlier by different genotyping methods

Aim: To evaluate whole cell protein profiling vis‐à‐vis genotyping to discern phylogenetic relationships among strains of Y. enterocolitica biovar 1A. Methods and Results: Whole cell protein profiling of Y. enterocolitica biovar 1A strains was performed using SDS–PAGE. Twenty‐one distinct protein profile types were obtained among a collection of 81 strains isolated from clinical and nonclinical sources. Whole cell protein profiling exhibited discriminatory index (DI) of 0·80 and clustered the strains into two distinct clonal groups. The clinical and the aquatic serotype O:6,30–6,31 strains were clustered into two discrete subgroups. Conclusions: Whole cell protein profiling displayed sufficient diversity among strains of Y. enterocolitica biovar 1A, and the phylogenetic relationships obtained were in good agreement with those established earlier by genotyping techniques. Significance and impact of the study: Whole cell protein profiling was as discriminatory as some of the genotyping methods and has the potentiality to be used as an adjunct tool to study epidemiology of Y. enterocolitica.