Real‐time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis

A multiplex real‐time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage λ; the latter was used as an internal amplification control. The Y. pestis‐specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10–100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.     

The structure of Yersinia pestis Caf1 polymer in free and adjuvant bound states

Caf1 of the plague bacterium, Yersinia pestis is a polymeric virulence factor and vaccine component, formed from monomers by a donor strand exchange (DSE) mechanism. Here, EM images of Caf1 reveal flexible polymers up to 1.5 μm long (4 MDa). The bead-like structures along the polymer are 5.8 ± 1 nm long and correspond to single Caf1 proteins. Short polymers often form circles, presumably by DSE. We also provide the first images of proteins bound to alhydrogel adjuvant. Caf1, hemocyanin and anthrax PA are all resolved clearly and Caf1 exhibits adjuvant bound stretches with long intervening loops draped from the edges.     

首次发现0:9血清型小肠结肠炎耶尔森氏菌引起的腹泻爆发流行

本文首次报告由0:9血清型、生物3型小肠结肠炎耶尔森氏菌引起的一起食物源性急性腹泻爆发流行。发病率为64.44%、病人的主要临床表现为腹痛(95.17%)、腹泻(56.25%)、头昏。头痛(53.40%)及发热(29.29%)。从33名病人粪便标本中分离出13株0:9血清型小肠结肠炎耶尔森氏菌。这些菌株具有典型的生化反应,大多数具有病原性。病人恢复期血清抗体滴度多在1:160以上。     

Cross-reaction between Yersinia outer membrane proteins and anti-Borrelia antibodies in sera of patients with Lyme disease

Diagnosis of Yersinia infections accompanied by reactive arthritis could be complicated by cross-reaction with other arthritogenic bacteria. The possible cross-reaction between Yersinia antigens and anti- Borrelia antibodies in blood sera of patients with Lyme disease was studied. The occurrence of specific IgA, IgG and IgM antibodies was analyzed in serum samples from 30 patients with Yersinia -triggered reactive arthritis, 30 patients with Lyme disease and five samples from healthy blood donors. For anti- Borrelia IgG antibodies, cross-reaction was detected with YopH, YopB, V-ag, YopD, YopN, YopP and YopE, and for IgA with YopD. For IgM, no cross-reaction was detected. Owing to cross-reactivity with Borrelia , the diagnosis of Yersinia -triggered reactive arthritis should be based on a combination of serological and clinical findings.     

Urinary Tract and Renal Findings in Acute Yersinia Infections

ABSTRACT We studied 71 patients with acute Yersinia infection for the occurrence of pathologic urinary and renal findings. Transient proteinuria and/or microhematuria was found in 17 patients (24%) and slightly elevated serum creatinine in seven patients (10%). Renal biopsy was done in two patients and revealed mild mesangial glomerulonephritis in both cases. One of these patients had IgA glomerulonephritis and Reiter's syndrome. Pyuria occurred in 16 patients (23%) and was frequently associated with Reiter's syndrome. Seventy-three patients with acute intrinsic renal failure were studied for the occurrence of acute Yersinia infection by determining Yersinia antibodies by ELISA. One out of 13 patients with acute glomerulonephritis but none of 60 patients with acute tubulointerstitial renal disease had acute Yersinia infection. Acute Yersinia infection seems to be rarely an etiologic factor in acute intrinsic renal failure. Our results indicate that transient proteinuria, microhematuria, pyuria or impaired renal function are frequent findings in patients with acute Yersinia infections. However, glomerulonephritis seems to be a rather infrequent and mild complication of acute Yersinia infection.     

TNFα and IFNγ contribute to F1/LcrV-targeted immune defense in mouse models of fully virulent pneumonic plague

Immunization with the Yersinia pestis F1 and LcrV proteins improves survival in mouse and non-human primate models of pneumonic plague. F1- and LcrV-specific antibodies contribute to protection, however, the mechanisms of antibody-mediated defense are incompletely understood and serum antibody titers do not suffice as quantitative correlates of protection. Previously we demonstrated roles for tumor necrosis factor-alpha (TNFα) and gamma-interferon (IFNγ) during defense against conditionally attenuated pigmentation (pgm) locus-negative Y. pestis. Here, using intranasal challenge with fully virulent pgm-positive Y. pestis strain CO92, we demonstrate that neutralizing TNFα and IFNγ interferes with the capacity of therapeutically administered F1- or LcrV-specific antibody to reduce bacterial burden and increase survival. Moreover, using Y. pestis strain CO92 in an aerosol challenge model, we demonstrate that neutralizing TNFα and IFNγ interferes with protection conferred by immunization with recombinant F1-LcrV fusion protein vaccine (p < 0.0005). These findings establish that TNFα and IFNγ contribute to protection mediated by pneumonic plague countermeasures targeting F1 and LcrV, and suggest that an individual's capacity to produce these cytokines in response to Y. pestis challenge will be an important co-determinant of antibody-mediated defense against pneumonic plague.     

Yersinia colitis

A 2-year-old child with a febrile, nonbloody diarrheal illness of acute onset with repeatedly negative stool and blood cultures for pathogenic bacteria is presented. Sigmoidoscopic and roentgenographic studies revealed an inflammatory colitis. Fortunately, diagnostic perserverance and a high index of suspicion resulted in a positive stool culture forYersinia enterocolitica. Serologic study and clinical course provided data consistent with the diagnosis of an infections colitis due toYersinia enterocolitica. This case demonstrates the necessity to considerYersinia enterocolitica in the radiographic differential diagnosis of Crohn's disease of the colon or ulcerative colitis, as well as intractable diarrhea of childhood.     

小肠结肠炎耶尔森菌O∶3血清型特异性单克隆抗体制备

目的　制备出小肠结肠炎耶尔森菌血清O∶3型特异的单克隆抗体。方法　用常规的骨髓杂交瘤融合技术。结果　筛选到了一株针对于小肠结肠炎耶尔森菌O∶3血清型特异性的克隆株 (编号 :Y6H8) ,且证实分泌的抗体属于针对于脂多糖免疫球蛋白IgG ,可用于玻片凝集法鉴定 0∶3血清型小肠结肠炎耶尔森菌。 结论　通过本实验证实小肠结肠炎耶尔森菌血清O∶3型菌株脂多糖存在其特异性成分 ,可用于该菌株的分型鉴定     

直接检测模拟阳性标本致病性小肠结肠耶尔辛菌

为了快速准确地检测致病性小肠结肠那尔辛菌（ＹＥ），根据该菌染色体上ａｉｌ基因顺序设计一时引物，通过聚合酶链反应直接检测模拟阳性粪便中致病性ＹＥ，该反应能扩增出１７０ｂｐ的ａｉｌ基因片段。在５００ｍｇ粪便中接种约１０￣３致病性ＹＥ即可得到阳性结果，而其他被测细菌均为阴性。运用Ｓａｎｇｅｒ双脱氧法测定了扩增产物的核苷酸顺序并证实其真实性。这一检测方法不仅特异性强、灵敏度高，而且能在１天之内出结果，还能准确地判断致病性ＹＥ的存在。