人tPA信号肽和Yersinia pestis保护性抗原F1-V融合基因的构建及其免疫效果观察

为获得含有鼠疫F1和V抗原编码基因,以及人tPA信号肽基因的重组质粒tPA-pVAX1/F1-V,测定其诱导特异性免疫应答的能力,用PCR扩增鼠疫菌F1和V编码基因,分别与pGEM-T连接测序.构建pVAX1/F1-V融合重组质粒,PCR扩增tPA信号肽片段,并将其插入到F1-V的上游,构建tPA-pVAX1/F1-V融合重组质粒;转染COS-7细胞,Western印迹法鉴定目的蛋白的表达,重组质粒tPA-pVAX1/F1-V加GM-CSF佐剂免疫BALB/c小鼠,观察免疫效果.400个LD50强毒鼠疫菌皮下攻毒观察保护率.结果表明,tPA-pVAX1/F1-V在COS-7细胞中表达;免疫鼠体内产生特异性抗体;抗体亚型分析、细胞因子等指标的测定表明,所构建DNA疫苗以诱发Th1型免疫为主;(攻毒保护率达90%.结果提示,已成功构建F1-V融合蛋白真核表达载体tPA-pVAX1/F1-V,它具有诱导特异性细胞免疫和体液免疫应答的能力,对强毒鼠疫菌皮下攻毒有一定的保护效力,为鼠疫菌新型疫苗研制奠定了基础.

Mesenteric adenitis caused by Yersinia pseudotubercolosis in a patient subsequently diagnosed with Crohn's disease of the terminal ileum

Although the association between inflammatory bowel disease and gastrointestinal infections has been suggested, the mechanisms involved in the pathogenesis of Crohn＇s disease （CD） are still undetermined. We report the case of a man, who presented with mesenteric adenitis initially due to a Yersinia pseudotubercolosis infection, who was later diagnosed with Crohn＇s disease. This case is in keeping with recent evidence in the literature which suggests that CD is a disease linked to abnormal immune responses to enteric bacteria in genetically susceptible individuals.

Yersinia type Ⅲ effectors perturb host innate immune responses

The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type Ⅲ secretion system(T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp.(Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gramnegative bacteria that share in common a 70 kb virulence plasmid which encodes the T3 SS. Translocation of the Yersinia effector proteins(YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector proteins and their contribution to Yersinia pathogenesis.

Different Strategies for Preparation of Non-tagged rV270 Protein and Its Efficacy against Yersinia Pestis Challenge

Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was prepared by different methods in this study.Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a,or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a.After Co2＋ affinity chromatography,a purification strategy was developed by cleavage of His tag on column,following Sephacryl S-200HR column filtration chromatography.Results Removal of His tag by thrombin,enterokinase and factor Xa displayed a yield of 99.5%,32.4% and 15.3%,respectively.Following Sephacryl S-200HR column filtration chromatography,above 97% purity of rV270 protein was obtained.Purified rV270 that was adsorbed to 25% （v/v） Al（OH）3 adjuvant in phosphate-buffered saline （PBS） induced very high titers of antibody to rV270 in BALB/c mice and protected them （100% survival） against subcutaneous challenge with 106 CFU of Y.pestis virulent strain 141.Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa,but they exhibited extremely low cleavage activity to the corresponding recognition site.Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy.The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Comparative Genomic Analysis of Gene Variations of Two Chinese Yersinia pestis Isolates from Vaccine Strain EV76

Objective To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China. Methods A microarray containing 12 000 probes covering the entire genome of seven Yersinie pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results. Results The gene variations detected included the absence of five genes related to the synthesis of betaine in both EV76 and another sequenced attenuated strain, KIM D27. Several genes related to phage-related membrane proteins were found to be absent in the Antiqua biovar Yunnan strain, 485, which was isolated from a rodent plague foci. Conclusion These findings provide initial insight into the distinct strains isolated from natural foci, within their genomic context, including Yunnan Y. pestis strains. This information will be used therefore to establish subsequent comparisons of these sequences with published complete genomes of other strains.

Identification and Antimicrobial Susceptibility of <i>Yersinia enterocolitica</i>Found in Chitterlings, Raw Milk and Swine Fecal Samples

Foodborne illness is an escalating concern upon public health. The prevalence of Yersinia enterocolitica was assessed in chitterlings, raw milk and swine fecal from North Carolina. Uncleaned thirty chitterling samples procured from a local grocery store, forty-five swine fecal samples, and forty unpasteurized cow milk samples supplied by the University farm were evaluated for the presence of Y. enterocolitica. Isolates identified as presumptive positive were characterized as colonies with a pink or deep-red center on MacConkey and CIN agar, and verified further through polymerase chain reaction (PCR) for the presence of 16S rRNA gene for the Yersinia genera. Results showed that 4.4% swine fecal samples, 7.5% milk samples and 11.3% chitterling samples were presumptive positive for Y. enterocolitica by the direct plating method on selective agars. Of the thirty-chitterling samples examined by PCR for the 16S rRNA gene, 26% samples contained the identification gene for the bacteria of interest. After conducting virulence tests, the fecal samples were revealed as non-pathogenic. Only one of the milk samples were considered pathogenic and consisted of the following virulent genes: Yersinia heat-stable toxin (yst), invasion (inv), attachment invasion locus (ail), virulence regulon transcriptional activator (virF), Yersinia adehesin A (yadA), and the O:3 antigen gene (rfbC). Seven out of the eight (87.5%) chitterling samples were shown to be pathogenic. Disc diffusion was conducted to determine the antimicrobial susceptibility of the isolates. Over half (55.5%) of the antimicrobial agents were found effective, with isolates being completely susceptible to ciprofloxacin, kanamycin, trimethoprim, cefotaxime, and gentamycin. Ampicillin was determined to be least effective, where 84.6% of the samples presented resistance to the drug. Random amplified polymorphic DNA (RAPD) analysis and ERIC-PCR techniques were used to evaluate genetic similarity among the Yersinia isolates. There was approximately 85% similarity between two chitterlings and a fecal isolate during RAPD testing. With ERIC-PCR the largest similarity among all samples was at 95%, which was found between isolates from a chitterling and milk sample. Chitterling samples showed the highest prevalence of Y. enterocolitica compared to the other samples. Cross contamination at the farm level could be the root cause of this pathogen being prevalent in farm animal and food sources, which does pose a risk to public human health when food is improperly prepared.

Yersinia辅助DHCP攻防实验的设计与实现

《网络安全技术》课程的实验环境中,需要大量硬件平台和软件系统的支持,其中安全攻防仿真系统由于数量众多,常常令许多任课教师难以选择。本文介绍了安全攻防软件Yersinia的功能,并以基于DHCP协议攻击的实验设计为例,介绍了如何使用Yersina辅助实现一个典型的安全攻防实验。

An ironic case of liver infections:Yersinia enterocolitis in the setting of thalassemia

A 49 years old Vietnamese male with a history of thalassemia,presented with gastrointestinal symptoms and signs of hemolysis.He was diagnosed with yersinia enterocolitis.Yersinia is a gram-negative rod that most frequently occurs in children especially during the winter months.In the current case,the bone marrow biopsy showed hemophagocytosis along with positive cultures for Yersinia.The microorganism likely triggered hemophagocytosis.This syndrome,also known as,hemophagocytic lymphohistiocytosis,is defined by fever for more than 7 d,cytopenia of two or more cell lines,hemophagocytosis,hepatitis,serum ferritin greater than500,jaundice,lymphadenopathy,and hepatosplenomegaly.This disorder can be either familial or secondary to a strong immunologic activation.Both have an overwhelming activation of T-cells and macrophages.

Experimental infection with Yersinia pseudotuberculosis in European brown hare(Lepus europaeus,Pallas)

Objective:To investigate clinicopathological,bacteriological and pathological aspects of an experimental infection with Yersinia pseudotuberculosis(Y.pseudotuberculosis)in hares in order to verify the efficacy of serology for the in vivo diagnosis.Moreover,the pathogenicity of two Y.pseudotuberculosis strains was investigated in order to detect potential differences.Methods:Twelve European brown hares(Lepus europaeus,Pallas)were experimentally infected per os and via conjunctival mucosae with Y.pseudotuberculosis:six subjects were infected with a strain isolated from a naturally infected hare(YpH)and six subjects with a strain isolated from a naturally infected rabbit(YpR).Two hares were used as negative controls.All animals were subjected to clinical,bacteriological and serological examinations during 9 weeks following the infection and,at the end of the control period,subjects still alive were euthanized and submitted to a complete post mortem examination.Results:All faecal samples collected during the control period were positive for bacteriological examinations and to a PCR for the inv gene of Y.pseudotuberculosis,while only one Yp H-infected hare showed a positive haemocultures.From the 2nd to the 9th week post infection(pi),serological analysis revealed specific antibodies with titers ranging from 1:10 to 1:160 in all YpH-infected and two YpR-infected subjects.All the Yp H-infected and two Yp R-infected hares scored positive for Y.pseudotuberculosis by means of bacteriological investigations.Grossly,suppurative multifocal lesions were detected in liver,spleen,kidney and sub-mandibular lymph nodes in both YpH-and YpR-infected hares and confirmed with histopathology.Pulmonary lesions were observed only in Yp H-infected subjects.Immunohistochemistry confirmed the presence of bacterial antigen in all infected animals.Conclusion:Results of this study revealed that YpH strain is more pathogenic for hares than the YpR strain;moreover the serological test performed in this study could be used for the diagnosis of pseudotuberculosis in hares,whereas post mortem diagnosis should be confirmed by means of bacteriological examination,PCR,histopathology and immunohistochemistry.

Use of Rich BHI Medium Instead of Synthetic TMH Medium for Gene Regulation Study in Yersinia pestis

Objective This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. Methods The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 ~C, and with temperature shifting from 26 to 37 ~C, the wild-type （WT） strain or its phoP or crp null mutant （AphoP or Acrp, respectively） was subject to RNA isolation, and then the promoter activity of rovA or plo in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and AphoP to measure the promoter activity of rovA in these two strains with the ^-Galactosidase enzyme assay system. Results When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that ofpla was stimulated by CRP. Conclusion The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions （which are essential to triggering relevant gene regulatory cascades）, can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.

Comparison of Lipopolysaccharide and Protein Immunogens from Pathogenic Yersinia enterocolitica Bio-serotype 1B/O:8 and 2/O:9 using SDS-PAGE

Objective Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitico. Methods We used SDS-PAGE and western blotting to characterize lipopolysaccharide （LPS）, Yersinio outer membrane proteins （Yops）, membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitico bio-serotype 2/0：9, isolated from China, and highly pathogenic bio-serotype 1B/O：8, isolated from Japan. Results These two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains. Conclusion The major antigens of the two strains e and membrane proteins, as shown by comparing preparations. citing the host immune protein samples with response were the LPS reference and purified

Pleiotropic Effect of tatC Mutation on Metabolism of Pathogen Yersinia enterocolitica

Objective To analyze the impact of depletion of the twin arginine translocation （TAT） system on virulence and physiology of Yersinia enterocolitica for a better understanding of its pathogenicity. Methods We constructed a △tatC：：Sp^R mutant of Yersinia enterocolitica by P1 phage mediated transduction using Escherichia coli K-12 △tatC：：Sp^R strain as a donor. Results A Pl-mediated genetic material transfer was found between the two species of enterobacteria, indicating a great potential of acquisition of antibiotic resistance in emergency of a new threatening pathogen by genetic material exchanges. Periplasmic trimethylamine N-oxidase reductase activity was detected in the wild type E enterocolitica strain and translocation of this enzyme was completely abolished by the △tatC：：Sp^R mutation. In addition, the △tatC：：Sp^R mutation showed a pleiotropic effect on the metabolism of E enterocolitica. However, the tat mutation did not seem to affect the mobility and virulence of Y. enterocolitica under the conditions used. Conclusion Unlike other pathogenic bacteria studied, the TAT system of E enterocolitica might play an important role in the pathogenic process, which is distinct from other pathogens, such as Pseudomonas aeruginosa and enterohemorrhagic E. coli O 157：H7.

Isolation and Pathogenicity Analyses on Yersinia enterocolitica from Pelteobagrus vachelli

Yersinia enterocolitica is an important zoonotic pathogen that can induce disease outbreaks in a wide host range. Strain YER6022 was isolated from Pelteobagrus vachelli and identified using bacterial morphology and 16S rDNA sequence analysis. Five virulence factors were detected, then artificial infection experiment and histopathological method were carried out. These results showed that strain YER6022 was one of Y. enterocolitica family members. In addition, ail, ystb, virF, yadA and HPIint were dectected. In artificial infection experiment, with 80% mortality and 100% morbidity, injected Pelteobagrus vachellis showed red swollen of the anus, abdomen swelling and fim bleeding. There existed serious hyperaemia and edema in kidney, spleen, intestine and liver at the light microscope. Ultrastructural observation indicated that mitochondria of the liver, kidney, spleen and intestine swelled and mitochondrial cristae broke. The data had further shed light on its pathogenicity in Pelteobagrus vachelli. It would benefit for further studies on pathogenesis ofPelteobagrus vachelli infected with Y. enterocolitica.

Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis,and Roles of RyhBs in Biofilm Formation

Objective To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis(Y.pestis),as well as the roles of RyhBs in biofilm formation.Methods Regulatory relationships were assessed by a combination of colony morphology assay,primer extension,electrophoretic mobility shift assay and DNase I footprinting.Results Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription,while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level.In addition,both RyhB1 and RyhB2 positively regulated Y.pestis biofilm exopolysaccharide(EPS)production and the expression of hmsHFRS and hmsT.Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation,and RyhB homologs have a positive regulatory effect on biofilm formation in Y.pestis.

Behavioral Pattern of Native Food Isolates of <i>Yersinia</i><i>enterocolitica</i>and <i>Yersinia</i><i>intermedia</i>under Simulated Time-Temperature Combinations of the Food Chain

The public health significance of Yersinia spp. gives a new dimension to the prevailing food chain, wherein the foods do get exposed to heat and cold treatments. In this study, the effect of heat treatment on the native isolates of Yersinia enterocolitica CFR 2301 and Y. intermedia CFR 2303 revealed the D-values ranging from the lowest of 0.08 min at 65℃ in skim milk/beef gravy to the highest of 18.52 min at 50℃ in beef gravy. The heat sensitivity of both these cultures was in the order of Milli-Q water > 0.85% saline > skim milk > beef gravy. The z-values of the test cultures ranged from 7.55℃ for Y. intermedia to 12.08℃ for Y. enterocolitica. The heat sensitivity in Y. enterocolitica appeared to be related with growth incubation temperatures and also fatty acid profile of cell membrane. The effect of low temperature treatments (–20 ℃, 0℃ and 4℃ for 20 d) in water, saline and skim milk revealed the ability of Y. enterocolitica to survive more efficiently at –20℃, while Y. intermedia was more tolerant at 0℃. In packaged drinking water, Y. enterocolitica could survive and grow at 4℃ and 16℃, while at 30℃, inactivation was rapid. The findings did indicate that heat and cold treatments would not always ensure safety from Y. enterocolitica and Y. intermedia in the food chain.

Investigation of <i>Yersinia enterocolitica</i>Bioserotype 4/O:3 Clusters in Finland, 2017-2018

In December 2017, two suspected outbreaks of Yersinia enterocolitica 4/O:3 were notified in Finland. We analyzed the surveillance and outbreak investigation data and genotyped Y. enterocolitica strains in order to understand Y. enterocolitica epidemiology in Finland and to find out whether the notified outbreaks were related. A total of 13,344 Y. enterocolitica cases were reported in 1995-2018 to the National Infectious Diseases Register. The mean annual incidence ranged from 7.9 to 15.9/100,000 inhabitants. The highest incidence was observed in young adults (14/100,000). Incidence varied geographically. The incidence was higher in spring compared to other seasons. The most common pathogenic bioserotype was 4/O:3 but the information on bio/serotype was missing in 64% of the surveillance cases. For most of the cases, 87%, no travel history was reported. In the YE 4/O:3 outbreak investigation, whole genome sequencing was performed for isolates from 29/69 (42%) of the outbreak cases. The sequencing results showed that cases consisted of six independent clusters with 2 - 6 isolates in each cluster, and 5 sporadic isolates. No food item was found to be common to all of the clusters. In order to improve monitoring and facilitate the detection and investigation of Y. enterocolitica outbreaks, more comprehensive information on bio/serotype and on travel history is required in the surveillance.

Yersinia enterocolitica Infection of a Prosthetic Knee Joint.Case Report and Review of the Literature on Deep Sited Infections Caused by Y.enterocolitica

Prosthetic joint infection is a rare manifestation of Yersinia enterocolitica. We report a case of a patient presenting with fever and a purulent infection in his prosthetic knee joint caused by Y. enterocolitica. He had been operated in 1990 for arthrosis of the right knee. Re-operation was performed in 2007 for loosening of the prosthesis. Seven months later, following progressively increasing knee pain, he became acutely febrile and a purulent knee joint infection was diagnosed. Y. enterocolitica was isolated from the joint fluid. Serum antibodies against Y. enterocolitica were also positive. He was treated with debridement, replacement of the liner component of the prosthesis and a long course of intravenous antimicrobial therapy. The infection was thought to be in a chronic suppressive state. The final outcome after all therapy was good.

Genotyping of strains of Yersinia pestis from Marmota baibacina-Spermophilus undulates Plague Focus of Tianshan Mountains in China

The aim of this study is to carry out genotyping of 61 Y. pestts strmns tsolated trom lwarmota baibacina-Spermophilus undulates Plague Focus of Tianshan Mountains in China. Primer pairs targeting the 22 DFRs were designed for detecting the genotypes of 61 strains. As a result, 61 strains of Y. pestis were divided into four genotypes 1, 2, 3, and 4. Genotypes 1, 2 and 3 were found from west part in Northern Tianshan Mountains Plague Focus, but type 1 was only from Nileke county. The type of strains from Aheqi was different from those of Atushi counties in Southern Tianshan Mountains and similar to strains in Northern Tianshan Mountains Plague Focus. The type 4 distributed over Atushi county, which was identical with that of strains from Marmota caudae Plague Focus of the Pamiri Plateau. It is concluded that geonotyping is identical with ecotyping made by Shuli Ji. Tianshan Mountains Plague Focus and Marmota caudae Plague Focus of the Pamiri Plateau have a cross spreading profile.

辽宁省市售生肉及冷冻鱼糜制品中小肠结肠炎耶尔森菌检测及病原特征分析

为了解辽宁省市售鸡肉、牛肉、猪肉和冷冻鱼糜制品中小肠结肠炎耶尔森菌的污染情况和病原特征。2018—2021年从辽宁省超市和农贸市场采集鸡肉250份、猪肉320份、牛肉178份以及冷冻鱼糜制品128份,进行小肠结肠炎耶尔森菌检测,对分离菌株进行生物分型、血清分型、毒力基因检测、药敏试验和质谱分析。结果表明:876份样品中共检出30株小肠结肠炎耶尔森菌,检出率为3.42%(30/876);其中鸡肉检出率为7.20%(18/250),牛肉检出率为4.49%(8/178),猪肉检出率为0.94%(3/320),冷冻鱼糜制品检出率为0.78%(1/128)。30株分离菌株的生物型均为1A型;血清型O:5型为27株,O:8型为3株;检测的5种毒力基因中,ail、ystA、yadA、virF均为阴性,ystB阳性为15株。30株分离株中,耐4种药物的分离株为4株(13.33%),耐6种药物的为1株(3.33%);30株小肠结肠炎耶尔森菌对头孢唑啉、氨苄西林和头孢噻肟3种药物的整体耐药程度较高,耐药率分别为93.33%、66.67%和53.33%,30株分离株均对头孢他啶、氯霉素和庆大霉素3种抗生素100%敏感。30株小肠结肠炎耶尔森菌质谱特征分析表明,聚类为3簇,其中携带ystB基因的分离株均分布在第3簇中。辽宁省市售鸡肉、牛肉、猪肉和冷冻鱼糜制品中小肠结肠炎耶尔森菌的污染是引起食源性疾病的潜在风险因素,分离株对头孢唑啉、氨苄西林和头孢噻肟有较高耐药率。

AN OUTBREAK OF A NEW EPIZOOTIC IN SILVER CARP AND BIGHEAD CARP——Yersinia ruckeri, A NEW PATHOGEN OF SILVER CARP AND BIGHEAD CARP

Silver carp, Hypophthalmichthys molitrix, and bighead carp, Aristichthys nobilis, habitually celebrated for their being cultured easily with fewer diseases and high-yield, have suffered from a new severe epidemic disease recently. The spread of the epizootic has been far and wide, from several provinces in South China to the Beijing and Tianjin area. The affected fish suffered heavy mortafity in one week, and even died off.