2004-2005年江苏省海安县小肠结肠炎耶尔森菌病监测分析

目的 了解2004-2005年江苏省海安县小肠结肠炎耶尔森菌人群带菌情况以及家禽、家畜肉污染情况.方法 采集动物和腹泻患者粪便及冷藏的禽、畜肉和苍蝇等样本进行小肠结肠炎耶尔森菌增菌培养,并对阳性菌株进行血清学分型和毒力基因检测.结果 所有样本中,仅160份动物粪便样本检测阳性,阳性率达17.19%(160/931),其中猪的带菌率较高.151株小肠结肠炎耶尔森菌共有29个血清型,其中血清型以O:16,29、O:6,30、O:5、O:7,8、O:12,26、O:4,33、O:8等为主,除61株ystB阳性外,其余毒力基因均为阴性.结论 小肠结肠炎耶尔森菌江苏省海安县家畜家禽中普遍携带菌,但这些菌株大多属于非致病菌株,对人群威胁不大.     

2011年江苏省东台市小肠结肠炎耶尔森菌监测分析

目的 了解江苏省东台市腹泻患者和不同宿主粪便小肠结肠炎耶尔森菌感染及其毒力基因携带情况。 方法 从所检标本中分离小肠结肠炎耶尔森菌,同时用聚合酶链反应方法检测分离菌株5种毒力基因携带状况。 结果 15种样品795份标本中,小肠结肠炎耶尔森菌检出率为3.77%,猪粪中的检出率最高,达22.50%;犬粪次之,为8.05%。猪粪中携带ail、 ystA、yadA 和virF 4种毒力基因的致病菌株检出率最高,均达12.50%;犬粪次之,分别为4.60%、4.60%、4.60%和4.60%。 结论 猪、犬是东台市小肠结肠炎耶尔森菌的重要携带者。     

云南部分鼠疫疫源地中小肠结肠炎耶尔森菌的分离及鉴定

目的 了解云南部分鼠疫疫源地中小肠结肠炎耶尔森菌的分布及病原学特征。方法 2012年5月至2014年4月采集剑川、梁河两县鼠类盲肠,对其进行培养,通过菌落聚合酶链反应初筛小肠结肠炎耶尔森菌,通过生化鉴定确定菌株并作毒力基因检测。结果 915份标本中分离到小肠结肠炎耶尔森菌35株,总检出率为3.83%,分离株包含1株致病株、34株非致病株。35株小肠结肠炎耶尔森菌全部为生物1A型,1株致病株为1A/O:9生物血清型,毒力基因为(ail+、ystA-、ystB-、yadA-、virF-、rbfc-),非致病株血清为O:5、O:8及未分型,毒力基因为(ail-、ystA-、ystB+、yadA-、virF-、rbfc-)。 结论 小肠结肠炎耶尔森菌在剑川野鼠鼠疫疫源地鼠间流行,分离菌株血清型别较为多样,分离到的致病株缺乏典型毒力基因;小肠结肠炎耶尔森菌在梁河家鼠鼠疫疫源地尚未发现鼠间的流行。     

聚合酶链反应法和分离培养法筛检小肠结肠炎耶尔森菌方法的比较研究

目的 比较针对小肠结肠炎耶尔森菌的两种不同检测方法,即ail基因联合foxA基因的聚合酶链反应(polymerase chain reaction,PCR)检测和分离培养法的差异。 方法 从生猪咽拭子和回盲部肠内容物标本中提取细菌基因组DNA并进行小肠结肠炎耶尔森菌保守基因foxA和粘附侵袭位点基因ail的PCR检测;菌株分离培养按常规方法进行。将两种方法的检出结果进行统计分析,以判定哪种更有优势。 结果 700份标本中,小肠结肠炎耶尔森菌特异性基因PCR检测阳性402份,阳性率57.43%;小肠结肠炎耶尔森菌分离培养阳性278份,阳性率39.71%。在PCR检测阳性的402份标本中,分离小肠结肠炎耶尔森菌271株,分离阳性率为67.41%;PCR检测阴性的298份标本中,分离小肠结肠炎耶尔森菌7株,分离阳性率仅为2.35%。 结论 本次实验证实,检测来源于生猪标本中的小肠结肠炎耶尔森菌,采用foxA和ail基因联合的PCR检测法,检出率明显高于小肠结肠炎耶尔森菌分离培养法(P0.05)。     

2013-2014年云南省剑川县野鼠鼠疫疫源地小肠结肠炎耶尔森菌的调查分析

目的 对剑川县野鼠鼠疫疫源地小肠结肠炎耶尔森菌分布情况调查分析。方法 从剑川采集鼠盲结肠标本进行小肠结肠炎耶尔森菌分离培养、表型鉴定和特异基因分析。结果 从采集的645份鼠类标本中,分离到34株小肠结肠炎耶尔森菌,分离率为5.27%(34/645);其中致病株1株、非致病株33株。鼠类标本分别是大绒鼠、贝氏树鼩、褐家鼠、齐氏姬鼠、小林姬鼠等,其中大绒鼠采集率和检出率最高(2.79%),其次为齐氏姬鼠(1.55%)。结论 剑川县野鼠鼠疫疫源地中,小肠结肠炎耶尔森菌的主要宿主与鼠疫菌主要宿主基本一致,推测小肠结肠炎耶尔森菌的存在与鼠疫的相对静息相关。     

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.     

小肠结肠炎耶尔森菌O:8血清型特异性单克隆抗体的制备

目的 利用杂交瘤技术制备小肠结肠炎耶尔森菌O:8血清型特异性单克隆抗体,用于小肠结肠炎耶尔森菌O:8血清型菌株的分型鉴定.方法 用小肠结肠炎耶尔森菌O:8血清型菌株作为抗原,以骨髓杂交瘤细胞融合技术制备小肠结肠炎耶尔森单克隆抗体,用玻片凝集筛选法进行单克隆抗体的特异性鉴定.结果 筛选到1株与小肠结肠炎耶尔森菌O:8血清型菌株呈现阳性反应,并与日本的抗血清检测结果相一致,而与被检测的其他血清型小肠结肠炎耶尔森菌和常见肠道致病菌无交叉凝集反应.结论 本实验筛选获得的单克隆抗体,用于小肠结肠炎耶尔森菌O:8血清型菌株鉴定,具有良好的特异性,避免了免疫血清难以避免的交叉凝集现象.     

肠聚集性大肠杆菌中小肠结肠炎耶尔森菌强毒力岛的结构和功能

目的:通过了解小肠结肠炎耶尔森菌强毒力岛在肠聚集性大肠杆菌中的结构和摄铁功能,从而进一步研究肠聚集性大肠杆菌的毒力进化及其致病机制。方法:实验于2005-08/2006-09在南方医科大学公共卫生与热带医学学院完成。6株肠聚集性大肠杆菌采自某部队腹泻患者粪便,并经血清学鉴定。采用聚合酶链反应方法观察肠聚集性大肠杆菌中强毒力岛irp2和fyua阳性检出率和强毒力岛在阳性菌株中的定位鉴定。原位杂交方法进行肠聚集性大肠杆菌中强毒力岛irp2和fyua基因序列分析。SDS-聚丙烯酰氨凝胶电泳方法观察小肠结肠炎耶尔森菌WA和毒力岛阳性的肠聚集性大肠杆菌HMWP1和HMWP2的表达。结果:6株肠聚集性大肠杆菌有5株检出了强毒力岛,检出率达到83.33%,而且这些阳性菌株中的毒力岛都位于天冬氨酸tRNA位点;在缺铁条件下,肠聚集性大肠杆菌能够表达和小肠结肠炎耶尔森菌相同的摄铁蛋白HMWP1和HMWP2。结论:小肠结肠炎耶尔森菌强毒力岛在肠聚集性大肠杆菌中具有阳性率较高的分布,并具有相同的摄铁功能,很可能肠聚集性大肠杆菌和小肠结肠炎耶尔森菌中的强毒力岛有相同的转移机制。     

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     

部分ESIEC菌株存在耶尔森氏菌HPI毒力岛

本文应用小肠结肠炎耶尔森氏菌毒力岛HPI上的irp2、fyuA基因和大肠杆菌染色体上asnT -tRNA位点检测的引物 ,对 43株肠产志贺样毒素且具侵袭力的大肠杆菌 (ESIEC)进行了PCR检测。发现约 34 9%的菌株irp2、fyuA引物扩增阳性 ,而这部分菌株asnT -tRNA位点引物扩增为阴性 ,证实ESIEC中确有耶尔森氏菌毒力岛的irp2、fyuA基因 ,并且也在染色体上asnT -tRNA位点插入。     

Identification of invasive Yersinia species using oligonucleotide probes.

Oligonucleotide probes directed to the inv and ail invasion genes of Yersinia species were used to analyse yersiniae and non-yersiniae isolates by colony hybridization. The INV-3 probe, targeted to the inv gene of Yersinia pseudotuberculosis, hybridized with all 48 HeLa cell-invasive Y. pseudotuberculosis isolates examined; the PF-13 probe, specific for the ail gene of Yersinia enterocolitica, identified all invasive strains (36 of 52) of Y. enterocolitica tested. Neither probe hybridized with non-yersinia isolates or other Yersinia species. Southern analyses of restriction enzyme-digested genomic DNA confirmed the specificity of both probes. INV-3 hybridized with a 4.5 kilobase (kb) Bam HI fragment known to carry the inv gene in Y. pseudotuberculosis. PF-13 was specific for a 1.2 kb Cla I-Ava I fragment in Y. enterocolitica that carried the ail locus. Reactivity with either probe correlated closely with the ability of Y. pseudotuberculosis and Y. enterocolitica isolates to invade HeLa cells.     

小肠结肠炎耶尔森菌菌种保存22年的跟踪调查

目的为了探索一种简便、实用并能长期保存小肠结肠炎耶尔森菌菌种的方法。方法 1988年将小肠结肠炎耶尔森菌菌种(血清O:3型和血清O:9型)接种于鸡蛋斜面培养基及半固体培养基复苏后加灭菌液体石蜡,置4℃普通冰箱保存,且每年进行复苏鉴定。结果 O:3型菌种可保存21年,O:9型可保存22年以上,O:9型到发稿为止,依然存活。结论鸡蛋斜面保存法和半固体穿刺法-4℃保存小肠结肠炎耶尔森菌菌种效果良好,可长时间保存,且不需要反复传代。     

区别检测小肠结肠炎耶尔森氏菌与布鲁氏菌LAMP引物的设计与分析

目的小肠结肠炎耶尔森氏菌（耶氏菌）和布鲁氏菌都是人兽共患细菌病的主要病原菌,血清学检测过程中,两者常出现交叉反应,尤其是O：9型耶氏菌与布鲁氏菌干扰更为明显。本实验目的是在DNA水平上区别检测这两种致病菌。方法根据耶氏菌outL基因设计一套可用于环介导等温扩增（LAMP）检测方法的引物,拟采用该项特异性强、灵敏性高、操作简单的核酸检测技术,对两种菌进行检测。结果LAMP方法检测耶氏菌结果为阳性,检测布鲁氏菌结果为阴性。结论成功地在DNA水平上区别检测这两种致病菌。并且为建立耶氏菌的非特异性干扰小的LAMP检测方法提供可应用的引物,同时也为LAMP技术的开发应用以及LAMP引物设计提供应用参考。     

Identification of Yersinia-infected blood donors by anti-Yop IgA immunoassay

BACKGROUND: From 1991 through 1996, nine transfusion-related cases of septicemia and endotoxemia occurred in New Zealand, a rate approximately 80 times that in the United States. Eight cases involved the transfusion of Yersinia enterocolitica-infected blood and one involved Serratia liquefaciens-infected blood. Six of the recipients died. Donor exclusion by recent gastrointestinal illness failed to prevent the four most recent such infections, and it has led to an estimated 3- to 5-percent rate of donor deferral. STUDY DESIGN AND METHODS: An antigen preparation containing the released proteins (Yops) of Y. enterocolitica was used to establish an EIA to detect IgA directed against these proteins in donated blood. The assay was tested with serum from donors in transfusion-related endotoxemia cases, subjects who were stool culture-positive for Y. enterocolitica, and 495 healthy volunteer blood donors. RESULTS: The assay detected anti-Yop IgA in the donors of all 6 infected units tested. Ninety-six percent of culture-positive subjects tested positive, whereas there was 70-percent positivity with a commercial immunoassay based on lipopolysaccharide. Five percent of random donors tested positive; only one of these had Y. enterocolitica present in a stool sample, and none were bacteremic. CONCLUSION: The anti-Yop immunoassay used in this study could be applied to reduce the risk of posttransfusion endotoxic shock caused by Y. enterocolitica.     

Chronic and lingering Yersinia ileitis

AIM: To study the incidence, clinical manifestations, and diagnosis of chronic and lingering Yersinia ileitis. MATERIALS AND METHODS: 82 patients with pains in the right iliac area. The coagglutination test was used to reveal Y. pseudotuberculosis and Y. enterocolitica antigens as part of circulating immune complexes in the serum and coprofiltrates. RESULTS: Correct diagnosis was established 3 months to 2 years after the acute period of the disease. After etiotropic treatment, 2 patients continued to have the steady pain syndrome in the right iliac area, which is likely to be associated with periileitis and, 46 patients developed chronic Yersinia ileitis, as evidenced by their positive serological reactions to antigens and iliac morphological findings. Development of a residual phase may be presumed in 2 patients who continued to have stool disorders and abdominal pain, but no pseudotuberculous antigens in the stool and blood. CONCLUSION: Patients with lingering (up to 3 months) and chronic (over 3 months) Yersinia ileitis were 38.1% of the total number of those with ileocecal pain. The chronic nature of Yersinia ileitis is associated with late diagnosis, developed periileitis, and immunity disorders.     

Specific detection of plasmid bearing Yersinia isolates by PCR

A total of 210 isolates belonging to 9 different species of the genus Yersinia (Y.) was investigated with three different PCR assays targeting two plasmoidal genes, the Yersinia adhesin gene (yadA) and the V-antigen gene. The yadA PCR assay described in 1995 by Blais and Phillipe, targeting a Y. enterocolitica specific gene region and a newly designed assay targeting the gene region functionally responsible for autoagglutination, were compared. Both assays identified the same Y. enterocolitica strains. To exclude the possibility that false negative results were obtained due to mutations that had occurred in parallel in both gene regions, a third PCR assay by Neubauer et al. (2000) targeting a conserved region of the V-antigen gene was used as control. Again, DNA of the same Y. enterocolitica strains was amplified. In contrast to the yadA PCR assay described by Blais and Phillipe, the newly established yadA and the V-antigen PCR assays amplified DNA from Y. pseudotuberculosis strains. Therefore, by using the PCR technique as a molecular tool spontaneous mutations could be excluded as the cause of anomalous reactions in PCR assays targeting genes of the Yersinia virulence plasmid. Based on these results, it can be assumed that all presumptive pathogenic Yersinia isolates can be identified on the basis of PCR analysis. These molecular assays may also produce fewer false positive reactions in comparison to phenotypic tests such as the autoagglutination test which depend heavily on the handler's experience. It has to be stressed that the PCR assays used in this study have not been evaluated for routine use. Therefore, standardization of the PCR methodology including sample preparation, primer target sequences and PCR reagents is needed for the reliable and safe diagnosis of pathogenic Yersinia spp. in future.