中国鼠疫自然疫源地分型研究Ⅲ.鼠疫耶尔森菌DFR/MLVA主要基因组型生物学特征

鼠疫菌基因组型与鼠疫生态地理景观型及其主要宿主、媒介密切相关,自然组合形成鼠疫生物群落,互相依赖,互相制约,相互适应,同步进化,维持鼠疫自然疫源性和生物群落的延续.鼠疫菌差异区段/多位点串联重复序列(DFR/MLVA)基因组型反映了鼠疫自然疫源地生物学特征.     

鼠疫噬菌体研究概述

鼠疫噬菌体是一种特异性很强的鼠疫耶尔森菌（Yersinia pestis,鼠疫菌）的细菌病毒,一直被用来进行鼠疫病原体的鉴定。鼠疫噬菌体的早期研究主要以如何在自然界分离、生物学特性、诊断和治疗鼠疫,近年来,随着微生物基因组和蛋白质组学技术的发展,揭示了鼠疫噬菌体在鼠疫菌进化过程中所发生的作用。现就鼠疫噬菌体的研究进展,尤其是基因组的结构、与宿主菌在致病和免疫相关研究做一些介绍,为研发新型分子生物学技术和鼠疫治疗药物提供帮助。     

中国鼠疫自然疫源地分型研究Ⅲ．鼠疫耶尔森菌DFR／MLVA主要基因组型生物学特征

鼠疫菌基因组型与鼠疫生态地理景观型及其主要宿主、媒介密切相关,自然组合形成鼠疫生物群落,互相依赖,互相制约,相互适应,同步进化,维持鼠疫自然疫源性和生物群落的延续。鼠疫菌差异区段／多位点串联重复序列(DF形ML讼)基因组型反映了鼠疫自然疫源地生物学特征。其型别对认识鼠疫菌自然疫源地生物学具有重要意义。通过对鼠疫菌基因组型的鉴定,可实现对鼠疫菌的追踪溯源,追溯出所代表的鼠疫主要宿主、主要媒介及其生态地理景观型的生物学特征。因此。鉴定鼠疫菌基因组型、主要基因组型对中国鼠疫预防控制、应急反恐、生物安全、监测预报及软件技术平台体系建设具有实践和理论指导意义。     

青藏高原鼠疫耶尔森菌基因型分布

目的研究青藏高原鼠疫耶尔森菌（鼠疫菌）基因组型分布特征.方法对分离到的青藏高原鼠疫菌297株,根据已经证实的22个差异区段设计引物,每株鼠疫菌的每个基因差异区段都采用PCR技术进行验证.结果在喜马拉雅旱獭鼠疫自然疫源地中,鼠疫菌基因组型有9种,分别为1、5、6、7、8、10、11、新基因组型和Ype-ancestor型,其中以5、8和10型为主,3种基因组型合计所占比例为80.6%（204/253）,而且不同地区鼠疫菌基因组型的分布也不一致.青藏高原青海田鼠鼠疫疫源地鼠疫菌基因组型全部为14型.结论青藏高原鼠疫菌基因组型分布具有明显的地理特征.根据基因组型的分布状况推测出了鼠疫菌在青藏高原的传播路径.     

马尔尼菲青霉菌的分子生物学研究进展

马尔尼菲青霉菌是东南亚国家和我国南方地区艾滋病机会性感染常见的侵袭性真菌,一般引起播散性感染.其感染确诊需要病原诊断,治疗难度较大,病死率高.该菌致病途径多样,发病机制复杂,与流行病学、基因组学、蛋白组学等因素相关.近年来,马尔尼菲青霉菌分子流行病学特点、基因组特征、致病与耐药分子机制及PCR快速诊断等方面的研究取得了初步进展,为有效防治艾滋病合并马尔尼菲青霉菌感染提供了可靠的理论依据.     

青藏高原鼠疫自然疫源地鼠疫耶尔森菌病原学研究

目的:了解青藏高原鼠疫自然疫源地鼠疫耶尔森菌（简称鼠疫菌）病原学特征。方法:以青藏高原1954-2016年不同地区及宿主、媒介体内分离的1 378株鼠疫菌作为实验对象,采用常规技术与分子生物学技术对其进行表型特征、质粒谱、基因组分型等研究,并对青藏高原鼠疫菌病原学、地理分布等特征进行探讨。结果:青藏高原鼠疫菌含6个生化...     

中国鼠疫耶尔森菌的分子生物学特征与遗传学意义

目的 根据中国鼠疫耶尔森菌(鼠疫菌)的分子生物学特征分析其遗传背景。方法利用实验研究中rRNA基因指纹图(ribotyping)、脉冲场凝胶电泳(PFGE)、随机扩增DNA多态性(RAPD)及插入序列(IS)等方面工作的原始数据,通过聚类分析等统计学手段,探究中国鼠疫菌株间的遗传学距离,分析各种方法作为鼠疫菌分子识别特征的意义。结果 rRNA基因指纹图型与脉冲场电泳型之间是相对应的,但同属于7拷贝rRNA基因的田鼠菌株与西藏仲巴菌株,其间的遗传学距离,却较6拷贝rRNA基因的菌株还远。而随机扩增型与rRNA基因型之间对应关系较不明确,有较多的例外情况。用相似值可以表示这些菌株之间的遗传距离。结论 中国的鼠疫菌具有多种独特的分子生物学特征,其中新疆灰旱獭疫源地和西藏南部冈底斯山地理环境复杂,有较多的自然屏障,使鼠疫菌在流行过程中,限于局部地区,各自独立进化,产生了相互混合存在的多种基因类型。     

中国鼠疫自然疫源地分型研究Ⅳ.鼠疫耶尔森菌生物型生物学特征的探讨

鼠疫菌生物型是鼠疫菌起源进化遗传性状最稳定的生物学标准,是鼠疫起源进化普系的“活化石”,也是该菌起源进化最具代表性的模式.依据其对甘油和阿拉伯糖的酵解和还原硝酸盐的能力,鼠疫菌可分为4种生物型,即古典型(甘油酵解阳性,阿拉伯糖酵解阳性,硝酸盐还原阳性)、中世纪型(甘油酵解阳性,阿拉伯糖酵解阳性,硝酸盐还原阴性)、东方型(甘油酵解阴性,阿拉伯糖酵解阳性,硝酸盐还原阳性)和田鼠型(甘油酵解阳性,阿拉伯糖酵解阴性,硝酸盐还原阴性).前三种生物型与人类历史3次鼠疫大流行相对应,最后一种生物型对人不致病,但能造成田鼠鼠疫并在其自然疫源地内流行.目前中国鼠疫自然疫源地有4种鼠疫菌生物型.     

中国鼠疫自然疫源地分型研究Ⅳ.鼠疫耶尔森菌生物型生物学特征的探讨

鼠疫菌生物型是鼠疫菌起源进化遗传性状最稳定的生物学标准, 是鼠疫起源进化普系的“活化石”, 也是该菌起源进化最具代表性的模式.依据其对甘油和阿拉伯糖的酵解和还原硝酸盐的能力, 鼠疫菌可分为4种生物型, 即古典型(甘油酵解阳性, 阿拉伯糖酵解阳性, 硝酸盐还原阳性)、中世纪型(甘油酵解阳性, 阿拉伯糖酵解阳性, 硝酸盐还原阴性)、东方型(甘油酵解阴性, 阿拉伯糖酵解阳性, 硝酸盐还原阳性)和田鼠型(甘油酵解阳性, 阿拉伯糖酵解阴性, 硝酸盐还原阴性).前三种生物型与人类历史3次鼠疫大流行相对应, 最后一种生物型对人不致病, 但能造成田鼠鼠疫并在其自然疫源地内流行.目前中国鼠疫自然疫源地有4种鼠疫菌生物型.     

中国鼠疫耶尔森菌差异区段分型及其地理分布特征

目的 研究中国鼠疫耶尔森菌差异区段（DFR）分型及地理分布特征。方法 对分离自中国11个疫源地3 044株鼠疫菌,应用23个DFR和质粒验证（PMT1）PCR扩增,进行鼠疫菌DFR基因组分型及地理分布特征分析。结果 3 044株鼠疫菌共包括52个基因组型,其中19个为主要基因组型、33个为次要基因组型。在以往鉴定的31个基因组型基础上又增加了 21个新基因组型。其中增加的3个新基因组型均为主要基因组型,即青藏高原喜马拉雅旱獭鼠疫自然疫源地新增加32型、44型,内蒙古高原长爪沙鼠鼠疫自然疫源地新增加50型。结论 在新增加的 21个新基因组型中有3个为主要基因组型,中国鼠疫菌DFR主要基因组型具有明显的地理分布特征。     

青海省三江源地区鼠疫病原学分析

目的 研究青海省三江源地区鼠疫菌株生物学特性并确定疫源地空间结构及性质.方法 对1954-2007年青海省三江源地区分离的鼠疫菌株进行生物学表型鉴定及分子生物学研究.结果 411株代表性菌株中,12株脱氮(-)阿胶糖(-)甘油(+)属田鼠型,399株为脱氮(+)阿胶糖(+)甘油(+)属古典型.411株均能产生F1和Pst I;vw+菌株占95.13%(391/411),VW-菌株占4.87%(20/411);Pgm+菌株占80.78%(332/411)、Pgm+菌株占9%(37/411)、Pgm-菌株占10.22%(42/411).220株代表株中96.82%(213/220)的菌株对小白鼠表现为强毒株,3.18%(7,220)为中等毒力,说明绝大多数具高致病性,其毒力很强.90.02%(307/341)菌株携带6×106 45×106 x65×106质粒.80株代表株包括8个基因组型,其中6个基因型与原分型相同,另有2个新的基因组型.结论 三江源地区鼠疫菌株具备青藏高原鼠疫病原体特性,人类一旦感染可导致发病急、病情重、传染性强、病死率高等特点.     

A procedure for monitoring the presence of the virulence plasmid (pYV) in Yersinia pestis under culture conditions

The pathogenicity of Yersinia pestis depends on the presence of a virulence plasmid (pYV). The unstable nature of pYV in Y. pestis leads to the eventual outgrowth of pYV-less cells due to its higher growth rate. Thus, it was necessary to develop procedures to monitor the presence of the plasmid during cultivation, storage, and laboratory manipulations. A procedure was developed to monitor the presence of pYV in Y. pestis by using low calcium response and Congo red binding techniques. The selection of pYV in the isolated clones was confirmed by polymerase chain reaction and by expression of pYV-associated phenotypes. Thus, using this procedure, low calcium response-Congo red-positive clones can be isolated for use in the development of growth models of virulent Y. pestis in food.     

河北省鼠疫自然疫源地鼠疫菌生物学特征的研究概述

为进一步研究河北省鼠疫疫源地的结构和性质,通过查阅大量资料,对该疫源地分离的鼠疫菌株的生物学特征做了总结,分别从生化特征、营养需求、质粒特征、毒力因子、毒力和毒素几方面进行介绍,发现河北省分离的鼠疫菌属于B群鄂尔多斯高原型,营养需求表现为对甘氨酸的半依赖和对色氨酸的营养依赖,含有4种质粒,其中13×106质粒是内蒙古长爪沙鼠疫源地所特有,有的菌株缺失45×106质粒,全部菌株表现为F1抗原和鼠疫菌素（PstⅠ）阳性,而鼠疫菌色素沉着（Pgm）和VW抗原阳性仅占一定比例,表现为遗传的不稳定性,中等毒力。     

Electrophoretic characterisation of the outer membrane proteins of Yersinia pestis isolated in north-east Brazil

The outer membrane proteins of 38 Yersinia pestis isolates from all known plague foci of north-east Brazil were analysed by SDS-PAGE. Approximately 20 bands were consistently found in all strains analysed and 11 were selected for comparative studies. Although qualitative differences among the electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates were not observed, quantitative alterations were clearly noted for most of these proteins. No particular quantitative alteration of the electrophoretic profile of outer membrane proteins could be associated with the period of isolation and geographic origin of the isolates. The 64 kDa outer membrane protein was significantly expressed in higher amounts among Y. pestis strains isolated from a recent plague outbreak. The possible use of electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates as a tool for epidemiological studies and for the analysis of virulence determinants is discussed.     

一起肺鼠疫暴发流行的病原学研究及流行病学分析

[目的]研究一起人间肺鼠疫暴发流行的鼠疫菌分离株其生物学特性、基因特征等。拟从分子流行病学的角度分析传染源和传播关系。[方法]对8株于鼠疫患者(尸体)分离的鼠疫菌进行糖醇类酵解试验;检测其毒力、毒力因子、质粒、生物型、基因型;运用分子流行病学方法分析本次流行的传染源。[结果]8株鼠疫菌全部为甘油+、阿胶糖+、脱氮+,生物型为古典型。具备强毒鼠疫菌的4个毒力决定因子,为FraI+、VW+、PstI+、Pgm+;对小白鼠的最小致死量介于50～1000个菌,为强毒株;具备我国多数鼠疫菌所携带的6、45和65Mdal质粒;基因组型全部为10型。[结论]根据分子流行病学和现场流行病学调查结果证实,此次肺鼠疫暴发流行来自同一个传染源。     

利用插入序列IS285对云南玉龙鼠疫耶尔森菌进行初步分析

目的了解新发现的玉龙鼠疫菌的遗传特征，分析其与中国各型鼠疫菌的亲缘天系。方法根据鼠疫菌C092株基因组DNA中的6个IS285位点设计引物，利用聚合酶链反应（PCR）对五龙鼠疫菌及我国其他各型鼠疫菌进行分析，通过SPSS软件对结果进行聚类。结果与CO92相比，我国鼠疫菌的某些IS285位点存在变异。5株玉龙鼠疫菌中有4株结果一致，另1株的一个位点发生变异。通过6个位点的PCR，可以将我国鼠疫菌聚为8类。所研究的5株玉龙鼠疫菌分别属于2种聚类，其中4株与滇西纵谷型、滇闽居民型及大部分灰旱獭菌株、全部喜马拉雅旱獭菌株聚为一类。结论对玉龙鼠疫菌的遗传特征获得初步了解，提示玉龙菌株在我国鼠疫菌传播演化过程中可能具有较重要地位．     

Role of immune response in Yersinia pestis infection

Yersinia pestis (Y. Pestis) is an infamous pathogen causing plague pandemics throughout history and is a selected agent of bioterrorism threatening public health. Y. pestis was first isolated by Alexandre Yersin in 1894 in Hong Kong and in the following years from all continents. Plague is enzootic in different rodents and their fleas in Africa, North and South America, and Asia, including the Middle/Far East and ex-USSR countries. Comprehending the multifaceted interaction between Y. pestis and the host immune system will enable us to design more effective vaccines. Innate immune response and both components (humoral and cellular) of adaptive immune response contribute to host defense against Y. pestis infection, but the bacterium possesses different mechanisms to counteract the immune response. The review aims to analyze the role of immune response versus Yersinia pestis infection and to highlight the various stratagems adopted by Y. pestis to escape the immunological defenses.     

Identification of encapsulated and non-encapsulated Yersinia pestis by immunofluorescence tests using polyclonal and monoclonal antibodies

Rabbit polyclonal hyperimmune antibodies to Yersinia pestis, and a mouse monoclonal antibody against the capsular antigen fraction 1 (F1) were compared in immunofluorescence (IF) tests. Fluorescent antibody conjugates were prepared from polyclonal antisera to four F1 positive Y. pestis strains; the conjugated antibody to strain A1122 gave the strongest IF staining of F1 positive and F1 negative Y. pestis strains. Indirect assays were rejected in favour of direct assays utilizing polyclonal and monoclonal reagents because the increased background staining reduced the effective contrast of bacterial visualisation. Polyclonal conjugates gave fairly homogeneous staining of Y. pestis bacterial populations, but in monoclonal assays a skew distribution of fluorescence intensity was observed, the majority of bacteria being poorly stained. The proportion of cells stained well by the monoclonal sufficed for easy identification of Y. pestis of the F1 positive phenotype however, and staining was not affected by washing the bacteria or treating them with formaldehyde. Y. pestis strains of the F1 positive genotype reacted with the monoclonal if bacteria were grown at 37 degrees C but not if the growth temperature was reduced to 25 degrees C thus preventing capsule production. The polyclonal conjugate reacted with bacteria of these strains that had been grown at either temperature. Strains of F1 negative genotype grown at either temperature reacted with the polyclonal conjugate but not with the monoclonal. Cross reactions between the polyclonal reagents and Y. enterocolitica biovar 2, serovar O 8 could not be removed by selective absorption; however, the monoclonal antibody gave no cross reaction. The F1 phenotypic status of bacterial preparations was verified by ELISA measurement of the fraction 1 antigen concentration. Antigen levels for F1 positive and F1 negative phenotypes differed by about three logs for suspensions of Y. pestis harvested from solid media. The polyclonal and monoclonal direct IF tests applied to spleen and blood smears of laboratory mice infected with Y. pestis were able to differentiate between lethal infection with an F1 positive strain carrying all four classical virulence determinants, an F1 positive vaccine strain, and an F1 negative strain.     

鼠疫耶尔森菌在自然疫源地保存机制的研究进展

通过阐明鼠疫的起源,分析鼠疫耶尔森菌（鼠疫菌）在鼠疫自然疫源地内的保存学说,描述动物间鼠疫流行特征和20世纪70年代的灭鼠拔源工作带给我们的启示,揭示鼠疫菌在自然界保存机制的研究进展情况,提出在鼠疫流行间歇期内,鼠疫菌主要以基因突变的方式长期保存在自然界中,形成鼠疫菌-宿主-媒介三位一体的鼠疫生态系统的新学说,为我国鼠疫的防治提供科学的参考依据。     

Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague

The current human plague vaccine, a killed Yersinia pestis whole-cell preparation, does not protect against aerosol challenge and is reactogenic and antigenically undefined. Live attenuated Y. pestis, such as pigmentation-deficient (Pgm-) strains, have been used frequently as vaccines and are efficacious. They are used widely in plague research and assumed to be safe. However, they can cause serious adverse reactions, and their aerosol infectivity is not known. We tested the virulence of a defined Pgm- variant of the C092 strain of Y. pestis in mouse and non-human primate models of pneumonic plague. The ten-fold lower median lethal dose by the aerosol compared to the subcutaneous (s.c.) routes of the Pgm- strain in mice suggested that the Pgm- strain might be less attenuated by the former than by the latter route. After exposure of 16 African green monkeys to inhaled doses ranging from 1.1 x 10(4) to 8.1 x 10(7)cfu, eight died and eight survived. The terminal cultures collected from five of the non-survivors were all positive for Y. pestis. Two of the remaining three non-survivors were culture-negative but had pathologic and immunologic evidence of infection with Y. pestis, specimens could not be obtained nor the cause of death determined for the third one. The deaths were not dose-related, and there were some differences in the pathology associated with infection by the Pgm- strain compared to the wild-type (wt) strain. However, the Pgm- derivative was clearly virulent for monkeys by the aerosol route. A mutant of the Pgm- strain, which has a deletion in the plasminogen activator (Pla) virulence locus (pla), appeared to be more attenuated than was either the Pgm- single mutant (in NHPs and mice) or the Pla- single mutant strain (in mice) and has potential as a live vaccine.