青藏铁路沿线鼠疫菌外膜蛋白种类的研究

目的研究并分析青藏铁路沿线鼠疫菌外膜蛋白种类及相对分子质量。方法鼠疫菌经破碎后采取超高速离心法提取外膜蛋白，利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）系统分离鼠疫菌外膜蛋白。结果在10％的SDS-PAGE上，37℃条件下的培养物可表达26条外膜蛋白带，28℃培养物可表达36条外膜蛋白。结论试验用鼠疫菌株可分泌标准的外膜蛋白，属YOP1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。     

Genotyping, Orientalis-like Yersinia pestis, and plague pandemics

Three pandemics have been attributed to plague in the last 1,500 years. Yersinia pestis caused the third, and its DNA was found in human remains from the second. The Antiqua biovar of Y. pestis may have caused the first pandemic; the other two biovars, Medievalis and Orientalis, may have caused the second and third pandemics, respectively. To test this hypothesis, we designed an original genotyping system based on intergenic spacer sequencing called multiple spacer typing (MST). We found that MST differentiated every biovar in a collection of 36 Y. pestis isolates representative of the three biovars. When MST was applied to dental pulp collected from remains of eight persons who likely died in the first and second pandemics, this system identified original sequences that matched those of Y. pestis Orientalis. These data indicate that Y. pestis caused cases of Justinian plague. The two historical plague pandemics were likely caused by Orientalis-like strains.     

Relative immunogenicity and protection potential of candidate Yersinia Pestis antigens against lethal mucosal plague challenge in Balb/C mice

Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine.     

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.     

随机扩增多态性DNA技术用于鼠疫耶尔森氏菌基因分型的研究

目的 对来自全国不同疫源地、不同生态型的１０３株鼠疫耶尔森氏菌进行基因分型研究。方法 用随机扩增多态性ＤＮＡ技术（ＲＡＰＤ）。结果 将鼠疫耶尔森氏菌分成两种基因型别：全国大部分菌株为ＲＡＰＤ－１型,青海省境内的大部分菌株为ＲＡＰＤ－２型。结论 不同生态型的鼠疫耶尔森氏菌基因结构存在差异,为鼠疫耶尔森氏菌的基础研究和防治提供依据。     

四川省132株鼠疫耶尔森菌差异区段基因分型及地理分布研究

目的 分析四川省鼠疫耶尔森菌（Y. pestis）差异区段分型（DFR）及地理分布,为四川鼠疫研究提供科学依据。方法 对分离自四川省鼠疫疫区的132株鼠疫菌,应用23个DFR和PMT1的扩增引物,对被试菌株DNA进行PCR扩增,并使用Mapinfo软件和Excel进行分型,确定四川省鼠疫耶尔森菌DFR基因型和地理分布特征。结果 四川省鼠疫耶尔森菌DFR分型分为3型,其中石渠县有两型,主要为G14型和G43型,其它疫源地为G05型。结论 四川省鼠疫耶尔森菌的DFR分型具有明显的地理分布特征。     

Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis

Plasmid-mediated high-level resistance to multiple antibiotics was reported in a clinical isolate of Yersinia pestis in Madagascar in 1997. We describe a second Y. pestis strain with high-level resistance to streptomycin, isolated from a human case of bubonic plague in Madagascar. The resistance determinants were carried by a self-transferable plasmid that could conjugate at high frequencies to other Y. pestis isolates. The plasmid and the host bacterium were different from those previously associated with multiple-drug resistance, indicating that acquisition of resistance plasmids is occurring in this bacterial species. Emergence of resistance to streptomycin in Y. pestis represents a critical public health problem since this antibiotic is used as the first-line treatment against plague in many countries.     

云南省自然感染鼠疫媒介生物的研究

目的掌握云南省家、野鼠两型鼠疫疫源地自然感染鼠疫媒介生物的地理分布及季节变化。方法整理核实我所1974—2006年有关鼠疫监测和调查资料，并通过SPSS13．0统计软件进行处理和分析。结果家鼠鼠疫疫源地自然感染鼠疫节肢动物有印鼠客蚤、缓慢细蚤、人蚤等8种，其中印鼠客蚤为该疫源地的主要媒介，1982—2006年云南省自印鼠客蚤累计分离鼠疫菌618株，占92．65％；野鼠鼠疫疫源地自然感染鼠疫的动物有特新蚤指名亚种、方叶栉眼蚤等7种，其中特新蚤指名亚种为主要媒介，最近在玉龙县发现其染疫蚤；棕形额蚤为次要媒介，1974—2006年自特新蚤指名亚种和棕形额蚤各分离鼠疫菌14株，各占15．56％。从家鼠鼠疫疫源地媒介生物中检出的鼠疫菌集中在6—8月，最高为8月。野鼠疫源地媒介昆虫季节变化调查和监测结果基本一致，呈双峰，集中于4月和12月。结论两型疫源地有着各自不同的染疫媒介，其地理分布有着明显区别，染疫情况和季节变化也有着相对的独立性。     

青藏铁路沿线鼠疫菌表型性状及对抗生素敏感性的研究

目的检测青藏铁路沿线鼠疫菌表型性状、质粒组成、外膜蛋白种类及对抗生素的敏感性。方法采用常规糖醇发酵管法检测生化等表型性状;采用反向血凝试验、用假结核菌为复盖种子层法、用草酸镁培养基和刚果红色素培养基检测FraI、Pstl、Vwa和Pgm4种毒力因子;用Lawton最小培养基测定营养型;采用常规的动物接种试验测定毒力;采用碱裂解、酚一氯仿抽提法提取鼠疫菌质粒;鼠疫菌经破碎后采取超高速离心法提取外膜蛋白;采用纸片法进行抗生素敏感性试验。结果实验用的青藏铁路沿线鼠疫菌株生化性状表现为甘油＋、鼠李糖一、阿胶糖＋、密二糖一、麦芽糖＋、脱氮＋的特征;具有鼠疫菌荚膜抗原（FraI＋）、毒力抗原（Vwa＋）、鼠疫菌素I（PstI＋）、色素沉着因子（Pgm＋）4种毒力因子;营养型表现为对Trp、Thr、Leu、Arg不依赖,而对Phe、Met、Cys依赖,对Ile和Glu表现出低营养;对小白鼠的LD100在10^2～10^3个菌,LDs。为7．94-31．62个菌,对豚鼠的LD100在10^5～10^7个菌,LD50为68．14-685．49个菌;携带Mr为6×10^6、45×10^6、52×10^6、65×10^6、92×10^6]质粒;在10％的SDS-PAGE上37℃条件下的培养物可表达26条外膜蛋白带,28℃培养物可表达36条外膜蛋白;部分头孢类和喹诺酮类药物对实验菌株的敏感性效果较好,20种抗生素均较链霉素为佳。结论青藏铁路沿线的鼠疫菌属于典型的青藏高原型菌株,都是强毒力鼠疫菌,临床治疗除常规的链霉素外可选用头孢类和喹诺酮类药物,该项研究可为青藏铁路沿线的鼠疫防治提供科学的依据。     

云南省鼠疫菌质粒特征及地理分布

目的探讨云南省鼠疫菌质粒分子流行病学意义.方法按Kado和Liu法提取云南省44个县市及中缅边境一带共1020株鼠疫菌的质粒,经琼脂糖凝胶电泳进行检测分析.结果云南省鼠疫菌具有相对分子质量(Mr)分别约3.93、6.05、22.97、35.65、45.35、64.82、74.59、111.36和129.55×106的9种质粒,其中3.93、35.65和111.36×106的3种质粒为云南省鼠疫菌特有.按质粒组成可分为10种不同类型的质粒谱,其中Ⅰ～Ⅴ种类型较为常见,且在分布上具有明显的地区集中现象.结论根据鼠疫菌质粒特征,可将云南省自1982年以来发生鼠疫流行的地区划分为多片相对独立的疫源地.认为80年代重新开始的家鼠鼠疫流行,可能来源于多片潜伏的鼠疫自然疫源地的复燃.     

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.     

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。     

青海省2004年人间鼠疫分离株基因分型及流行病学意义

目的探讨鼠疫耶尔森菌（鼠疫菌）基因分型在鼠疫暴发中的流行病学意义.方法根据已经证实的23个差异区段设计引物,对2004年青海省人间鼠疫流行期间分离到的13株鼠疫菌进行PCR扩增.结果13株鼠疫菌可分为4个基因组型,即Genomovar 8、10、15和16.其中囊谦的8株菌全部为Genomovar 10;乌兰的2株菌分别为Genomovar 8和15;祁连、曲麻莱、称多的3株鼠疫菌的基因组型均为Genomovar 16.结论鼠疫菌基因分型是鼠疫疫情暴发流行病学调查的有力工具.     

Epidemiologic features of four successive annual outbreaks of bubonic plague in Mahajanga, Madagascar

From 1995 to 1998, outbreaks of bubonic plague occurred annually in the coastal city of Mahajanga, Madagascar. A total of 1,702 clinically suspected cases of bubonic plague were reported, including 515 laboratory confirmed by Yersinia pestis isolation (297), enzyme-linked immunosorbent assay, or both. Incidence was higher in males and young persons. Most buboes were inguinal, but children had a higher frequency of cervical or axillary buboes. Among laboratory-confirmed hospitalized patients, the case-fatality rate was 7.9%, although all Y. pestis isolates were sensitive to streptomycin, the recommended antibiotic. In this tropical city, plague outbreaks occur during the dry and cool season. Most cases are concentrated in the same crowded and unsanitary districts, a result of close contact among humans, rats, and shrews. Plague remains an important public health problem in Madagascar, and the potential is substantial for spread to other coastal cities and abroad.     

用TaqMan探针实时定量PCR法快速检测鼠疫耶尔森菌

目的建立一种快速、准确、特异的定量检测鼠疫耶尔森菌的方法。方法针对鼠疫耶尔森菌特异的染色体标识序列设计引物和TaqMan探针，进行实时定量聚合酶链反应(polymerase chain reaction，PCR)分析，优化反应体系，检测其灵敏度、特异性和重复性。结果以EV76灭活培养物为对象，检测灵敏度可达每反应体系0．08CfU；30株非鼠疫菌株的检测结果表明，对照菌株均为阴性，检测特异性良好；对同一样品进行17次重复检测，其循环阈值的标准偏差为0．35。结论TaqMan探针法能够灵敏、特异、稳定地对鼠疫耶尔森氏菌进行定量检测，为鼠疫监控、诊断提供有力手段。     

青海高原藏系绵羊鼠疫菌系统发育关系和流行特征的研究

  目的  对青海高原藏系绵羊引发的人间鼠疫疫情流行病学和相关鼠疫菌遗传特征进行研究,为制定出适合青海省藏系绵羊鼠疫防控提供科学依据。  方法  汇总分析青海省1958―2020年藏系绵羊引发的人间鼠疫疫情资料,描述其时间、地区和人群分布及其他流行病学特征;对38株分离自人、藏系绵羊和喜马拉雅旱獭的鼠疫杆菌进行测序,再结合其他参考菌株的基因序列分析菌株的全基因组单核苷酸多态性特征,构建菌株之间的系统发育关系。  结果  1958―2020年共发生藏羊为疫情传染源的人间鼠疫疫情16起,发病69人,死亡42人,病死率60.86%。与藏系绵羊相关的鼠疫杆菌都位于系统发育树的同一进化分支1.IN2中,并具有明显的地域性特征。同一鼠疫事件中人、藏系绵羊和周边喜马拉雅旱獭分离菌株位于同一簇群,与流行病学调查资料相符。  结论  流行病学结合系统发育关系分析,证实了青海省人间鼠疫来自藏系绵羊,而藏系绵羊鼠疫则起源于旱獭。藏系绵羊是青海高原上重要的家畜,因此不应低估藏系绵羊鼠疫的危害。     

云南省鹤庆县2017年分离鼠疫菌分子溯源

目的 了解2017年云南省鹤庆县新分离的鼠疫菌的基因分型,为该地的鼠疫防控提供科学依据。方法 采用差异片段（DFR）、规律成簇的间隔短回文重复序列（CRISPRs）和多位点可变数目串联重复序列分析（MLVA）3种方法对10株鹤庆县新分离鼠疫菌进行分型,并将10株鼠疫菌及邻近疫源地鼠疫菌株93株纳入聚类分析。结果 鹤庆鼠疫菌株与丽江鼠疫疫源地菌株具有相同的DFR型（Genomovar 05型）及CRISPRs型（Ca7簇,22型）,在MLVA聚类分析中,鹤庆鼠疫菌株与丽江野鼠鼠疫菌株位于同一个簇,两者之间仅有2个位点（N2117,M23）的差异。结论 2017年云南省鹤庆鼠疫菌株与丽江野鼠鼠疫疫源地菌株具有很高的同源性,鹤庆县疫情可能是丽江鼠疫进一步向南扩散的结果。     

Antibodies and cytokines independently protect against pneumonic plague

Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFalpha) and gamma-interferon (IFNgamma) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFalpha and IFNgamma confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFalpha and IFNgamma upon exposure to Y. pestis.     

Developing live vaccines against plague

Three great plague pandemics caused by the gram-negative bacterium Yersinia pestis have killed nearly 200 million people and it has been linked to biowarfare in the past. Plague is endemic in many parts of the world. In addition, the risk of plague as a bioweapon has prompted increased research to develop plague vaccines against this disease. Injectable subunit vaccines are being developed in the United States and United Kingdom. However, the live attenuated Y. pestis-EV NIIEG strain has been used as a vaccine for more than 70 years in the former Soviet Union and in some parts of Asia and provides a high degree of efficacy against plague. This vaccine has not gained general acceptance because of safety concerns. In recent years, modern molecular biological techniques have been applied to Y. pestis to construct strains with specific defined mutations designed to create safe, immunogenic vaccines with potential for use in humans and as bait vaccines to reduce the load of Y. pestis in the environment. In addition, a number of live, vectored vaccines have been reported using attenuated viral vectors or attenuated Salmonella strains to deliver plague antigens. Here we summarize the progress of live attenuated vaccines against plagu.