4种病原菌特异基因片段阳性蜱的16S rRNA基因克隆文库分析

Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, Bartonella henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus-or species-bacteria were detected in 103 valid sequences. Eight species were edge type (Clone No. > 5). Three kinds of pathogens were identified (Borrelia burgdorferi, Bartonella henselae and Rickettsia sp). Three kinds of pathogens were not edge type(Clone No. < 5). Coverage value was 96.11%, and Shannon-Wiener index was 2.40. Analysis results of cloning sequence showed that tick-parasitic bacteria mainly were α and γ deformation mycetes which accounted for 56.25% (9/16). Conclusions The 16S rRNA gene sequences technology could make relative quantitative of bacterial flora, and detect many kinds of pathogens in tick. It's a good method for detection of pathogens and bacterial flora analysis.     

蚤、蜱中巴尔通体的分离培养及检测鉴定

目的了解蚤、蜱在我国作为巴尔通体传播媒介的可能性，获得其自然状态下存在于吸血节肢动物中的证据。方法采集家猫、狗、牛及鼠类动物体表寄生蚤、蜱，采用含5％去纤维兔血脑心浸液（brain heart infusion，BHI）培养基置于35℃含5％ CO2培养箱中从蚤、蜱中分离巴尔通体，疑似菌落应用聚合酶链反应技术（Polymerase Chain Reaction，PCR）扩增巴尔通体特异基因片段，阳性产物测序并用同源性比较及系统发育分析等方法分析确定巴尔通体的亲缘关系。结果分别从猫栉首蚤、微小牛蜱中各分离出1株巴尔通体，用5对巴尔通体属特异性引物进行PCR反应，扩增产物均产生阳性目标带，证实为巴尔通体属微生物。序列同源性比较发现蚤、蜱分离株之间同源性较小，tRNA^Ile-tRNA^Ala地基因间隔区序列为58．2％、ftsZ基因序列为72．8％；这两株菌分别与云南鼠类宿主血液中分离的巴尔通体菌株RT222SM、RT221SM同源性最大，与其它已知巴尔通体同源性均较小。蚤培养物与RT222SM的tRNA^Ile-tRNA^Ala基因间隔区的同源性是77．9％、与ftsZ是96．2％；蜱培养物与RT221SM的tRNA^Ile-tRNA^Ala基因间隔区的同源性是99．4％，与云南鼠血中分离的Rf1561yn的gltA基因338bp核酸序列同源性为99．1％，基于gltA种系发育分析提示与Rfl561yn亲缘关系最近。结论从猫栉首蚤、微小牛蜱中分离培养出巴尔通体，为蚤、蜱作为巴尔通体传播媒介提供了线索，同时为在我国进一步开展媒介生物中巴尔通体感染情况调查提供了实验方法的参考依据。同源性搜索、比较及系统发育分析支持这2株巴尔通体与中国云南分离的巴尔通体基因型相似，而与国外的巴尔通体分离株亲缘关系较远。     

用PCR方法检出蚤类携带巴尔通体

目的调查我国巴尔通体宿主动物体表寄生蚤类是否携带巴尔通体。方法2003年6～7月在云南省大理州云龙县居民区采集家猫、狗、鼠类等体表寄生蚤，用3对巴尔通体属特异性引物BhCS．781p—BhCS．1137n、Bh．311p—Bh．452n和TI1e．455p—TA1a．885n进行聚合酶链反应(PCR)，扩增巴尔通体gltA和16S～23S rRNA ITS中部分核酸片段，检测采集的蚤类是否感染巴尔通体。结果共采集251只寄生蚤，包括猫栉首蚤、人蚤、缓慢细蚤等7个常见蚤种，从1组猫栉首蚤和1组缓慢细蚤中扩增出目标带，证实有巴尔通体感染。结论猫栉首蚤和缓慢细蚤能够感染巴尔通体，是该种病原体的潜在传播媒介，间接表明当地家猫和鼠类动物存在巴尔通体感染。     

一次不明原因发热疫情中病人血清的Q热抗体检测

目的:从不明原因发热病人血清中筛查Q热抗体.方法:用间接免疫荧光法对一次不明原因发热疫情中的9位病人共15份血清进行Q热IgG检测.结果:9例患者的血清中共检出6例Q热抗体阳性,阳性率达66.7%.结论:Q热在过去的疫区可能仍有较频繁的发生,各地相关部门应加强对Q热的调查和应急储备.     

云南玉龙及古城区鼠疫自然疫源地判定及初步研究

目的确定云南玉龙县及古城区鼠疫自然疫源地的存在,提出防控策略。方法采用常规调查方法,通过血清学、细菌学、基因诊断等技术进行判定。结果共分离到5株鼠疫杆菌,均酵解甘油、麦芽糖、阿胶糖,不酵解鼠李糖、密二糖,脱氮试验阳性。均带有Pgm 、PstI 、FI 、Vwa 等毒力决定因子;疫区犬血清阳性率为23.5%;猫血清阳性率为26.7%。结论细菌学证实玉龙县为鼠疫自然疫源地,目前鼠疫流行处于活跃期;血清学证实古城区为新的鼠疫疫源区(县);阳性血清动物分布面积210km2,并有进一步扩散趋势;齐氏姬鼠及大绒鼠是本地区优势鼠种,玉龙绒鼠有局部分布。特新蚤指明亚种,方叶栉眼蚤为优势蚤种;分离的鼠疫菌与滇西纵谷型及滇闽居民区型菌株生化特性不同,与西藏北部青藏高原型鼠疫菌的生化特性接近。     

4种病原菌特异基因片段阳性蜱的16S rRNA基因克隆文库分析

Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, Bartonella henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus-or species-bacteria were detected in 103 valid sequences. Eight species were edge type (Clone No. > 5). Three kinds of pathogens were identified (Borrelia burgdorferi, Bartonella henselae and Rickettsia sp). Three kinds of pathogens were not edge type(Clone No. < 5). Coverage value was 96.11%, and Shannon-Wiener index was 2.40. Analysis results of cloning sequence showed that tick-parasitic bacteria mainly were α and γ deformation mycetes which accounted for 56.25% (9/16). Conclusions The 16S rRNA gene sequences technology could make relative quantitative of bacterial flora, and detect many kinds of pathogens in tick. It's a good method for detection of pathogens and bacterial flora analysis.     

中国鼠疫监测的现状与发展

人们希望掌握疾病流行的规律,以便采取正确的控制措施,减轻人类所受到的疾病危害。只有通过疾病监测才能达到这样的目的。     

鼠疫形势与我国鼠疫控制策略

鼠疫在全球范围内进入一个新的活跃时期,中国也出现同样的形势。在过去的20年里,人问鼠疫发病率上升,在逐步扩大的鼠疫疫源地中越来越频繁地发生动物问鼠疫,新的鼠疫自然疫源地类型陆续发现。加上交通的改善以及恐怖主义兴起,鼠疫对人类构成更大的威胁。在这种新形势下需要建立新的国家鼠疫控制策略,这一策略由4部分组成：①需要改善应急反应系统,通过引人新技术缩短鼠疫诊断时间,并将控制措施与疫源地调查与监测联系起来;②需要加强阻断鼠疫由动物向人的传播环节,在我国西部的旱獭鼠疫地区,这一措施不应为灭蚤而应为禁猎;③在不同类型的鼠疫疫源地中应当采取不同的疫源地干预措施,例如,在家鼠鼠疫地区应当降低鼠类密度,而在旱獭鼠疫地区则应当发展獭洞灭蚤技术;④生态改造应为最根本的鼠疫控制措施,在我国灌溉和造林起最重要的作用。应当将这些措施列入国家的生态恢复规划。鼠疫也是地球生态系统的一个组成部分,新的鼠疫控制策略应当给予生态平衡更多的考虑。     

我国秋季型恙虫病地方特点及流行状况分析

恙虫病是由恙虫病东方体引起的自然疫源性疾病,根据其流行的季节特征大致分为夏季型、秋季型和冬季型3种类型,各种类型的恙虫病流行特征存在较大的差异。本文根据我国文献报道秋季型恙虫病疫情资料进行分析,归纳总结了我国秋季恙虫病的一些主要的流行特征,为制定秋季型恙虫病预防控制措施提供参考和依据。     

用多引物聚合酶链反应检测鼠疫耶尔森菌

目的建立多引物检测鼠疫耶尔森菌（鼠疫菌）的PCR（M-PCR）方法.方法合成4对引物,分别来源于质粒和染色体DNA上F1、pla、Hms、Inv基因,对164株鼠疫菌进行扩增.结果在164株鼠疫菌中,有152株菌4种基因扩增均阳性,仅云南省12株Hms基因扩增为阴性.结论采用M-PCR方法检测鼠疫菌DNA具有较好的敏感性、特异性和稳定性,其可作为检测与鉴别鼠疫菌和疫情监测方面快速诊断的方法.