用PCR/RFLP技术对黑龙江沿岸蜱类和鼠类中北亚蜱传斑点热的检测

为了解黑龙江沿岸部分地区斑点热自然疫源地存在情况，作者用PCR／RFLP的方法检测该地区蜱类及鼠类中的斑点热群（spottedfevergroup，SFG）立克次体。结果从该地区的森林革蜱、嗜群血蜱、黑线姬鼠、东方田鼠、麝鼠及棕背中均扩增出了斑点热群立克次体的特异片段，而对斑疹伤寒立克次体、恙虫病立克欢体、Q热立克次体则为阴性。PCR产物经PstⅠ及RsaⅠ酶切后发现它们的酶切图谱与西伯利亚立克次体完全相同，而有别于其它斑点热群立克次体标准株。作者认为黑龙江沿岸调查点可能存在北亚蜱传斑点热的自然疫源地。     

浙江省在蜱中检测到斑点热群——立克次体DNA片段

目的 了解蜱、鼠中自然感染斑点热群立克次体的状况。方法 用聚合酶链反应（PCR）检测鼠类脾脏和蜱类中的斑点热DNA片段。结果 在金华市金东区捕获社鼠2只，黄毛鼠10只，黑线姬鼠4只，用PCR检测结果均为阴性。用PCR检测49只中华硬蜱，阳性8只，阳性率16．33％。结论 初步认定浙江省存在斑点热病原，尚需进一步调查。     

东北部分地区蜱携带几种病原立克次体的分子检测

目的为了解东北地区蜱中携带立克次体的情况。方法应用人工小时布旗法采集东北部分地区不同生境的游离蜱,采用PCR作斑点热立克次体、查菲埃立克体、人粒细胞无形体DNA检测。结果从东北地区全沟硬蜱和森林革蜱检出查菲埃立克体DNA,全沟硬蜱平均阳性率为3.29%,森林革蜱为1.88%;从全沟硬蜱检出人粒细胞无形体DNA,平均阳性率为2.98%;从嗜群血蜱检出斑点热立克次体DNA,其平均阳性率(6.67%)明显高于全沟硬蜱(1.69%)和森林革蜱(2.36%)。结论东北地区全沟硬蜱和森林革蜱可能是查菲埃立克体、人粒细胞无形体的主要携带媒介,嗜群血蜱主要携带斑点热立克次体,提示东北地区存在三种蜱媒立克次体病的自然疫源地。     

2005-2010年北京市立克次体病流行病学特征分析

立克次体(Rickettsia)是一类严格细胞内寄生的原核细胞型微生物,人类通过节肢动物人虱、鼠蚤、蜱或螨的叮咬而感染立克次体.近几年,恙虫病、人粒细胞无形体病等立克次体病在我国部分地区发病呈上升趋势,目前我国至少存在10余种立克次体病,其中包括流行性斑疹伤寒、地方性斑疹伤寒、恙虫病、北亚蜱传斑点热、黑龙江蜱传斑点热、内蒙古蜱传斑点热、Q热、人单核细胞埃立克体病、人粒细胞埃立克体病、人粒细胞无形体病,以及巴尔通体病等疾病[1-3].为了解北京市立克次体病的发病趋势及流行规律,为合理制订防控措施提供科学依据,笔者对2005 -2010年北京市恙虫病、斑疹伤寒和人粒细胞无形体病等3种立克次体病病例的流行病学特征进行分析,现报告如下.     

一组长角血蜱中检出莱姆病螺旋体和斑点热群立克次体复合感染

目的 了解浙江省山区野生动物和蜱中莱姆病、斑点热、埃立克体病(无形体病)的感染情况.方法 采用巢式PCR对采集的鼠、蜱标本进行莱姆病伯氏疏螺旋体、斑点热群立克次体、埃立克体(无形体)特异性核酸片段检测分析.结果 从121份鼠标本和105组蜱标本中检出阳性结果 14份.鼠标本中检出伯氏疏螺旋体5S～23S rDNA间隔区片段1份和埃立克体(无形体)16SrDNA 5'端片段2份.蜱标本中检出阳性11份,包括伯氏疏螺旋体5S～23S rDNA间隔区片段3份和斑点热群立克次体外膜蛋白OmpA基因5'端片段8份.其中1组长角血蜱成虫标本为伯氏疏螺旋体和斑点热群立克次体复合感染,5S～23S rRNA基因间隔区和ompA基因片段均阳性,分别与伯氏疏螺旋体法雷氏基因型和马赛立克次体株等关系较近.结论 在同一组长角血蜱成虫中同时检出莱姆病疏螺旋体和斑点热群立克次体复合感染.     

新疆尉犁荒漠地区蜱中主要蜱传疾病病原检测

目的 调查尉犁荒漠地区蜱的种群组成和分布及自然感染莱姆病、Q热、巴贝西原虫病、斑点热和人粒细胞无形体病病原体情况.方法 采用布旗法和动物体表搜集法采集蜱标本,PCR法进行病原检测.结果 在尉犁地区共采集蜱519只,隶属2属3种,分别为短小扇头蜱、图兰扇头蜱和亚洲璃眼蜱;短小扇头蜱为该地区的优势种,占捕获总数的88.25％.只在短小扇头蜱中检出2种蜱传疾病病原体,分别是斑点热群立克次体和无形体,检出率分别为6.27％和3.53％.结论 斑点热群立克次体和无形体在尉犁地区蜱中有不同程度的自然感染.     

中国东北地区远东蜱传斑点热流行病学调查

目的 了解远东蜱传斑点热在东北地区感染情况、分布及其流行规律.方法 选择东北三省9市(县)作为调查地区,采集正常人群血清、鼠血清、游离蜱及疑似患者血液等标本,用间接免疫荧光法检测蜱传斑点热抗体,豚鼠接种法分离立克次体.结果 调查地区总体人群蜱传斑点热检出率17.46%,除瑷辉县外其余8市(县)人群以黑龙江立克次体抗体为优势;人群当年蜱传斑点热感染率为9.4%,2年内蜱叮咬率林业工人为92.8%,军人为33.5%,抗体阳性率与蜱叮咬率呈正比关系;鼠种调查经鉴定为3科12种,以黑线姬鼠为优势种占58.6%,大林姬鼠占13.7%,其他鼠种占27.7%.鼠血清阳性率为27.3%,不同鼠种阳性率各有差异,黑线姬鼠为21.9%,大林姬鼠23.1%,棕背<鼠,平>33.3%,大仓鼠55.6%.从蜱及疑似病例血液标本中分离到黑龙江立克次体共20株,证实远东蜱传斑点热病例16例.结论 东北三省东部与北部普遍存在远东蜱传斑点热感染,存在蜱传斑点热的自然疫源地.     

2016—2019年内蒙古大兴安岭林区立克次体关联细菌群监测

目的了解内蒙古大兴安岭林区蜱中立克次体关联细菌群的流行情况和分子特征。方法 2016—2019年,在内蒙古东部林区采集游离蜱样本,利用形态学特征和18S rRNA分型法鉴定蜱种。提取蜱DNA,对斑点热立克次体(gltA和rOmpA)、无形体属(msp2/p44)和埃立克体属(omp1/p28)等特异性基因进行PCR扩增。扩增序列测序后进行同源性比对和系统进化分析。结果共采集成蜱3 251只,隶属于3属4种。PCR检测结果显示,斑点热立克次体阳性率为43.3%,扩增出的gltA序列与已注册Candidatus R. tarasevichiae(MN450397)和R.heilongjiangensis(AY285776)的同源性为100%,检出率分别为40.1%和3.1%。无形体msp2阳性率为8.1%,埃立克体omp1阳性率为7.0%。2016—2019年连续监测的结果显示,Candidatus R. tarasevichiae一直保持较高的检出率,为31.0%～46.6%,R.heilongjiangensis的检出率为2.6%～3.5%,无形体和埃立克体的阳性率分别为5.7%～10.6%、6.7%～7.9%。其中,全沟硬蜱Candidatus R. tarasevichiae的阳性率基本呈现上升的趋势,且感染率均高于其它蜱类。结论该地区全沟硬蜱为优势蜱种,Candidatus R. tarasevichiae和R.heilongjiangensis为优势菌种,且在该地区蜱中的高感染率,表明人类接触该菌的风险很高,需加强监控。     

2009年卢氏县人和家畜立克次体病血清流行病学调查

目的了解2009年卢氏县正常人及主要家畜(羊、牛、犬)立克次体血清流行病学特点。方法根据地理位置及农村牲畜饲养分布特点,选择官道口、范里、文峪、东明4个乡镇为调查点。以间接免疫荧光法检测人和家畜斑疹伤寒群立克次体、斑点热群立克次体、恙虫病东方体、贝氏苛克斯氏体、查菲埃立克体、人粒细胞无形体、巴尔通体、海南斑点热分离株等8种常见立克次体血清抗体。结果在4个调查点244份人血清和97份家畜血清中8种立克次体抗体均有检出。人群血清学检测阳性率最高为人粒细胞无形体(26.23%),最低为恙虫病东方体(0.82%);畜群感染率最高为海南斑点热分离株(90.72%),最低为贝氏苛克斯氏体(8.25%)。人群人粒细胞无形体感染率有明显地区间差异;羊的斑疹伤寒群立克次体和恙虫病东方体感染率,牛的巴尔通体感染率,犬的斑点热群立克次体感染率明显高于其它畜种。结论卢氏县人畜间广泛存在多种立克次体感染,局部地区高发、多发,应作进一步调查研究并加强防控。     

逊克县火山熔岩地区蜱携带新发蜱传病原体及复合感染调查

目的调查黑龙江省逊克县火山熔岩地区蜱携带新发蜱传病原体及复合感染情况。方法 2015年4-6月采集活蜱样本257只,利用PCR方法分别扩增6种新发蜱传病原体的特异性核酸片段,通过基因测序进一步鉴定。结果共检出斑点热群立克次体、巴尔通体和嗜吞噬细胞无形体核酸阳性样本分别为86、5和11例,感染率分别为33.46%、1.94%和4.28%。伯氏疏螺旋体、巴贝西虫和埃立克体的DNA核酸检测结果均为阴性。森林革蜱、嗜群血蜱和全沟硬蜱体内共检出复合感染8例,感染率为3.11%,证实该地区森林革蜱中存在斑点热群立克次体及巴尔通体,嗜群血蜱和全沟硬蜱存在斑点热群立克次体及嗜吞噬细胞无形体复合感染的情况。结论应加强黑龙江省逊克县火山熔岩地区蜱的防控。     

浙江省人和家畜蜱媒传染病血清流行病学调查

目的 了解浙江省农村人群与主要家畜蜱媒传染病病原体感染的情况.方法 按地理位置和蜱媒传染病病原体历史检测情况,选择浙西北丘陵区的安吉县、浙中丘陵盆地区的金东区和浙东盆地低山区的天台县为调查点,采集3个调查点农村人群和家畜血标本,以间接免疫荧光法检测黑龙江立克次体、恙虫病东方体、莫氏立克次体、嗜吞噬无形体、查菲埃立克体、巴尔通体、海南立克次体和贝纳柯克斯体8种常见蜱媒传染病病原体IgG抗体.结果 3个调查点共检测683份血清标本,包括579份人标本和104份家畜标本(牛53份、羊51份),不同地区恙虫病东方体、莫氏立克次体、查菲埃立克体、贝纳柯克斯体4种病原体抗体阳性率差异有统计学意义(P值均等于0.000).人血清标本8种病原体IgG抗体均检出,阳性率最高的分别是莫氏立克次体(20.7%)、巴尔通体(10.9%)和贝纳柯克斯体(5.5%),其中莫氏立克次体抗体阳性率随年龄增长而增加.家畜血清标本除嗜吞噬无形体外其余7种病原体IgG抗体均检出,阳性率最高的是莫氏立克次体(69.2%)、巴尔通体(51.0%)和海南立克次体(22.1%),不同家畜抗体阳性情况不同.结论 浙江省不同地区农村人群与家畜中广泛存在多种蜱媒传染病病原体的感染,特别是莫氏立克次体、巴尔通体、海南立克次体和贝纳柯克斯体抗体阳性率较高.     

Rickettsia sp. and Anaplasma spp. in Haemaphysalis longicornis from Shandong province of China,with evidence of a novel species “Candidatus Anaplasma Shandongensis”

Haemaphysalis longicornis is one of the most dominant and widespread tick species in China. This species mainly infests wild animals and occasionally attacks humans, and has been associated with the transmission of a variety of zoonotic pathogens including spotted fever group Rickettsia (SFGR), severe fever with thrombocytopenia syndrome virus (SFTSV), Anaplasma phagocytophilum, Babesia spp. and Theileria spp.. Although there are increasing reports of various pathogens associated with H. longicornis, some neglected pathogens in certain areas still need to be studied. In this study, a total of 171 H. longicornis ticks were collected from goats in three locations of Shandong Province, Eastern China (Zibo, Linyi, and Qingdao cities), and subsequently screened for the presence of Rickettsia, Anaplasma, and Ehrlichia bacteria. A total of four bacterial species were identified and characterized. “Candidatus Rickettsia jingxinensis” was detected in one tick specimen from Zibo city. Of 98 ticks from Linyi city, 63.27% (62/98) were tested positive for Anaplasma capra and 5.10% (5/98) were positive for Anaplasma bovis. Interestingly, a novel Anaplasma species was detected and characterized in one tick specimen from Zibo and one other from Linyi, respectively. Genetic and phylogenetic analysis based on the 16S, gltA, groEL, and msp4 genes indicated that it was divergent from all known Anaplasma species but mostly related to A. phagocytophilum and “Cadidatus Anaplasma boleense”. Based on where it was first detected, we named it “Candidatus Anaplasma shandongensis”.     

云南省保山市一起地方性斑疹伤寒暴发的调查

目的 了解云南省保山市一起地方性斑疹伤寒暴发的流行病学特征.方法 收集患者的流行病学资料.应用外斐反应和间接免疫荧光方法同时检测患者血清中莫氏立克次体、恙虫病东方体IgG抗体.用PCR检测鼠类脾脏标本中莫氏立克次体、普氏立克次体gltA基因,恙虫病东方体56kDa蛋白基因,斑点热群立克次体ompA基因,埃立克体16S rRNA基因和无形体16SrRNA基因.结果 2009年7-8月保山市隆阳区该起地方性斑疹伤寒累计发病58例,其中48例为临床诊断病例,10例为实验室诊断病例.3例地方性斑疹伤寒实验室诊断病例存在Karp型恙虫病东方体感染.PCR检测黄胸鼠脾脏85份,其中莫氏立克次体gltA片段阳性3份(阳性率为3.5%),其序列与莫氏立克次体Wilmington株(GenBank U59714.1)同源性为100%;普氏立克次体、恙虫病东方体、斑点热群立克次体、无形体和埃立克体均为阴性.结论 经流行病学、临床资料和实验室检测证实,保山市隆阳区该起疫情为地方性斑疹伤寒.从当地优势鼠种黄胸鼠中检测到莫氏立克次体核酸及序列,表明当地存在地方性斑疹伤寒疫源地.

Abstract：

Objective To understand the epidemiologic characteristics of endemic typhus in Baoshan city. Methods Epidemiological data were collected and characteristics were analyzed. IgG antibody(Ab) of Rickettsia mooseri and Orientia tsutsuganushi in serum of patients were tested using both Weil-Felix and IFA method. The Rickettsia mooseri gltA gene, Rickettsia prowazekii gltA gene,Orientia tsutsugamushi 56 kDa protein gene, SFGR ompA gene, Ehrlichia sp. 16S rRNA gene and Anaplasma sp. 16S rRNA gene in spleen of mice were examined by PCR. Results Fifty- eight endemic typhus cases were found in Longyang district of Baoshan city, during July to August, 2009.Among them, 48 cases were confirmed by clinical diagnosis and 10 cases by laboratory tests. The Ab of Orientia tsutsugamushi Karp serotype was detected in 3 cases from laboratory diagnosis. The spleen samples from 85 Rattns flavipectus were tested using PCR. Of them, 3 samples for Rickettsia mooseri gltA gene showed positive (positive rate was 3.5% ), and the homology of 3 Rickettsia mooseri and Rickettsia mooseri Wilmington strain (GenBank U59714.1) was 100% through comparing gene sequence. The results of PCR for detecting Rickettsia prowazekii, Orientia tsutsugamushi, SFGR,Anaplasma sp. and Ehrlichia. sp were all negative. Conclusion The outbreak of endemic typhus was confirmed in Longyang district of Baoshan city through epidemiological data, clinical diagnosis and laboratory tests. Rickettsia mooseri DNA was detected in the dominant Raw flavipectus, suggesting that endemic typhus did exist in the local areas.     

浙江省蜱标本中斑点热群立克次体rOmpA和gltA基因检测

目的了解浙江省部分山区蜱中感染斑点热群立克次体的状况,探讨以外膜蛋白A（rOmpA）和柠檬酸合成酶基因（gltA）为靶基因的PCR方法可行性。方法利用PCR方法,检测天台县左溪镇和临安县西天目地区共46组蜱类标本中rOmpA和gltA基因特异片段。对所检测到的阳性结果进行克隆与序列测定,并进行聚类分析。结果从46组蜱标本中检测发现2组长角血蜱中斑点热群立克次体的rOmpA和gltA基因片段均为阳性,核酸序列基本一致,但推测斑点热群立克次体种的进化位置存在差异。结论浙江省部分山区存在蜱中感染斑点热群立克次体的状况。利用rOmpA和gltA基因能从标本中检测到斑点热群立克次体,但从2个结果推测立克次体的分类位置存在差异。     

三种蜱媒传染病在媒介蜱和鼠类中复合感染的研究

目的 了解中国部分地区埃立克体病和其他蜱媒传染病病原体的复合感染情况。方法 运用聚合酶链反应方法对内蒙古自治区、黑龙江省、北京市的蜱和鼠类标本粒细胞埃立克体、莱姆病螺旋体、斑点热群立克次体的感染进行检测，对福建省的蜱和鼠脾脏标本中查菲埃立克体、斑点热立克次体及莱姆病螺旋体的感染情况进行检测。结果 内蒙古采集的全沟硬蜱408只，人粒细胞埃立克体感染率68％，莱姆病螺旋体感染率7．8％，斑点热群立克次体感染率45．6％，其中粒细胞埃立克体与莱姆病螺旋体复合感染标本5份(5／408)，粒细胞埃立克体与斑点热群立克次体复合感染标本1份(1／408)。黑龙江采集的46只全沟硬蜱中人粒细胞埃立克体感染率6．5％，莱姆病螺旋体感染率10．8％，斑点热群立克次体感染率34．8％，其中粒细胞埃立克体与莱姆病螺旋体复合感染标本1份(1／46)。北京市采集的蜱类标本922只，有2只检测到莱姆病螺旋体的DNA片段。福建省宁化地区采集的优势蜱种越原血蜱283组(659只)中，查菲埃立克体最小阳性率为3．8％(25组阳性)，38份野鼠脾脏的阳性率为564％(21只阳性)，检测出有2只(2／659)蜱同时感染了粒细胞埃立克体和斑点热群立克次体，野鼠的脾脏标本中，也发现有一只同时感染了查菲埃立克体和斑点热立克次体。结论 人粒细胞埃立克体和莱姆病螺旋体复合感染以及查菲埃立克体和斑点热群立克次体复合感染可能在特定地区发生；其地区的蜱类、鼠类存在着极低的复合感染。     

Antigenic relationships between the typhus and spotted fever groups of rickettsiae.

Paired sera from cases of epidemic typhus in Ethiopia and from probable cases of Rocky Mountain spotted fever (RMSF) in the United States were examined by microagglutination (MA) and microimmunofluorescence (micro-IF) tests for antibodies against Rickettsia prowazekii, Rickettsia typhi, Rickettsia canada, Rickettsia rickettsii, Rickettsia conorii and Ricksettsia akari. IgG and IgM antibodies against the various rickettsiae were titrated with specific fluorescein-conjugated anti-IgG and anti-IgM sera. Purified, particulate rickettsial antigens were employed in all tests. A majority of patients acutely ill with epidemic typhus produced both IgG and IgM antibodies against R. prowazekii, R. typhi and R. canada. Concurrently they produced IgG (but seldom IgM) antibodies against members of the spotted fever group. In contrast, patients ill with probably spotted fever, while producing IgG and IgM antibodies against R. rickettsii, R. conorii and R. akari, also produced both IgG and IgM antibodies with about equal frequency against members of the typhus group. It was concluded that a relatively broad antigenic relationship exists between rickettsiae of the typhus and spotted fever groups.     

广东省斑点热群立克次体自然疫源地调查

目的　了解广东省是否存在斑点热自然疫源地。方法　用血清流行病学方法调查广东省人群、宿主动物感染状况 ;用聚合酶链反应初筛 ,鸡胚卵黄囊培养法直接从标本中分离立克次体 ;用血清学试验、序列测定对分离株进行鉴定。结果　检测 86 0份自然人群及 32 1份鼠类血清标本 ,发现健康人群平均阳性率为 3.84 % ,各调查点之间的阳性率差异有显著性 (χ2 =6 0 2 .39,df =8,P <0 .0 1) ,山区和平原阳性率差异无显著性 (χ2 =0 .32 ,df =1,P >0 .0 5 ) ;鼠类阳性率为 4 .6 7% ,针毛鼠、白腹巨鼠、板齿鼠的阳性率分别为 11.5 9%、12 .90 %和 3.13% ;在针毛鼠、白腹巨鼠、板齿鼠发现斑点热自然感染抗体是国内首次报道 ;采集鼠脾标本 32 1份 ,未分离出菌株 ;自鼠体表采集到蜱 394匹 ,从2只针毛鼠体表采集的蜱中分离到 2株斑点热群立克次体 ,命名为GDFK5 8 2 0 0 0株、GDFK5 9 2 0 0 0株 ,经血清学鉴定为西伯利亚种 ,对GDFK5 8 2 0 0 0和GDFK5 9 2 0 0 0的OmpA基因起始部位 5 33bp片段进行克隆和测序 ,测序结果与Genbank中其他斑点热群立克次体相应基因片段的核苷酸进行比较 ,GDFK5 8 2 0 0 0、GDFK5 9 2 0 0 0和西伯利亚立克次体之间的核苷酸同源性为 99.6 %～ 10 0 % ,推断氨基酸同源性为 10 0 %。结论　从宿主动     

Rickettsial infections and fever, Vientiane, Laos

Rickettsial diseases have not been described previously from Laos, but in a prospective study, acute rickettsial infection was identified as the cause of fever in 115 (27%) of 427 adults with negative blood cultures admitted to Mahosot Hospital in Vientiane, Laos. The organisms identified by serologic analysis were Orientia tsutsugamushi (14.8%), Rickettsia typhi (9.6%), and spotted fever group rickettsia (2.6% [8 R. helvetica, 1 R. felis, 1 R. conorii subsp. indica, and 1 Rickettsia "AT1"]). Patients with murine typhus had a lower frequency of peripheral lymphadenopathy than those with scrub typhus (3% vs. 46%, p<0.001). Rickettsioses are an underrecognized cause of undifferentiated febrile illnesses among adults in Laos. This finding has implications for the local empiric treatment of fever.     

Serosurvey of wild rodents for Rickettsioses (spotted fever, murine typhus and Q fever) in Java Island, Indonesia

The prevalence of antibodies against spotted fever group rickettsia (SFGR), murine typhus and Q fever were investigated in wild rats captured in Indonesia. Sera of 327 rats were collected from Jakarta and Boyolali on Java Island. The prevalences of antibodies against SFGR and murine typhus were 128 (39.1%) and 48 (14.7%), respectively. Antibodies against Q fever were not detected in these serum samples. Antibodies against SFGR were found in all species of rats (20.8–51.9%). The antibody positive rate against murine typhus in Rattus norvegicus (38.0%) was significantly higher than that in other rat species (0–4.8%, p < 0.01). The antibody positive rates against SFGR and murine typhus in rats captured in Jakarta were significantly higher than those in rats captured in Boyolali (p < 0.01). In this survey, all species of rats had antibodies against SFGR, indicating that the 4 species of tested rats (R. norvegicus, R. rattus, R. exulans, R. tiomanicus) were infected with SFGR and that SFGR may infest the whole of Java Island. Most of the rats that were antibody-positive against murine typhus were captured in Jakarta. Therefore, R. norvegicus and R. rattus are likely to be important hosts of murine typhus in Jakarta. The antibody-positive rates against SFGR and murine typhus in rats captured in the dry season were significantly higher than those in rats captured in the rainy season. This may coincide with the active periods of ticks and fleas in Indonesia.