新疆石河子地区绵羊嗜吞噬细胞无形体的检测和16S rRNA序列分析

目的 调查新疆石河子地区绵羊嗜吞噬细胞无形体感染状况以及分析其病原16S rRNA序列特征.方法 在石河子地区采集羊血,提取DNA,巢式PCR扩增16S rRNA基因并与GenBank中相应基因序列进行比对分析.结果 在109份羊血样本中,检测出阳性样本37份,阳性率为33.94％,检测出的16S rRNA(524 bp)基因序列与部分GenBank中嗜吞噬细胞无形体16S rRNA基因序列同源性达99％.结论 新疆石河子地区绵羊中存在嗜吞噬细胞无形体的感染.     

中国西南横断山区小型兽类嗜吞噬细胞无形体基因的检测及序列测定

目的 了解中国西南横断山区小型兽类自然感染嗜吞噬细胞无形体的情况.方法 采集位于滇西北横断山区的高黎贡山山脉、香格里拉雪山等山地(海拔1000～4500m)林区的小型兽类脏器标本以低温保存和运输,所获标本在实验室应用聚合酶链反应(PCR)方法对嗜吞噬细胞无形体16S rRNA基因和Msp4基因片段进行扩增测序,并将所测序列与GenBank中注册的基因序列进行相似性比较.结果 共检测小型兽类5目18属35种共436只,从6属11种小型兽类中发现阳性标本32份,总阳性率为7.34%.其中,高黎贡山林区检测小型兽类标本25种301只,阳性26份,阳性率为8.64%(26/301);香格里拉雪山等林区检测小型兽类标本19种135只,阳性6份,阳性率为4.44%(6/135);阳性标本绝大部分发现于小型兽类中的啮齿类动物.序列比较分析表明:不同小型兽类间的16S rRNA基因序列相似性为99%～100%,且与吉林野鼠中检测的无形体相对应片段(GenBank:DQ449948)最相近,相似性达99%～100%.对其Msp4基因核苷酸序列进一步分析发现与GenBank中相应片段相似性为95%～97%,提示中国西南横断山区小型兽类所感染无形体株变异较大.结论 首次证实和发现中国西南横断山区6属11种小型兽类自然感染嗜吞噬细胞无形体,其中啮齿类动物可能是这一地区嗜吞噬细胞无形体的主要宿主.     

安徽省广德县山区易感动物无形体检测结果分析

目的了解安徽省广德县鼠、狗、羊中自然感染埃立克体及无形体的状况。方法用巢式PCR扩增鼠、狗、羊血块中埃立克体属及无形体属16SrRNA5’末端片段,用嗜吞噬细胞无形体与嗜单核细胞埃立克体种特异引物扩增阳性产物做测序分析。结果在安徽省广德县捕捉的5只鼠、11只狗、10只羊,用标本血块做核酸检测,其中鼠阳性0份,狗阳性0份,羊阳性4份,阳性率40%,将属特异性引物扩增产物做测序分析。于Genbank上做比对分析,4份均与牛株无形体相同基因区最为接近,属无形体科,无形体属。结论安徽省广德县存在无形体病原,羊可能为无形体宿主,其他宿主需进一步调查研究。     

黑龙江密山口岸三种蜱媒传染病在媒介蜱中复合感染的调查

目的调查黑龙江密山口岸三种蜱媒传染病在媒介蜱中复合感染的情况。方法采集蜱类标本44份,建立伯氏疏螺旋体(Borrelia burgdorferi)、斑点热群立克次体(spotted fever group rickettsia)、嗜吞噬细胞无形体(anaplasma phagocytophilum)三种新发蜱传疾病病原体特异性通用引物,利用PCR方法对所有标本进行特异性片段扩增,对阳性标本测序并进行序列分析。结果在所检测的44份标本中,共检测出伯氏疏螺旋体阳性标本4份,斑点热群立克次体阳性标本6份,嗜吞噬细胞无形体阳性标本2份,感染率分别为13.63%、9.10%和4.55%。在全沟硬蜱检测到两份分别是莱姆病螺旋体与斑点热群立克次体及嗜吞噬细胞无形体和斑点热群立克次体的复合感染,复合感染率为4.55%。结论黑龙江密山地区至少存在两种莱姆病螺旋体(Borrelia garinii,Borrelia afzelii),三种斑点热群立克次体(Rickettsia raoultii,Candidatus Rickettsia tarasevichiae,Rickettsia heilongjiangensis),和嗜吞噬细胞无形体(anaplasma phagocytophilum);全沟硬蜱存在复合感染。     

安徽省广德县山区易感动物无形体检测结果分析

目的了解安徽省广德县鼠、狗、羊中自然感染埃立克体及无形体的状况。方法用巢式PCR扩增鼠、狗、羊血块中埃立克体属及无形体属16SrRNA5’末端片段,用嗜吞噬细胞无形体与嗜单核细胞埃立克体种特异引物扩增阳性产物做测序分析。结果在安徽省广德县捕捉的5只鼠、11只狗、10只羊,用标本血块做核酸检测,其中鼠阳性0份,狗阳性0份,羊阳性4份,阳性率40％,将属特异性引物扩增产物做测序分析。于Genbank上做比对分析,4份均与牛株无形体相同基因区最为接近,属无形体科,无形体属。结论安徽省广德县存在无形体病原,羊可能为无形体宿主,其他宿主需进一步调查研究。     

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。     

浙江省家畜动物无形体分子流行病学调查

目的调查浙江省天台、安吉及金华县家畜动物牛及山羊无形体感染状况。方法以在册户籍为准,采用随机方法抽取调查点每农户5只山羊及5头牛,采集5ml静脉血分离血清,采用WHO推荐的微量间接免疫荧光方法（mIFA）进行血清IgG抗体检测;动物血球标本用于提取DNA进行无形体16SrRNA基因扩增。PCR产物测序并进行同源分析比较,同时进行遗传变异分析。结果当地家畜动物存在较高无形体携带率（39.42%）,主要分布在天台县;无形体流行株16SrRNA基因存在较大变异性,除发现15种独立核酸变异序列外,至少存在5种优势序列变异株。结论浙江省家畜动物普遍存在无形体携带状况;进一步开展人群无形体病调查及分子流行病学研究十分必要。     

蜱传嗜吞噬细胞无形体分子生物学检测方法的研究进展

嗜吞噬细胞无形体(anaplasma phagocytophilum)是一种经蜱传播的病原体,可侵染人和动物的中性粒细胞而引起无形体病。开展精准、快速的嗜吞噬细胞无形体检测,对无形体病的早诊断、早预防、早控制等具有重要意义。目前,嗜吞噬细胞无形体感染的诊断方法主要有流行病学史、临床症状表现及实验室检测等,其中分子生物学检测技术具有特异性高、操作方法简单、快速等特点,是对该病原体检测的重要方法。我们综述了近年来常见的嗜吞噬细胞无形体分子检测技术的应用进展,如巢式PCR法,荧光定量PCR法、多重PCR法、环介导等温扩增技术以及单细胞测序技术等,为后续的嗜吞噬细胞无形体研究和发展方向提供参考依据,也为无形体病的诊断提供借鉴。     

牡丹江镜泊湖区蜱中伯氏疏螺旋体与嗜吞噬细胞无形体感染的研究

目的了解牡丹江镜泊湖区伯氏疏螺旋体和嗜吞噬细胞无形体的感染情况。方法运用聚合酶链方法(PCR)对牡丹江镜泊湖区采集的蜱标本,进行伯氏疏螺旋体5S~23S基因间隔区基因片段与嗜吞噬细胞无形体柠檬酸盐合成酶基因(glt A)片段的检测。结果 2013年5月,在牡丹江镜泊湖北湖区附近共采集嗜群血蜱和全沟硬蜱643只,其中嗜群血蜱573只,占89.11%;全沟硬蜱70只,占10.89%。嗜群血蜱和全沟硬蜱伯氏疏螺旋体感染率分别为0.17%和15.71%,嗜吞噬细胞无形体感染率分别为0和2.86%;全沟硬蜱中伯氏疏螺旋体和嗜吞噬细胞无形体的复合感染率为1.43%。结论牡丹江镜泊湖区采集的蜱中检测到伯氏疏螺旋体和嗜吞噬细胞无形体的感染,并在全沟硬蜱中检测到复合感染情况,但复合感染的感染率较低。     

西藏亚东县西藏血蜱携带微生物群落情况及主要病原体进化分析

目的 了解西藏自治区亚东县西藏血蜱携带微生物群落的情况及主要病原体无形体的分型。方法 2022年5月在亚东县采集寄生于牦牛身上的蜱虫样本141只。根据硬蜱线粒体16S rDNA基因扩增序列进行分子鉴定以确定蜱种。采用Illumina Novaseq 6000平台对西藏血蜱进行高通量测序，将测序得到的原始数据处理后对其进行组装、基因预测、丰度分析和分类学预测。利用巢式PCR扩增无形体的16S rDNA基因片段并测序及进行序列的遗传进化分析。结果 121只成蜱样本鉴定为西藏血蜱。非冗余数据集经NCBI-NR数据库比对进行物种注释后，最常见的微生物种类是真核生物（19.78%）和细菌（13.54%）。在细菌中，优势门分别是变形杆菌门、拟杆菌门和厚壁菌门；优势属分别是无形体属、锥虫属、冷杆菌属和立克次体属；优势种分别是嗜吞噬细胞无形体、泰勒虫和斑点热群立克次体。无形体的系统发育树与绵羊无形体遗传距离最近，具有较高的同源性。结论 西藏自治区亚东县地区优势蜱种为西藏血蜱。无形体的主要分型为绵羊无形体。当地人畜暴露在多种蜱传病原体中，存在潜在蜱传疾病的威胁，应针对性加强对蜱的监测和预防控制工作。     

东北林区鼠类粒细胞埃立克体感染的调查

目的了解东北林区啮齿动物感染粒细胞埃立克体病原体的情况。方法运用聚合酶链反应方法对吉林、黑龙江、内蒙古林区采集的啮齿动物标本粒细胞埃立克体的16S rRNA和gltA基因片段进行检测并测序,将所测序列与GenBank中注册的基因序列进行比较分析。结果共检测鼠标本276份,其中吉林长白山林区102份,黑龙江小兴安岭林区61份,内蒙古大兴安岭林区113份,阳性率分别为8.82%、1.64%及0.00%。体表有寄生蜱的鼠感染危险性是体表无寄生蜱鼠的11.30倍(P=0.002)。吉林、黑龙江林区鼠标本及其寄生蜱标本16S rRNA基因序列完全相同,与美国、瑞典、日本等国家检出的粒细胞埃立克体对应序列的同源性为97%-99%。gltA基因核苷酸序列与GenBank注册的相应片段比较相似性为87%-97%,推导的氨基酸序列相似性为84%-99%。结论吉林及黑龙江林区存在粒细胞埃立克体宿主动物感染。     

Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setubal District, mainland Portugal

A total of 278 Ixodes ticks, collected from Madeira Island and Setubal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.     

Circulation of four species of Anaplasmataceae bacteria in ticks in Harbin,northeastern China

Ticks play an important role in the evolution and transmission of Anaplasmataceae bacteria which are agents of emerging infectious diseases. In this study, a total of 1286 adult ticks belonging to five species were collected from cattle, goats, horses and vegetation in Harbin area, Heilongjiang province, northeastern China. The tick-borne Anaplasmataceae bacteria were identified by amplifying and sequencing the 16S rRNA (rrs) and heat shock protein-60 encoding (groEL) genes. The results showed that Ixodes persulcatus was dominant (38.8%, 499/1283) among the five tick species, and Anaplasmataceae bacteria were detected in all tick species with an overall prevalence of 7.4%. Four species of Anaplasmataceae bacteria (Anaplasma phagocytophilum, Anaplasma ovis, Anaplasma bovis, and “Candidatus Neoehrlichia mikurensis”), which are pathogenic to humans and/or animals, were identified from tick samples by phylogenetic analyzes of the rrs and groEL gene sequences. Interestingly, the cluster 1 strains were first identified in Asian, and a novel cluster was also detected in this study. These data revealed the genetic diversity of Anaplasmataceae bacteria circulating in ticks in Harbin area, highlighting the need to investigate these tick-borne pathogens and their risks to human and animal health.     

浙江首例人粒细胞无形体病患者病原学分析

目的 对浙江首例疑似人粒细胞无形体病(HGA)患者进行病原学诊断与分子生物学分析.方法 运用分子生物学方法检测吞噬细胞无形体16S rRNA,间接免疫荧光法(IFA)检测患者临床症状期和恢复健康3个月后血清吞噬细胞无形体IgG抗体.结果 患者发热期血液PCR获得的16S rRNA基因片段:282 bp和389 bp,符...     

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”.     

山东省烟台市人与动物新型布尼亚病毒感染调查及同源性分析

目的 了解山东省烟台市人和动物发热伴血小板减少综合征布尼亚病毒 （SFTSV）感染及流行情况。方法 2011年4－11月分别在烟台市的蓬莱和莱州2个SFTS病例高发地区连续采集与人密切接触的5种家养动物 （羊、 牛、 猪、 犬、 鸡） 血清标本3 576份,应用双抗原夹心ELISA方法和Real time RT-PCR方法检测血清中SFTSV总抗体和病毒核酸; 观察不同月份感染情况; 采集两地人群血清2 590份,应用间接ELISA方法检测SFTSV IgG抗体; 用Vero细胞从核酸阳性的人和动物血清中分离病毒,通过RT-PCR方法对 SFTSV S片段进行序列扩增、 同源性分析。结果 3 576份动物血清标本中SFTSV血清总抗体阳性1 439份,阳性率为40.24%,病毒核酸阳性163份,阳性率为4.56%。其中羊、 牛、 鸡、 犬、 猪抗体阳性率分别为62.78%、 52.97%、 45.56%、28.73%和1.45%,核酸阳性率分别为5.72%、 4.63%、 3.02%、 5.25%和3.73%。动物体内的抗原抗体随季节消长而变化。2 590份人群血清SFTSV IgG抗体阳性率为5.41%。对10株来自人的毒株和3株来自动物的毒株进行S片段基因序列扩增分析,显示其同源性在95.23%～100.00%,与国内其他省市分离毒株比较,其同源性在94.72%～99.13%,高度同源。结论 烟台地区存在SFTSV流行,人与家养动物普遍易感,其基因序列高度同源,提示家养动物可能作为SFTSV的增殖宿主和扩散宿主,应引起高度重视。     

2010-2011无年形我体国的东感北染林情区况蜱调中查嗜吞噬细胞

目的调查我国东北林区蜱标本中嗜吞噬细胞无形体的感染情况。方法2010-2011年在东北林区采集蜱标本,用聚合酶链反应方法扩增gltA基因片段,分析东北林区蜱中无形体的感染情况,代表阳性片段联用长片段16S rRNA进行扩增测序确认;并将所得序列与GenBank中已注册的序列进行比对分析。结果共检测蜱2293只,其中2010年1161只,阳性率4.91%,2011年1132只,阳性率4.24%,2年的感染率差异无统计学意义(χ2=0.373,P=0.830;χ2=1.789,P=0.409)。全沟硬蜱阳性率4.86%(91/1872),长角血蜱为3.41%(14/411),森林革蜱为0(0/10),3个蜱种的感染率差异无统计学意义(P>0.05)。测得的gltA序列中,NE-gltA-1与Hc346株(GenBank:GU935788)最相似,仅差2 bp,相似性99%;NE-gltA-2与MDJ-Ip92株(HQ396224)序列一致,相似性100%。与2个序列相似性最远的是ZJ-HGA-72株(DQ458811),相差53 bp,相似性84%。测得的16S rRNA序列与我国从东北鼠源分离株China-C-T(tGQ412339)最接近,相似性为99%,与Florida株(AF309867)相似性最远,相似性97%。结论我国东北林区蜱中普遍存在嗜吞噬细胞无形体的感染,全沟硬蜱和长角血蜱可能为无形体的媒介宿主。     

Survey of tickborne infections in Denmark

We conducted a study of the distribution and prevalence of tickborne infections in Denmark by using roe deer as sentinels. Blood samples from 237 roe deer were collected during the 2002-2003 hunting season. Overall, 36.6% of deer were Borrelia seropositive, while 95.6% were Anaplasma phagocytophilum positive; all animals were negative for Bartonella quintana and B. henselae by indirect immunofluorescence assay. When a hemagglutination-inhibition test was used, 8.7% of deer were found positive for tickborne encephalitis (TBE)-complex virus. A total of 42.6% were found positive by polymerase chain reaction (PCR) for A. phagocytophilum with significant seasonal variation. All were PCR negative for Rickettsia helvetica. PCR and sequencing also showed a novel bacterium in roe deer previously only found in ticks. The study showed that the emerging pathogen A. phagocytophilum is widely distributed and that a marked shift has occurred in the distribution of TBE-complex virus in Denmark. This finding supports studies that predict alterations in distribution due to climatic changes.