两种虫媒传染病自然疫源地调查与病原特征研究
|
摘要
近年来，全球面临着新传染病不断出现、旧传染病死灰复燃的严峻形势。除大家熟知的SARS、高致病性禽流感外，新旧虫媒传染病的巨大现实危害也不容忽视。虫媒传染病多为自然疫源性疾病，其动物宿主、传播媒介分布广泛，易在自然界流行，当人进入疫源地就容易被感染并发病，因而这些疾病对在野外从事生产活动、经济开发、国防建设、旅游以及军事训练等人群存在极大威胁，暴发流行时有发生。虫媒传染病的病原体种类繁多，包括病毒、细菌、立克次体、螺旋体以及寄生虫等，并且一种媒介或动物宿主可携带两种或两种以上病原体，使其传播变得更加复杂。此外，自然灾害的发生、自然环境的破坏以及局部环境的改变都会影响虫媒传染病的发生及流行。
由于我国自然地理条件复杂，环境多样，动植物区系丰富，所以虫媒传染病的种类也十分丰富。近年来，新发虫媒传染病的出现和流行给我国公共卫生领域造成了严重威胁，尤其是蜱媒传染病。如嗜吞噬细胞无形体病的出现及其疫源地的活跃，新型布尼亚病毒的发现及其引起的发热伴血小板减少综合症的暴发流行。因此，针对这些新发或再发的虫媒传染病开展深入研究，摸清其疫源地本底情况及流行规律，掌握病原特征及其与宿主、媒介之间的相互作用，才能为疾病的预防和控制提供科学依据和理论支持。
无形体病是无形体科病原微生物感染而引起的相关疾病的统称，是一种蜱媒传染病，主要包括人单核细胞埃立克体病、人嗜吞噬细胞无形体病以及人埃文氏埃立克体病。2006年我国安徽某地区出现人嗜吞噬细胞无形体病小暴发，报道了10例疑似病例，并怀疑该病原体可能通过人—人传播。但此后国内相关研究并未发现无形体病确诊病例。我国东北林区是一个重要的虫媒传染病自然疫源地，媒介和动物宿主分布广泛，虫媒病原体种类丰富。本研究室于2009年从吉林省的鼠和羊中分离培养到嗜吞噬细胞无形体，提示当地可能存在无形体感染病例。
本研究于2010年在黑龙江省牡丹江地区开展流行病学调查，主要针对有蜱咬史并有发热症状的病例，结果在当地发现了7例新埃立克体感染病例。新埃立克体是隶属于无形体科的一种新发蜱传病原体，目前仅在欧洲有少数散发病例报道，本研究首次在我国乃至亚洲地区报道了一组新埃立克体感染病例。本研究还进一步开展了新埃立克体自然疫源地调查并分析了其病原特征。
在使用恒河猴分离培养新埃立克体的试验中，本研究发现恒河猴感染了五日热巴尔通体。五日热巴尔通体是战壕热的致病菌，是一种潜在的生物战剂，也是一种再发的虫媒病原体。人是五日热巴尔通体的主要宿主，体虱为主要传播媒介，蜱能否传播该病原体尚存争议。本研究进一步调查了猴中五日热巴尔通体的感染情况，证明了猴是该病原体的动物宿主，并鉴定了传播媒介。此外，通过多位点序列分型和全基因组测序方法分析了五日热巴尔通体的遗传进化特征。
主要研究结果：
1我国东北地区人群新埃立克体感染调查研究
本研究采用哨点医院监测和实验室研究相结合的方法开展人群蜱媒病原体感染调查。2010年5月至7月期间，在我国东北部的黑龙江省牡丹江市林业中心医院开展调查，共纳入628例有蜱咬史并有发热症状病例。采用针对rrs基因的通用PCR和针对groEL基因的特异性PCR方法，从7例病例的急性期血液标本中检测到新埃立克体。序列比对发现本研究获得的新埃立克体rrs和groEL基因序列与俄罗斯远东地区及西伯利亚地区已报道的序列一致。
临床资料分析发现7例新埃立克体感染病例均有发热、头痛和乏力症状，其它常见症状包括恶心、呕吐、肌痛和颈痛，偶见症状包括关节炎、咳嗽、腹泻、意识模糊和红斑；实验室化验结果显示1例白细胞减少，1例白细胞升高，2例血小板减少，2例贫血；血清丙氨酸氨基转移酶和天冬氨酸氨基转移酶均正常。欧洲报道的散发病例多为免疫受损者，感染新埃立克体后临床症状均较重，而本研究发现的7例病例既往健康，其临床特征更能代表健康人群感染新埃立克体后的症状。
本研究在发现新埃立克体感染病例后，对病例居住地周围的媒介蜱和鼠开展了流行病学调查。2010年5月至7月期间，共调查516只游离蜱，总感染率为1.6%，其中全沟硬蜱为1.9%（6/316），嗜群血蜱为0.8%（2/187），森林革蜱为0（0/13）；共调查211只鼠，总感染率为3.8%，其中棕背平鼠为4.6%（5/109），褐家鼠为5.7%（2/35），花鼠为33.3%（1/3），大林姬鼠、黑线姬鼠及小家鼠感染率均为0。媒介蜱和鼠中新埃立克体感染的发现证明我国东北地区为新埃立克体自然疫源地。
2我国鼠中新埃立克体感染情况调查及遗传进化特征分析
本研究从我国11个地区的15个调查点共采集841只野鼠，涵盖10个属22个种。其中黑线姬鼠所占比率最高，占14.6%；其次是褐家鼠和棕背平鼠，分别占10.3%和9.9%。通过对鼠脾脏组织提取的基因组DNA进行针对16S rRNA和groEL基因的巢氏PCR检测，发现34只鼠感染了新埃立克体，涵盖9个鼠种，包括西南绒鼠、花鼠、中华姬鼠、小林姬鼠、黑线姬鼠、大林姬鼠、棕背平鼠、社鼠和褐家鼠，感染率为1.1%~25.0%。其中西南绒鼠（25.0%，P=0.038）、小林姬鼠（12.5%，P=0.019）和黑线姬鼠（12.0%，P<0.001）感染率较其它鼠种高，差异具有统计学意义。在本研究的15个调查点中，有10个调查点均检测到新埃立克体，涵盖7个调查地区，分别是黑龙江、吉林、河南、浙江、福建、云南和内蒙古，感染率为1.5%~13.8%。Fisher’s精确检验表明不同地区鼠中新埃立克体感染率有差异（P<0.001），其中吉林地区鼠的感染率最高，为13.8%。
经序列比对分析，发现我国存在三种新埃立克体基因型，分别来自东北、东南和西南地区。选择GenBank数据库中所有已注册的新埃立克体groEL基因较长核苷酸序列（874bp）构建系统发育树，发现新埃立克体共有四类基因型，并且其分布与地理来源相关联。我国东北及俄罗斯亚洲地区来源的新埃立克体归为Cluster I。我国西南和东南地区来源的新埃立克体分别归为Cluster III和ClusterIV，并且日本来源的也被划分到这两类。此外，归类到Cluster II的新埃立克体均来自欧洲。基于rrs（1303bp）基因的遗传进化分析同样显示新埃立克体有四类基因型，结果与基于groEL基因的遗传进化分析一致。
3猴中五日热巴尔通体感染与传播调查研究
采用PCR扩增、血涂片染色、透射电子显微镜观察以及血培养方法从10只恒河猴中检测到4只感染了五日热巴尔通体。本研究进一步采用PCR和血培养方法调查了我国5个地区2种猴中五日热巴尔通体感染情况，共调查636只猴，包括恒河猴328只、食蟹猴308只。经PCR检测发现有96只猴（15.1%）感染五日热巴尔通体，包括恒河猴59只、食蟹猴37只；经血培养检测发现有53只猴（8.3%）感染五日热巴尔通体，包括恒河猴37只、食蟹猴16只。恒河猴的感染率高于食蟹猴，但两种猴的血液带菌量无显著性差异（P>0.05）。5个地区的猴中均有检测到五日热巴尔通体，并且南阳市的猴中五日热巴尔通体感染率较其它地区高（P<0.001）。卡方检验分析发现幼年猴（≤3岁）、青年猴（4~6岁）较成年猴（≥7岁）的感染率高（P<0.05）；猴中五日热巴尔通体的感染率与性别无明显关联（P>0.05）。
通过对10只恒河猴的连续监测，发现猴中存在五日热巴尔通体感染的传播，检查恒河猴体表，发现均有体虱寄生，且体虱均感染了五日热巴尔通体，推断可能为猴中五日热巴尔通体的传播媒介。经系统发育分析和形态学观察，鉴定该传播媒介为钝猴虱。本研究将五日热巴尔通体分离株接种到本底干净的实验恒河猴，连续监测发现恒河猴无明显临床症状，仅表现为长期慢性感染，表明猴能较好适应该病原体。这些研究结果表明猴是五日热巴尔通体的动物宿主，钝猴虱可能为传播媒介。此外，通过对8名猴饲养员的两次调查，发现4名出现五日热巴尔通体抗体阳转现象，表明五日热巴尔通体可能会经猴传播给人，需对其潜在危险加以重视。
4五日热巴尔通体的遗传进化特征分析
本研究采用MLST技术分析了53株猴来源五日热巴尔通体，与已报道的人来源五日热巴尔通体相关数据对比分析，发现44个新的核苷酸多态性位点，25个新的等位基因型，14个新的序列型（ST8~ST21），表明猴来源的五日热巴尔通体基因多样性更为丰富。采用eBURST软件分析各个序列型之间的亲缘关系，结果发现了2个新的克隆复合体以及3个新的单个型。基于MLST数据的系统发育分析发现人来源、恒河猴来源以及食蟹猴来源的五日热巴尔通体分别聚在不同分枝，并且两种猴来源的菌株进化关系更为接近，表明五日热巴尔通体的基因多样性与宿主种类存在关联。采用Splitstree4软件的splits decomposition analysis和phi test方法分析五日热巴尔通体各ST型的串联序列，发现五日热巴尔通体并未发生基因重组。
为进一步了解五日热巴尔通体的遗传进化特征，本研究采用罗氏454测序和Solexa测序相结合的方法测定了一株猴来源菌株（str. RM-11）的全基因组序列，拼接、组装、注释并提交GenBank数据库，注册号为CP003784。全基因组序列初步比对分析发现猴来源菌株与人来源菌株（str. Toulouse）存在一定差异，但需要进一步深入分析探讨其科学意义。
主要结论：
首次在我国发现了一组新埃立克体感染病例，确认我国东北地区为新埃立克体自然疫源地。流行病学调查发现我国多个地区多个鼠种均存在新埃立克体自然感染，表明该病原体在我国分布广泛。遗传进化分析发现新埃立克体的基因多样性可能与地理来源相关联。
流行病学调查发现我国多个地区猴中均存在五日热巴尔通体感染。首次发现猴中五日热巴尔通体感染的传播，并确认钝猴虱是其传播媒介。多方面研究结果有力证明猴是五日热巴尔通体的动物宿主。血清学调查发现猴携带的五日热巴尔通体可能会感染人。MLST分析发现14个新的五日热巴尔通体序列型，表明猴来源菌株基因多样性较人来源菌株更为丰富。遗传进化分析发现五日热巴尔通体的基因多样性与宿主种类存在关联，提示了五日热巴尔通体可能的起源。此外，本研究还首次测定了一株猴来源五日热巴尔通体的全基因组序列，初步比对分析发现与人来源菌株存在一定差异。
Emerging and re-emerging infectious diseases are an important issue in global health.The well-known SARS and highly pathogenic avian influenza cause great hazards insome regions, while the huge hazards from emerging and re-emerging vector-borneinfectious diseases can not be ignored. A disease that is transmitted to humans or otheranimals by an insect or other arthropod is called a vector-borne disease. Manyvector-borne diseases are natural focus diseases, and the etiologic pathogens includeviruses, rickettsiae, bacteria, parasites and so on. Vector-borne diseases are easilyprevalent in nature due to the widely distribution of vectors and reservoir hosts. Theoccurrence of natural disasters and the destruction of the natural environment willaffect the occurrence and prevalence of vector-borne diseases.
Because of the complex natural and geographical conditions, diverseenvironment, as well as rich flora and fauna, there are many kinds of vector-borneinfectious diseases in China. Recently, the emergence and prevalence of emergingtick-borne infectious diseases poses a serious threat to public health in China, such asthe emergence of anaplasmosis and the outbreak of severe fever withthrombocytopenia syndrome. Therefore, to find out the natural foci and epidemiologicfeatures, and to understand the pathogenic characteristics and the interaction betweenhosts and vectors, in-depth investigations on emerging and re-emerging vector-borneinfectious diseases should be performed. The findings from these researches willprovide a scientific basis and theoretical support for prevention and control ofdiseases.
“Anaplasmosis” is a generic name for infections caused by obligate intracellularbacteria in the family Anaplasmataceae, which is a kind of tick-borne disease andmainly includes human granulocytic anaplasmosis (HGA), human monocyticehrlichiosis and human ewingii ehrlichiosis. An outbreak of HGA including10caseswere reported in Anhui province in2006, and this study suggested human-to-humantransmission of HGA. Subsequently, no confirmed cases have been reported in China.Our previous surveys detected various tick-borne agents in ticks and animal hosts from forest areas of northeastern China, such as Anaplasma, Borrelia and rickettsia.In addition, our laboratory successfully isolated A. phagocytophilum from rodents andsheep in northeastern China, suggesting the potential existence of human cases ofHGA in this area.
In the current study, we initiated a surveillance project in Mudanjiang ForestryCentral Hospital, one of the largest hospitals treating patients with tick bites innortheastern China, and found7cases infected with Candidatus Neoehrlichiamikurensis. Field investigation on rodents and ticks confirmed the existence of localnatural foci.
When we used rhesus macaques for isolation of Candidatus Neoehrlichiamikurensis, Bartonella quintana infection in the macaques were unexpectedlyobserved. B. quintana was firstly recognized as the pathogenic bacteria of trenchfever. In China, few studies focus on B. quintana, which is recognized as are-emerging vector-borne pathogen now. The study was then aimed to identify theanimal hosts and vectors of B. quintana, and to investigate the genetic characteristicsof this agent.
The main results:
1Investigations on human infection with Candidatus Neoehrlichiamikurensis in eastern China
During May-July in2010, a total of628febrile patients who had a history ofrecent tick bites and sought treatment at Mudanjiang Forestry Central Hospital ofHeilongjiang Province were enrolled into the study. The peripheral blood sampleswere collected from the patients on acute phase of the illness. A board-range PCRtargeting rrs gene and a species-specific PCR targeting groEL gene were used todetect the infections of tick-borne agents. As a result,7patients were found to beinfected with Candidatus Neoehrlichia mikurensis through the amplifications of bothrrs and groEL. The amplified nearly entire rrs (1501bp) and partial groEL (914bp)nucleotide sequences from the7patients were identical, and same to those ofCandidatus Neoehrlichia mikurensis detected from I. persulctus ticks and field micein the Asian part of Russia.
Clinical symptoms of fever, headache and malaise were present in all7patients.Other major manifestations included nausea (5/7), vomiting (5/7), myalgia (4/7) andstiff neck (4/7). The less common symptoms were arthralgias (2/7) and cough (2/7),diarrhea (1/7), confusion (1/7) and erythema (1/7). Laboratory test results showed leucopenia in one patient, leukocytosis in one patient, thrombocytopenia in twopatients and anemia in two patients; serum levels of alanine aminotransferase andaspartate aminotransferase within reference ranges in all patients. Unlike previouslyreported cases with old age or immunocompromised conditions who developedoverwhelming disease, all7patients in the current study presented with relativelymild disease. The clinical manifestations might be typical of Candidatus Neoehrlichiamikurensis infection in otherwise healthy population.
A total of516host-seeking ticks, including316Ixodes persulcatus,187Haemaphysalis concinna and13Dermacentor silvarum, were collected on vegetationand individually examined. Candidatus Neoehrlichia mikurensis DNA was detected in6I. persulctus and2H. conicinna with an overall infection rate of1.6%, whereas noDNA from the agent was detected in D. silvarum. A total of211wild rodents from6species were captured and tested. Eight rodents from3species, includingClethrionomys rufocanus, Rattus norvegicus and Tamias sibiricus, were foundnaturally infected, with an overall infection rate of3.8%. These results suggest theexistence of natural foci of Candidatus Neoehrlichia mikurensis in Mudanjiang Cityof Heilongjiang Province.
2Prevalence and genetic diversity of Candidatus Neoehrlichia mikurensis inrodents from China
Totally,841rodents of22species from10genera were collected at15study sitesin11regions at local peak seasons of tick activities. The predominant species wereApodemus agrarius (14.6%) and the other species accounted for0.8%-10.3%. Eachregion had3-9species of rodents sampled. Candidatus Neoehrlichia mikurensis wasdetected in34(4.0%) of the841rodents by amplifications of both groEL and rrs. The34rodents with positive results represented9species, with the prevalence in eachspecies ranging from1.1%to25.0%. When each rodent species was compared withall others, the infection rates in Eothenomys custos (25.0%), Apodemus agrarius(12.0%) and A. sylvaticus (12.5%) were significantly higher. Candidatus Neoehrlichiamikurensis was detected in10of the15study sites, representing Heilongjiang, Jilin,Henan, Zhejiang, Fujian, Yunnan Provinces and Inner Mongolia Autonomous Region.The prevalence of Candidatus Neoehrlichia mikurensis in the7regions ranged from1.5%to13.8%and showed significant difference. Rodents collected from JilinProvince were at significantly higher risk for infection in contrast to other regions(P<0.001).
Sequence comparisons revealed three different Candidatus Neoehrlichiamikurensis variants that were consistent with the three geographic regions, NortheastChina, Southwest China and Southeast China. Phylogenetic analysis based on allavailable and comparably long groEL nucleotide sequences (874bp) revealed fourdistinct clusters of Candidatus Neoehrlichia mikurensis strains that related togeographic origins. Sequences obtained from Northeast China were identical to thosefrom the Asia part of Russia and included in Cluster I. Sequences obtained fromSouthwest China and Southeast China were separately clustered in Cluster III andCluster IV. No sequence of the present study was identified in Cluster II, which wasexclusively restricted to Europe. Similar to the phylogenetic analysis of groELsequences, neighbor-joining tree based on all available and nearly entire rrs (1303bp)also separated these sequences into four different clusters.
3Investigations on infection and transmission of Bartonella quintana inmacaques
Four of10one-year old male macaques housed in the Laboratory Animal Centerof the Academy of Military Medical Sciences were found to be infected with B.quintana by PCR, blood smear staining, transmission electron microscopy and bloodculture. Then, we performed an active surveillance of five primate centers acrossmainland China to identify prevalence and distribution of B. quintana infection incaptive monkeys. Blood samples were collected from328rhesus macaques and308cynomolgus macaques. A total of96(15.1%) macaques were positive for B. quintanaby PCR assay, including59rhesus macaques and37cynomolgus macaques; B.quintana isolates were successfully cultured from the blood of8.3%(53/636) of themacaques tested, including37rhesus macaques and16cynomolgus macaques.Prevalence of B. quintana in rhesus macaques was significantly higher than that incynomolgus macaques. B. quintana was detected in the macaques from each region,but the monkey colony from Nanyang City was at significantly higher risk (P<0.001).In both rhesus macaques and cynomolgus macaques, the B. quintana infection rates injuvenile (≤3years) and young animals (4~6y) were significantly higher than those inadult animals (≥7y)(P<0.05). No significant association was observed between theinfection and gender of the monkeys (P>0.05).
In three consecutive screening tests of a group of10monkeys, an increasedprevalence of B. quintana was observed with4of them being found positive at day1,7positive at day15, and all ten positive at day35during the period of observation. All10monkeys were found to be infested with lice at the last day of the observation(day36). Lice from each infested monkey were positive for detection of B. quintanaDNA, suggesting the lice might act as efficient vectors. By phylogenetic analysis andmorphologic features, the lice were identified as Pedicinus obtusus.
After intravenous inoculation of the Bartonella-negative macaques with B.quintana strain RM-11, the macaques presented a long-lasting chronic bacteremia, butdid not show abnormal clinical features. In addition, we found4workers involvedwith the macaques care showed seroconversion to antigens derived from our strain,suggesting that macaques could be a source for B. quintana human infection.
4Clonal and phylogenetic analysis of B. quintana
The current study identified44new nucleotide variable sites,25newly allelesand14novel sequence types (STs8-21) among53B. quintana isolates by MLSTanalysis. In contrast with the relatively low level of sequence divergence of B.quintana reported in humans, our investigation revealed much higher genetic diversityin non-human primates. To display the relationships between related STs, all macaqueisolates were analysed by eBURST v3in combination with the data from humanisolates. Two novel clonal complexes and three new singletons were found.Phylogenetic analysis based on the concatenated MLST sequences revealed3distinctclusters. Sequences (STs1–7) from B. quintana isolates recovered from humans wereincluded in Group1. Sequences (STs8–14) from16isolates cultured fromcynomolgus macaques belonged to Group2. Sequences (STs15–21) from37isolatescultured from rhesus macaques were found exclusively in Group3, which displayed ahigher homology to Group2than to Group1. The clustering of concatenatedsequences resembles the relationship between STs and host species, suggesting thegenetic diversity and host-cospeciation among the B. quintana populations. Inaddition, splits decomposition analysis and phi test were performed using Splitstree4,and revealed no apparent recombination among B. quintana isolates.
To further investigate genetic characteristics, the complete genome of B.quintana strain RM-11, which was isolated from rhesus macaques, was obtained by acombined strategy involving Solexa and Roche/454. A total of1204predicted proteincoding genes,42tRNA genes, and two copies each of the5S,16S, and23S rRNAgenes were identified. The genome sequence and annotation information areaccessible in the GenBank database with accession number CP003784. Using areciprocal best BLAST hit strategy, we identified a total of1096common genes between strains RM-11and Toulouse, a human B. quintana isolate whose genome hasbeen sequenced.
The main conclusions:
This study is the first report on human infection of the Candidatus Neoehrlichiamikurensis variant in the world other than Europe. Field survey on ticks and rodentssuggested that the existence of natural foci of Candidatus Neoehrlichia mikurensis innortheastern China. We detected Candidatus Neoehrlichia mikurensis in diversespecies of rodents from various regions, indicating the agent is widely distributed inChina. We also found that the Candidatus N. mikurensis gene clusters correlated withdistinct geographic origins.
The study demonstrated the prevalence of B. quintana infection was common inmacaques from main primate centers in mainland China. We firstly reported thetransmission of B. quintana in macaques, and identified Pedicinus obtusus lice as thevectors. These results indicated that macaques may serve as reservoir hosts for B.quintana. Serologic test suggested that macaques could be a source for B. quintanahuman infection. MLST analysis of the B. quintana isolates revealed14novelsequence types, suggesting much higher genetic diversity in non-human primates thanin humans. Phylogenetic analysis provided the evidence of host-cospeciation of B.quintana. These findings suggested that trench fever may primarily be a zoonoticdisease with macaques as the natural hosts. In addition, we sequenced the genome of aB. quintana strain, which was isolated from the blood of a rhesus macaque.
由于我国自然地理条件复杂，环境多样，动植物区系丰富，所以虫媒传染病的种类也十分丰富。近年来，新发虫媒传染病的出现和流行给我国公共卫生领域造成了严重威胁，尤其是蜱媒传染病。如嗜吞噬细胞无形体病的出现及其疫源地的活跃，新型布尼亚病毒的发现及其引起的发热伴血小板减少综合症的暴发流行。因此，针对这些新发或再发的虫媒传染病开展深入研究，摸清其疫源地本底情况及流行规律，掌握病原特征及其与宿主、媒介之间的相互作用，才能为疾病的预防和控制提供科学依据和理论支持。
无形体病是无形体科病原微生物感染而引起的相关疾病的统称，是一种蜱媒传染病，主要包括人单核细胞埃立克体病、人嗜吞噬细胞无形体病以及人埃文氏埃立克体病。2006年我国安徽某地区出现人嗜吞噬细胞无形体病小暴发，报道了10例疑似病例，并怀疑该病原体可能通过人—人传播。但此后国内相关研究并未发现无形体病确诊病例。我国东北林区是一个重要的虫媒传染病自然疫源地，媒介和动物宿主分布广泛，虫媒病原体种类丰富。本研究室于2009年从吉林省的鼠和羊中分离培养到嗜吞噬细胞无形体，提示当地可能存在无形体感染病例。
本研究于2010年在黑龙江省牡丹江地区开展流行病学调查，主要针对有蜱咬史并有发热症状的病例，结果在当地发现了7例新埃立克体感染病例。新埃立克体是隶属于无形体科的一种新发蜱传病原体，目前仅在欧洲有少数散发病例报道，本研究首次在我国乃至亚洲地区报道了一组新埃立克体感染病例。本研究还进一步开展了新埃立克体自然疫源地调查并分析了其病原特征。
在使用恒河猴分离培养新埃立克体的试验中，本研究发现恒河猴感染了五日热巴尔通体。五日热巴尔通体是战壕热的致病菌，是一种潜在的生物战剂，也是一种再发的虫媒病原体。人是五日热巴尔通体的主要宿主，体虱为主要传播媒介，蜱能否传播该病原体尚存争议。本研究进一步调查了猴中五日热巴尔通体的感染情况，证明了猴是该病原体的动物宿主，并鉴定了传播媒介。此外，通过多位点序列分型和全基因组测序方法分析了五日热巴尔通体的遗传进化特征。
主要研究结果：
1我国东北地区人群新埃立克体感染调查研究
本研究采用哨点医院监测和实验室研究相结合的方法开展人群蜱媒病原体感染调查。2010年5月至7月期间，在我国东北部的黑龙江省牡丹江市林业中心医院开展调查，共纳入628例有蜱咬史并有发热症状病例。采用针对rrs基因的通用PCR和针对groEL基因的特异性PCR方法，从7例病例的急性期血液标本中检测到新埃立克体。序列比对发现本研究获得的新埃立克体rrs和groEL基因序列与俄罗斯远东地区及西伯利亚地区已报道的序列一致。
临床资料分析发现7例新埃立克体感染病例均有发热、头痛和乏力症状，其它常见症状包括恶心、呕吐、肌痛和颈痛，偶见症状包括关节炎、咳嗽、腹泻、意识模糊和红斑；实验室化验结果显示1例白细胞减少，1例白细胞升高，2例血小板减少，2例贫血；血清丙氨酸氨基转移酶和天冬氨酸氨基转移酶均正常。欧洲报道的散发病例多为免疫受损者，感染新埃立克体后临床症状均较重，而本研究发现的7例病例既往健康，其临床特征更能代表健康人群感染新埃立克体后的症状。
本研究在发现新埃立克体感染病例后，对病例居住地周围的媒介蜱和鼠开展了流行病学调查。2010年5月至7月期间，共调查516只游离蜱，总感染率为1.6%，其中全沟硬蜱为1.9%（6/316），嗜群血蜱为0.8%（2/187），森林革蜱为0（0/13）；共调查211只鼠，总感染率为3.8%，其中棕背平鼠为4.6%（5/109），褐家鼠为5.7%（2/35），花鼠为33.3%（1/3），大林姬鼠、黑线姬鼠及小家鼠感染率均为0。媒介蜱和鼠中新埃立克体感染的发现证明我国东北地区为新埃立克体自然疫源地。
2我国鼠中新埃立克体感染情况调查及遗传进化特征分析
本研究从我国11个地区的15个调查点共采集841只野鼠，涵盖10个属22个种。其中黑线姬鼠所占比率最高，占14.6%；其次是褐家鼠和棕背平鼠，分别占10.3%和9.9%。通过对鼠脾脏组织提取的基因组DNA进行针对16S rRNA和groEL基因的巢氏PCR检测，发现34只鼠感染了新埃立克体，涵盖9个鼠种，包括西南绒鼠、花鼠、中华姬鼠、小林姬鼠、黑线姬鼠、大林姬鼠、棕背平鼠、社鼠和褐家鼠，感染率为1.1%~25.0%。其中西南绒鼠（25.0%，P=0.038）、小林姬鼠（12.5%，P=0.019）和黑线姬鼠（12.0%，P<0.001）感染率较其它鼠种高，差异具有统计学意义。在本研究的15个调查点中，有10个调查点均检测到新埃立克体，涵盖7个调查地区，分别是黑龙江、吉林、河南、浙江、福建、云南和内蒙古，感染率为1.5%~13.8%。Fisher’s精确检验表明不同地区鼠中新埃立克体感染率有差异（P<0.001），其中吉林地区鼠的感染率最高，为13.8%。
经序列比对分析，发现我国存在三种新埃立克体基因型，分别来自东北、东南和西南地区。选择GenBank数据库中所有已注册的新埃立克体groEL基因较长核苷酸序列（874bp）构建系统发育树，发现新埃立克体共有四类基因型，并且其分布与地理来源相关联。我国东北及俄罗斯亚洲地区来源的新埃立克体归为Cluster I。我国西南和东南地区来源的新埃立克体分别归为Cluster III和ClusterIV，并且日本来源的也被划分到这两类。此外，归类到Cluster II的新埃立克体均来自欧洲。基于rrs（1303bp）基因的遗传进化分析同样显示新埃立克体有四类基因型，结果与基于groEL基因的遗传进化分析一致。
3猴中五日热巴尔通体感染与传播调查研究
采用PCR扩增、血涂片染色、透射电子显微镜观察以及血培养方法从10只恒河猴中检测到4只感染了五日热巴尔通体。本研究进一步采用PCR和血培养方法调查了我国5个地区2种猴中五日热巴尔通体感染情况，共调查636只猴，包括恒河猴328只、食蟹猴308只。经PCR检测发现有96只猴（15.1%）感染五日热巴尔通体，包括恒河猴59只、食蟹猴37只；经血培养检测发现有53只猴（8.3%）感染五日热巴尔通体，包括恒河猴37只、食蟹猴16只。恒河猴的感染率高于食蟹猴，但两种猴的血液带菌量无显著性差异（P>0.05）。5个地区的猴中均有检测到五日热巴尔通体，并且南阳市的猴中五日热巴尔通体感染率较其它地区高（P<0.001）。卡方检验分析发现幼年猴（≤3岁）、青年猴（4~6岁）较成年猴（≥7岁）的感染率高（P<0.05）；猴中五日热巴尔通体的感染率与性别无明显关联（P>0.05）。
通过对10只恒河猴的连续监测，发现猴中存在五日热巴尔通体感染的传播，检查恒河猴体表，发现均有体虱寄生，且体虱均感染了五日热巴尔通体，推断可能为猴中五日热巴尔通体的传播媒介。经系统发育分析和形态学观察，鉴定该传播媒介为钝猴虱。本研究将五日热巴尔通体分离株接种到本底干净的实验恒河猴，连续监测发现恒河猴无明显临床症状，仅表现为长期慢性感染，表明猴能较好适应该病原体。这些研究结果表明猴是五日热巴尔通体的动物宿主，钝猴虱可能为传播媒介。此外，通过对8名猴饲养员的两次调查，发现4名出现五日热巴尔通体抗体阳转现象，表明五日热巴尔通体可能会经猴传播给人，需对其潜在危险加以重视。
4五日热巴尔通体的遗传进化特征分析
本研究采用MLST技术分析了53株猴来源五日热巴尔通体，与已报道的人来源五日热巴尔通体相关数据对比分析，发现44个新的核苷酸多态性位点，25个新的等位基因型，14个新的序列型（ST8~ST21），表明猴来源的五日热巴尔通体基因多样性更为丰富。采用eBURST软件分析各个序列型之间的亲缘关系，结果发现了2个新的克隆复合体以及3个新的单个型。基于MLST数据的系统发育分析发现人来源、恒河猴来源以及食蟹猴来源的五日热巴尔通体分别聚在不同分枝，并且两种猴来源的菌株进化关系更为接近，表明五日热巴尔通体的基因多样性与宿主种类存在关联。采用Splitstree4软件的splits decomposition analysis和phi test方法分析五日热巴尔通体各ST型的串联序列，发现五日热巴尔通体并未发生基因重组。
为进一步了解五日热巴尔通体的遗传进化特征，本研究采用罗氏454测序和Solexa测序相结合的方法测定了一株猴来源菌株（str. RM-11）的全基因组序列，拼接、组装、注释并提交GenBank数据库，注册号为CP003784。全基因组序列初步比对分析发现猴来源菌株与人来源菌株（str. Toulouse）存在一定差异，但需要进一步深入分析探讨其科学意义。
主要结论：
首次在我国发现了一组新埃立克体感染病例，确认我国东北地区为新埃立克体自然疫源地。流行病学调查发现我国多个地区多个鼠种均存在新埃立克体自然感染，表明该病原体在我国分布广泛。遗传进化分析发现新埃立克体的基因多样性可能与地理来源相关联。
流行病学调查发现我国多个地区猴中均存在五日热巴尔通体感染。首次发现猴中五日热巴尔通体感染的传播，并确认钝猴虱是其传播媒介。多方面研究结果有力证明猴是五日热巴尔通体的动物宿主。血清学调查发现猴携带的五日热巴尔通体可能会感染人。MLST分析发现14个新的五日热巴尔通体序列型，表明猴来源菌株基因多样性较人来源菌株更为丰富。遗传进化分析发现五日热巴尔通体的基因多样性与宿主种类存在关联，提示了五日热巴尔通体可能的起源。此外，本研究还首次测定了一株猴来源五日热巴尔通体的全基因组序列，初步比对分析发现与人来源菌株存在一定差异。
Emerging and re-emerging infectious diseases are an important issue in global health.The well-known SARS and highly pathogenic avian influenza cause great hazards insome regions, while the huge hazards from emerging and re-emerging vector-borneinfectious diseases can not be ignored. A disease that is transmitted to humans or otheranimals by an insect or other arthropod is called a vector-borne disease. Manyvector-borne diseases are natural focus diseases, and the etiologic pathogens includeviruses, rickettsiae, bacteria, parasites and so on. Vector-borne diseases are easilyprevalent in nature due to the widely distribution of vectors and reservoir hosts. Theoccurrence of natural disasters and the destruction of the natural environment willaffect the occurrence and prevalence of vector-borne diseases.
Because of the complex natural and geographical conditions, diverseenvironment, as well as rich flora and fauna, there are many kinds of vector-borneinfectious diseases in China. Recently, the emergence and prevalence of emergingtick-borne infectious diseases poses a serious threat to public health in China, such asthe emergence of anaplasmosis and the outbreak of severe fever withthrombocytopenia syndrome. Therefore, to find out the natural foci and epidemiologicfeatures, and to understand the pathogenic characteristics and the interaction betweenhosts and vectors, in-depth investigations on emerging and re-emerging vector-borneinfectious diseases should be performed. The findings from these researches willprovide a scientific basis and theoretical support for prevention and control ofdiseases.
“Anaplasmosis” is a generic name for infections caused by obligate intracellularbacteria in the family Anaplasmataceae, which is a kind of tick-borne disease andmainly includes human granulocytic anaplasmosis (HGA), human monocyticehrlichiosis and human ewingii ehrlichiosis. An outbreak of HGA including10caseswere reported in Anhui province in2006, and this study suggested human-to-humantransmission of HGA. Subsequently, no confirmed cases have been reported in China.Our previous surveys detected various tick-borne agents in ticks and animal hosts from forest areas of northeastern China, such as Anaplasma, Borrelia and rickettsia.In addition, our laboratory successfully isolated A. phagocytophilum from rodents andsheep in northeastern China, suggesting the potential existence of human cases ofHGA in this area.
In the current study, we initiated a surveillance project in Mudanjiang ForestryCentral Hospital, one of the largest hospitals treating patients with tick bites innortheastern China, and found7cases infected with Candidatus Neoehrlichiamikurensis. Field investigation on rodents and ticks confirmed the existence of localnatural foci.
When we used rhesus macaques for isolation of Candidatus Neoehrlichiamikurensis, Bartonella quintana infection in the macaques were unexpectedlyobserved. B. quintana was firstly recognized as the pathogenic bacteria of trenchfever. In China, few studies focus on B. quintana, which is recognized as are-emerging vector-borne pathogen now. The study was then aimed to identify theanimal hosts and vectors of B. quintana, and to investigate the genetic characteristicsof this agent.
The main results:
1Investigations on human infection with Candidatus Neoehrlichiamikurensis in eastern China
During May-July in2010, a total of628febrile patients who had a history ofrecent tick bites and sought treatment at Mudanjiang Forestry Central Hospital ofHeilongjiang Province were enrolled into the study. The peripheral blood sampleswere collected from the patients on acute phase of the illness. A board-range PCRtargeting rrs gene and a species-specific PCR targeting groEL gene were used todetect the infections of tick-borne agents. As a result,7patients were found to beinfected with Candidatus Neoehrlichia mikurensis through the amplifications of bothrrs and groEL. The amplified nearly entire rrs (1501bp) and partial groEL (914bp)nucleotide sequences from the7patients were identical, and same to those ofCandidatus Neoehrlichia mikurensis detected from I. persulctus ticks and field micein the Asian part of Russia.
Clinical symptoms of fever, headache and malaise were present in all7patients.Other major manifestations included nausea (5/7), vomiting (5/7), myalgia (4/7) andstiff neck (4/7). The less common symptoms were arthralgias (2/7) and cough (2/7),diarrhea (1/7), confusion (1/7) and erythema (1/7). Laboratory test results showed leucopenia in one patient, leukocytosis in one patient, thrombocytopenia in twopatients and anemia in two patients; serum levels of alanine aminotransferase andaspartate aminotransferase within reference ranges in all patients. Unlike previouslyreported cases with old age or immunocompromised conditions who developedoverwhelming disease, all7patients in the current study presented with relativelymild disease. The clinical manifestations might be typical of Candidatus Neoehrlichiamikurensis infection in otherwise healthy population.
A total of516host-seeking ticks, including316Ixodes persulcatus,187Haemaphysalis concinna and13Dermacentor silvarum, were collected on vegetationand individually examined. Candidatus Neoehrlichia mikurensis DNA was detected in6I. persulctus and2H. conicinna with an overall infection rate of1.6%, whereas noDNA from the agent was detected in D. silvarum. A total of211wild rodents from6species were captured and tested. Eight rodents from3species, includingClethrionomys rufocanus, Rattus norvegicus and Tamias sibiricus, were foundnaturally infected, with an overall infection rate of3.8%. These results suggest theexistence of natural foci of Candidatus Neoehrlichia mikurensis in Mudanjiang Cityof Heilongjiang Province.
2Prevalence and genetic diversity of Candidatus Neoehrlichia mikurensis inrodents from China
Totally,841rodents of22species from10genera were collected at15study sitesin11regions at local peak seasons of tick activities. The predominant species wereApodemus agrarius (14.6%) and the other species accounted for0.8%-10.3%. Eachregion had3-9species of rodents sampled. Candidatus Neoehrlichia mikurensis wasdetected in34(4.0%) of the841rodents by amplifications of both groEL and rrs. The34rodents with positive results represented9species, with the prevalence in eachspecies ranging from1.1%to25.0%. When each rodent species was compared withall others, the infection rates in Eothenomys custos (25.0%), Apodemus agrarius(12.0%) and A. sylvaticus (12.5%) were significantly higher. Candidatus Neoehrlichiamikurensis was detected in10of the15study sites, representing Heilongjiang, Jilin,Henan, Zhejiang, Fujian, Yunnan Provinces and Inner Mongolia Autonomous Region.The prevalence of Candidatus Neoehrlichia mikurensis in the7regions ranged from1.5%to13.8%and showed significant difference. Rodents collected from JilinProvince were at significantly higher risk for infection in contrast to other regions(P<0.001).
Sequence comparisons revealed three different Candidatus Neoehrlichiamikurensis variants that were consistent with the three geographic regions, NortheastChina, Southwest China and Southeast China. Phylogenetic analysis based on allavailable and comparably long groEL nucleotide sequences (874bp) revealed fourdistinct clusters of Candidatus Neoehrlichia mikurensis strains that related togeographic origins. Sequences obtained from Northeast China were identical to thosefrom the Asia part of Russia and included in Cluster I. Sequences obtained fromSouthwest China and Southeast China were separately clustered in Cluster III andCluster IV. No sequence of the present study was identified in Cluster II, which wasexclusively restricted to Europe. Similar to the phylogenetic analysis of groELsequences, neighbor-joining tree based on all available and nearly entire rrs (1303bp)also separated these sequences into four different clusters.
3Investigations on infection and transmission of Bartonella quintana inmacaques
Four of10one-year old male macaques housed in the Laboratory Animal Centerof the Academy of Military Medical Sciences were found to be infected with B.quintana by PCR, blood smear staining, transmission electron microscopy and bloodculture. Then, we performed an active surveillance of five primate centers acrossmainland China to identify prevalence and distribution of B. quintana infection incaptive monkeys. Blood samples were collected from328rhesus macaques and308cynomolgus macaques. A total of96(15.1%) macaques were positive for B. quintanaby PCR assay, including59rhesus macaques and37cynomolgus macaques; B.quintana isolates were successfully cultured from the blood of8.3%(53/636) of themacaques tested, including37rhesus macaques and16cynomolgus macaques.Prevalence of B. quintana in rhesus macaques was significantly higher than that incynomolgus macaques. B. quintana was detected in the macaques from each region,but the monkey colony from Nanyang City was at significantly higher risk (P<0.001).In both rhesus macaques and cynomolgus macaques, the B. quintana infection rates injuvenile (≤3years) and young animals (4~6y) were significantly higher than those inadult animals (≥7y)(P<0.05). No significant association was observed between theinfection and gender of the monkeys (P>0.05).
In three consecutive screening tests of a group of10monkeys, an increasedprevalence of B. quintana was observed with4of them being found positive at day1,7positive at day15, and all ten positive at day35during the period of observation. All10monkeys were found to be infested with lice at the last day of the observation(day36). Lice from each infested monkey were positive for detection of B. quintanaDNA, suggesting the lice might act as efficient vectors. By phylogenetic analysis andmorphologic features, the lice were identified as Pedicinus obtusus.
After intravenous inoculation of the Bartonella-negative macaques with B.quintana strain RM-11, the macaques presented a long-lasting chronic bacteremia, butdid not show abnormal clinical features. In addition, we found4workers involvedwith the macaques care showed seroconversion to antigens derived from our strain,suggesting that macaques could be a source for B. quintana human infection.
4Clonal and phylogenetic analysis of B. quintana
The current study identified44new nucleotide variable sites,25newly allelesand14novel sequence types (STs8-21) among53B. quintana isolates by MLSTanalysis. In contrast with the relatively low level of sequence divergence of B.quintana reported in humans, our investigation revealed much higher genetic diversityin non-human primates. To display the relationships between related STs, all macaqueisolates were analysed by eBURST v3in combination with the data from humanisolates. Two novel clonal complexes and three new singletons were found.Phylogenetic analysis based on the concatenated MLST sequences revealed3distinctclusters. Sequences (STs1–7) from B. quintana isolates recovered from humans wereincluded in Group1. Sequences (STs8–14) from16isolates cultured fromcynomolgus macaques belonged to Group2. Sequences (STs15–21) from37isolatescultured from rhesus macaques were found exclusively in Group3, which displayed ahigher homology to Group2than to Group1. The clustering of concatenatedsequences resembles the relationship between STs and host species, suggesting thegenetic diversity and host-cospeciation among the B. quintana populations. Inaddition, splits decomposition analysis and phi test were performed using Splitstree4,and revealed no apparent recombination among B. quintana isolates.
To further investigate genetic characteristics, the complete genome of B.quintana strain RM-11, which was isolated from rhesus macaques, was obtained by acombined strategy involving Solexa and Roche/454. A total of1204predicted proteincoding genes,42tRNA genes, and two copies each of the5S,16S, and23S rRNAgenes were identified. The genome sequence and annotation information areaccessible in the GenBank database with accession number CP003784. Using areciprocal best BLAST hit strategy, we identified a total of1096common genes between strains RM-11and Toulouse, a human B. quintana isolate whose genome hasbeen sequenced.
The main conclusions:
This study is the first report on human infection of the Candidatus Neoehrlichiamikurensis variant in the world other than Europe. Field survey on ticks and rodentssuggested that the existence of natural foci of Candidatus Neoehrlichia mikurensis innortheastern China. We detected Candidatus Neoehrlichia mikurensis in diversespecies of rodents from various regions, indicating the agent is widely distributed inChina. We also found that the Candidatus N. mikurensis gene clusters correlated withdistinct geographic origins.
The study demonstrated the prevalence of B. quintana infection was common inmacaques from main primate centers in mainland China. We firstly reported thetransmission of B. quintana in macaques, and identified Pedicinus obtusus lice as thevectors. These results indicated that macaques may serve as reservoir hosts for B.quintana. Serologic test suggested that macaques could be a source for B. quintanahuman infection. MLST analysis of the B. quintana isolates revealed14novelsequence types, suggesting much higher genetic diversity in non-human primates thanin humans. Phylogenetic analysis provided the evidence of host-cospeciation of B.quintana. These findings suggested that trench fever may primarily be a zoonoticdisease with macaques as the natural hosts. In addition, we sequenced the genome of aB. quintana strain, which was isolated from the blood of a rhesus macaque.
引文
1. Anderson BE, Dawson JE, Jones DC, Wilson KH. Ehrlichia chaffeensis, a newspecies associated with human ehrlichiosis. J Clin Microbiol.1991;29:2838–42.
2. Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of agranulocytotropic Ehrlichia species as the etiologic agent of human disease. J ClinMicrobiol.1994;32:589–95.
3. Buller RS, Arens M, Hmiel SP, et al. Ehrlichia ewingii, a newly recognized agentof human ehrlichiosis. N Eng J Med.1999;341:148–55.
4. Perez M, Rikihisa Y, Wen B. Ehrlichia canis-like agent isolated from a man inVenezuela: antigenic and genetic characterization. J Clin Microbiol.1996;34:2133–9.
5. Perez M, Bodor M, Zhang C, Xiong Q, Rikihisa Y. Human infection withEhrlichia canis accompanied by clinical signs in Venezuela. Ann N Y Acad Sci.2006;1078:110–7.
6. Newton PN, Rolain JM, Rasachak B, et al. Sennetsu neorickettsiosis: a probablefish-borne cause of fever rediscovered in Laos. Am J Trop Med Hyg.2009;81:190-4.
7. Schouls LM, Van De Pol I, Rijpkema SG, Schot CS. Detection and identificationof Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in DutchIxodes ricinus ticks. J Clin Microbiol.1999;37:2215-22.
8. Kawahara M, Rikihisa Y, Isogai E, et al. Ultrastructure and phylogenetic analysisof 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolatedfrom wild rats and found in Ixodes ovatus ticks. Int J Syst Evol Microbiol.2004;54:1837-43.
9. Welinder-Olsson C, Kjellin E, Vaht K, Jacobsson S, Wenneras C. First case ofhuman "Candidatus Neoehrlichia mikurensis" infection in a febrile patient withchronic lymphocytic leukemia. J Clin Microbiol.2010;48:1956-9.
10. von Loewenich FD, Geissdorfer W, Disque C, et al. Detection of "CandidatusNeoehrlichia mikurensis" in two patients with severe febrile illnesses: evidencefor a European sequence variant. J Clin Microbiol.2010;48:2630-5.
11. Fehr JS, Bloemberg GV, Ritter C, et al. Septicemia caused by tick-borne bacterialpathogen Candidatus Neoehrlichia mikurensis. Emerg Infect Dis.2010;16:1127-9.
12. Pekova S, Vydra J, Kabickova H, et al. Candidatus Neoehrlichia mikurensisinfection identified in2hematooncologic patients: benefit of molecular techniquesfor rare pathogen detection. Diagn Microbiol Infect Dis.2011;69:266-70.
13. Wen BH, Cao WC, Pan H. Ehrlichiae and ehrlichial diseases in China. Ann N YAcad Sci.2003;990:45–53.
14. Zhang LJ, Liu Y, Ni D, et a1. Nosocomial transmission of human granulocyticanaplasmosis in China. JAMA.2008;300:2263-70.
15. Cao WC, Zhao QM, Zhang PH, et al. Prevalence of Anaplasma phagocytophilaand Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China. AmJ Trop Med Hyg.2003;68:547-50.
16. Cao WC, Zhan L, De Vlas SJ, et al. Molecular detection of spotted fever groupRickettsia in Dermacentor silvarum from a forest area of northeastern China. JMed Entomol.2008;45:741-4.
17. Zhan L, Cao WC, Jiang JF, et al. Anaplasma phagocytophilum from Rodents andSheep, China. Emerg Infect Dis.2010;16:764-8.
18. Wen B, Jian R, Zhang Y, Chen R. Simultaneous detection of Anaplasmamarginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis bysequence analyses of16S ribosomal DNA in Boophilus microplus ticks from Tibet.J Clin Microbiol.2002;40:3286-90.
19. Parola P, Roux V, Camicas JL, et al. Detection of ehrlichiae in African ticks bypolymerase chain reaction. Trans R Soc Trop Med Hyg.2000;94:707-8.
20. Liz JS, Anderes L, Sumner JW, et al. PCR detection of granulocytic ehrlichiaeinIxodes ricinus ticks and wild small mammals in western Switzerland. J ClinMicrobiol.2000;38:1002–7.
21. Alekseev AN, Dubinina HV, Van De PI, Schouls LM. Identification of Ehrlichiaspp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J ClinMicrobiol.2001;39:2237–42.
22. Pan H, Liu S, Ma Y, Tong S, Sun Y. Ehrlichia-like organism gene found in smallmammals in the suburban district of Guangzhou of China. Ann N Y Acad Sci.2003;990:107-11.
23. Brouqui P, Sanogo YO, Caruso G, Merola F, Raoult D. Candidatus Ehrlichiawalkerii: a new Ehrlichia detected in Ixodes ricinus tick collected fromasymptomatic humans in Northern Italy. Ann N Y Acad Sci.2003;990:134–40.
24. Bakken JS, Dumler JS. Human granulocytic ehrlichiosis. Clin Infect Dis2000;31:554–60.
25. Horowitz HW, Aguero-Rosenfeld ME, McKenna DF, et al. Clinical and laboratoryspectrum of culture-proven human granulocytic ehrlichiosis: comparison withculture-negative cases. Clin Infect Dis.1998;27:1314–7.
26. Standaert SM, Yu T, Scott MA, et al. Primary isolation of Ehrlichia chaffeensisfrom patients with febrile illnesses: clinical and molecular characteristics. J InfectDis.2000;181:1082–8.
27. Olano JP, Masters E, Hogrefe W, Walker DH. Human monocytotropic ehrlichiosis,Missouri. Emerg Infect Dis.2003;9:1579–86.
1. Schouls LM, Van De Pol I, Rijpkema SG, Schot CS. Detection and identificationof Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in DutchIxodes ricinus ticks. J Clin Microbiol.1999;37:2215-22.
2. Kawahara M, Rikihisa Y, Isogai E, et al. Ultrastructure and phylogenetic analysisof 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolatedfrom wild rats and found in Ixodes ovatus ticks. Int J Syst Evol Microbiol.2004;54:1837-43.
3. Welinder-Olsson C, Kjellin E, Vaht K, Jacobsson S, Wenneras C. First case ofhuman "Candidatus Neoehrlichia mikurensis" infection in a febrile patient withchronic lymphocytic leukemia. J Clin Microbiol.2010;48:1956-9.
4. von Loewenich FD, Geissdorfer W, Disque C, et al. Detection of "CandidatusNeoehrlichia mikurensis" in two patients with severe febrile illnesses: evidencefor a European sequence variant. J Clin Microbiol.2010;48:2630-5.
5. Fehr JS, Bloemberg GV, Ritter C, et al. Septicemia caused by tick-borne bacterialpathogen Candidatus Neoehrlichia mikurensis. Emerg Infect Dis.2010;16:1127-9.
6. Pekova S, Vydra J, Kabickova H, et al. Candidatus Neoehrlichia mikurensisinfection identified in2hematooncologic patients: benefit of molecular techniquesfor rare pathogen detection. Diagn Microbiol Infect Dis.2011;69:266-70.
7. Brouqui P, Sanogo YO, Caruso G, Merola F, Raoult D. Candidatus Ehrlichiawalkerii: a new Ehrlichia detected in Ixodes ricinus tick collected fromasymptomatic humans in Northern Italy. Ann N Y Acad Sci.2003;990:134–40.
8. Andersson M, Raberg L. Wild rodents and novel human pathogen CandidatusNeoehrlichia mikurensis, southern Sweden. Emerg Infect Dis.2011;17:1716-18.
9. Fertner ME, Molbak L, Boye Pihl TP, Fomsgaard A, Bodker R. First detection oftick-borne “Candidatus Neoehrlichia mikurensis” in Denmark2011.2012;17:pii=20096.
10. Pan H, Liu S, Ma Y, Tong S, Sun Y. Ehrlichia-like organism gene found in smallmammals in the suburban district of Guangzhou of China. Ann N Y Acad Sci.2003;990:107-11.
11. Rar VA, Livanova NN, Panov VV, et al. Genetic diversity of Anaplasma andEhrlichia in the Asian part of Russia. Ticks Tick Borne Dis.2010;1:57-65.
12. Alekseev AN, Dubinina HV, Van De PI, Schouls LM. Identification of Ehrlichiaspp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J ClinMicrobiol.2001;39:2237–42.
13. Yabsley MJ, Murphy SM, Luttrell MP, et al. Characterization of “CandidatusNeoehrlichia lotoris”(family Anaplasmataceae) from raccoons (Procyon lotor).Int J Syst Evol Microbiol.2008;58:2794–8.
14. Maurer FP, Keller PM, Beuret C, et al. Close geographic association of humanneoehrlichiosis and tick populations carrying “Candidatus Neoehrlichia lotoris” ineastern Switzerland. J Clin Microbiol.2013;51:169-76.
15. Tabara K, Arai S, Kawabuchi T, et al. Molecular survey of Babesia microti,Ehrlichia species and Candidatus Neoehrlichia mikurensis in wild rodents fromShimane Prefecture, Japan. Microbiol Immunol.2007;51:359–67.
16. Dumler JS, Barbet AF, Bekker CPJ, et al. Reorganization of genera in the familiesRickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification ofsome species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichiawith Neorickettsia, descriptions of six new species combinations and designationof Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichiaphagocytophila. Int J Syst Evol Microbiol.2001;51:2145-65.
1. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. ClinMicrobiol Rev.1996;9:273–92.
2. McNee JW, Renshaw A, Brunt EH.“Trench fever”: a relapsing fever occurringwith the British forces in France. BMJ.1916;12:225–34.
3. Byam W, Carroll JH, Churchill JH, et al. Trench fever. Oxford (UK): OxfordUniversity Press;1919.
4. Vinson JW. In vitro cultivation of the rickettsial agent of trench fever. Bull WorldHealth Organ.1966;35:155–64.
5. Brouqui P, Lascola B, Roux V, Raoult D. Chronic Bartonella quintana bacteremiain homeless patients. N Engl J Med.1999;340:184–9.
6. Drancourt M, Mainardi JL, Brouqui P, et al. Bartonella (Rochalimaea) quintanaendocarditis in three homeless men. N Engl J Med.1995;332:419–23.
7. Koehler JE, Quinn FD, Berger TG, LeBoit PE, Tappero JW. Isolation ofRochalimaea species from cutaneous and osseous lesions of bacillaryangiomatosis. N Engl J Med.1992;327:1625–31.
8. Raoult D, Roux V. The body louse as a vector of reemerging human diseases. ClinInfect Dis.1999;29:888–911.
9. Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of Bartonellaquintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, andWolbachia pipientis in cat fleas, France. Emerg Infects Dis.2003;9:338-42.
10. La VD, Tran-Hung L, Aboudharam G, Raoult D, Drancourt M. Bartonellaquintana in domestic cat. Emerg Infect Dis.2005;11:1287-9.
11. Kelly P, Rolain JM, Maggi R, Sontakke S, Keene B. Bartonella quintanaendocarditis in dogs. Emerg Infect Dis.2006;12:1869-72.
12. O'Rourke LG, Pitulle C, Hegarty BC, et al. Bartonella quintana in cynomolgusmonkey (Macaca fascicularis). Emerg Infect Dis.2005;11:1931-4.
13. Huang RT, Liu QY, Li G, et al. Bartonella quintana infections in captive monkeys,China. Emerg Infect Dis.2011;17:1707-9.
14.杨发莲,杨慧,白鹤鸣,于彬彬,张青.不明原因发热病人中五日热巴尔通体感染调查.地方病通报.2007;22:90.
15.杨慧,白鹤鸣,杨发莲,于彬彬.云南巴尔通体感染的血清学调查.中国自然医学杂志.2007;9:277-80.
16. Jensen WA, Fall MZ, Rooney J, Kordick DL, Breitschwerdt EB. Rapididentification and differentiation of Bartonella species using a single-step PCRassay. J Clin Microbiol.2000;38:1717-22.
17. Birtles RJ, Raoult D. Comparison of partial citrate synthase gene (gltA) sequencesfor phylogenetic analysis of Bartonella species. Int J Syst Bacteriol.1996;46:891-7.
18. Roux V, Raoult D. The16S-23S rRNA intergenic spacer region of Bartonella(Rochalimaea) species is longer than usually described in other bacteria. Gene.1995;156:107-11.
19. Roux V, Raoult D. Inter-and intraspecies identification of Bartonella(Rochalimaea) species. J Clin Microbiol.1995;33:1573-9.
20. Pitulle C, Strehse C, Brown JW, Breitschwerdt EB. Investigation of thephylogenetic relationships within the genus Bartonella based on comparativesequence analysis of the rnpB gene,16S rDNA and23S rDNA. Int J Syst EvolMicrobiol.2002;52:2075-80.
21. Ohl ME, Spach DH. Bartonella quintana and Urban Trench Fever. Clin Infect Dis.2000;31:131-5.
22. Guptill L. Bartonellosis. Vet Microbiol,2010;140:347-59.
23. Chomel BB, Boulouis HJ, Breitschwerdt EB, et al. Ecological fitness andstrategies of adaptation of Bartonella species to their hosts and vectors. Vet Res.2009;40:29.
24. Bass JW, Vincent JM, Person DA. The expanding spectrum of Bartonellainfections: I. Bartonellosis and trench fever. Pediatr Infect Dis J.1997;16:2-10.
1. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. ClinMicrobiol Rev.1996;9:273–92.
2. Raoult D, Roux V. The body louse as a vector of reemerging human diseases. ClinInfect Dis.1999;29:888–911.
3. O'Rourke LG, Pitulle C, Hegarty BC, et al. Bartonella quintana in cynomolgusmonkey (Macaca fascicularis). Emerg Infect Dis.2005;11:1931-4.
4. Huang RT, Liu QY, Li G, et al. Bartonella quintana infections in captive monkeys,China. Emerg Infect Dis.2011;17:1707-9.
5. Alsmark CM, Frank AC, Karlberg EO, et al. The louse-borne human pathogenBartonella quintana is a genomic derivative of the zoonotic agent Bartonellahenselae. PNAS.2004;101:9716–21.
6. Spach DH, Kanter AS, Dougherty MJ, et al. Bartonella (Rochalimaea) quintanabacteremia in inner-city patients with chronic alcoholism. N Engl J Med.1995;332:424–8.
7. Rodriguez-Barradas MC, Hamill RJ, Houston ED, et al. Genomic fingerprintingof Bartonella species by repetitive element PCR for distinguishing species andisolates. J Clin Microbiol.1995;33:1089–93.
8. Roux V, Raoult D. Inter-and intraspecies identification of Bartonella(Rochalimaea) species. J Clin Microbiol.1995;33:1573-9.
9. Foucault C, La Scola B, Lindroos H, Andersson SG, Raoult D. Multispacer typingtechnique for sequence-based typing of Bartonella quintana. J Clin Microbiol.2005;43:41–8.
10. Feil EJ, Enright MC. Analyses of clonality and the evolution of bacterialpathogens. Curr Opin Microbiol.2004;7:308–13.
11. Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol.2006;60:561–88.
12. Iredell J, Blanckenberg D, Arvand M, et al. Characterization of the naturalpopulation of Bartonella henselae by multilocus sequence typing. J ClinMicrobiol.2003;41:5071–9.
13. Arvand M, Viezens J. Evaluation of pulsed-field gel electrophoresis andmulti-locus sequence typing for the analysis of clonal relatedness amongBartonella henselae isolates. Int J Med Microbiol.2007;297:255–62.
14. Arvand M, Feil EJ, Giladi M, Boulouis HJ, Viezens J. Multi-locus sequencetyping of Bartonella henselae isolates from three continents reveals hypervirulentand feline-associated clones. PLoS ONE.2007;2:e1346.
15. Arvand M, Raoult D, Feil EJ. Multi-locus sequence typing of a geographically andtemporally diverse sample of the highly clonal human pathogen Bartonellaquintana. PLoS ONE.2010;5:e9765.
16. Rozas J, Rozas R. DnaSP version3: an integrated program for molecularpopulation genetics and molecular evolution analysis. Bioinformatics.1999;15:174-5.
17. Tamura K, Peterson D, Peterson N, et al. MEGA5: Molecular EvolutionaryGenetics Analysis using maximum likelihood, evolutionary distance, andmaximum parsimony methods. Mol Biol Evoln.2011;28:2731–2739.
18. Huson DH, Bryant D. Application of phylogenetic networks in evolutionarystudies. Mol Biol Evol.2006;23:254–267.
19.王中强,邱少富,王勇,孙岩松,宋宏彬.多位点序列分型技术及其研究进展.军事医学.2010;34:76-9.
20. Drancourt M, Tran-Hung L, Courtin J, de Lumley H, Raoult D. Bartonellaquintana in a4000-year-old human tooth. J Infect Dis.2005;191:607-11.
21. Raoult D, Dutour O, Houhamdi L, et al. Evidence for louse-transmitted diseasesin soldiers of Napoleon's Grand Army in Vilnius. J Infect Dis.2006;193:112-20.
22. Chomel BB, Boulouis HJ, Breitschwerdt EB, et al. Ecological fitness andstrategies of adaptation of Bartonella species to their hosts and vectors. Vet Res.2009;40.
23. Engel P, Salzburger W, Liesch M, et al. Parallel evolution of a type IV secretionsystem in radiating lineages of the host-restricted bacterial pathogen Bartonella.PLoS Genetics.2011;7:e1001296.
24. Saenz HL, Engel P, Stoeckli MC, et al. Genomic analysis of Bartonella identifiestype IV secretion systems as host adaptability factors. Nature.2007;39:1469-76.
25. Greger M. The human/animal interface: emergence and resurgence of zoonoticinfectious diseases. Crit Rev Microbiol.2007;33:243-99.
1. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. ClinMicrobiol Rev.1996;9:273–92.
2. McNee JW, Renshaw A, Brunt EH.“Trench fever”: a relapsing fever occurringwith the British forces in France. BMJ.1916;12:225–34.
3. Byam W, Carroll JH, Churchill JH, et al. Trench fever. Oxford (UK): OxfordUniversity Press;1919.
4. Vinson JW. In vitro cultivation of the rickettsial agent of trench fever. Bull WorldHealth Organ.1966;35:155–64.
5. Kostrzewski J. The epidemiology of trench fever. Bull Acad Pol Sci (Med).1949;7:233–63.
6. Raoult D, Roux V. The body louse as a vector of reemerging human diseases. ClinInfect Dis.1999;29:888–911.
7. Brouqui P, Lascola B, Roux V, Raoult D. Chronic Bartonella quintana bacteremiain homeless patients. N Engl J Med.1999;340:184–9.
8. Spach DH, Kanter AS, Dougherty MJ, et al. Bartonella (Rochalimaea) quintanabacteremia in inner-city patients with chronic alcoholism. N Engl J Med.1995;332:424–8.
9. Brenner DJ, O’Connor SP, Winkler HH, Steigerwalt AG. Proposals to unify thegenera Bartonella and Rochalimaea, with descriptions of Bartonella quintanacomb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., andBartonella elizabethae comb. nov., and to remove the family Bartonellaceae fromthe order Rickettsiales. Int J Syst Bacteriol.1993;43:777–86.
10. Birtles RJ, Harrison TG, Saunders NA, Molyneux DH. Proposals to unify thegenera Grahamella and Bartonella, with descriptions of Bartonella talpae comb.nov., Bartonella peromysci comb. nov., and three new species, Bartonellagrahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov. IntJ Syst Bacteriol.1995;45:1–8.
11. Guptill L. Bartonellosis. Vet Microbiol,2010;140:347-59.
12.杨发莲,白鹤鸣,杨慧,张青.巴尔通体分离培养特性观察.微生物学杂志.2006;26:6-9.
13.杨发莲,白鹤鸣,杨慧,董兴齐,宋志忠.巴尔通体I、II代扫描透射电镜观察.中国病原生物学杂志.2007;2:168-169.
14. Alsmark CM, Frank AC, Karlberg EO, et al. The louse-borne human pathogenBartonella quintana is a genomic derivative of the zoonotic agent Bartonellahenselae. PNAS.2004;101:9716–21.
15. Rodriguez-Barradas MC, Hamill RJ, Houston ED, et al. Genomic fingerprintingof Bartonella species by repetitive element PCR for distinguishing species andisolates. J Clin Microbiol.1995;33:1089–93.
16. Roux V, Raoult D. Inter-and intraspecies identification of Bartonella(Rochalimaea) species. J Clin Microbiol.1995;33:1573-9.
17. Foucault C, La Scola B, Lindroos H, Andersson SG, Raoult D. Multispacer typingtechnique for sequence-based typing of Bartonella quintana. J Clin Microbiol.2005;43:41–8.
18. Arvand M, Raoult D, Feil EJ. Multi-locus sequence typing of a geographically andtemporally diverse sample of the highly clonal human pathogen Bartonellaquintana. PLoS ONE.2010;5:e9765.
19. Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of Bartonellaquintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, andWolbachia pipientis in cat fleas, France. Emerg Infects Dis.2003;9:338-42.
20. La VD, Tran-Hung L, Aboudharam G, Raoult D, Drancourt M. Bartonellaquintana in domestic cat. Emerg Infect Dis.2005;11:1287-9.
21. Kelly P, Rolain JM, Maggi R, Sontakke S, Keene B. Bartonella quintanaendocarditis in dogs. Emerg Infect Dis.2006;12:1869-72.
22. O'Rourke LG, Pitulle C, Hegarty BC, et al. Bartonella quintana in cynomolgusmonkey (Macaca fascicularis). Emerg Infect Dis.2005;11:1931-4.
23. Huang RT, Liu QY, Li G, et al. Bartonella quintana infections in captive monkeys,China. Emerg Infect Dis.2011;17:1707-9.
24. Mooser H, Weyer F. Experimental infection of Macacus rhesus with Rickettsiaquintana (trench fever). Proc Soc Exp Biol Med.1953;83:699-701.
25. Zhang P, Chomel BB, Schau MK, et al. A family of variably expressedouter-membrane proteins (Vomp) mediates adhesion and autoaggregation inBartonella quintana. PNAS.2004;101:13630-5.
26. Sasaki T, Poudel SK, Isawa H, et al. First molecular evidence of Bartonellaquintana in Pediculus humanus capitis (Phthiraptera: Pediculidae), collected fromNepalesechildren. J Med Entomol.2006;43:110–2.
27. Bonilla DL, Kabeya H, Henn J, Kramer VL, Kosoy MY. Bartonella quintana inbody lice and head lice from homeless persons, San Francisco, California, USA.Emerg Infect Dis.2009;15:912-5.
28. Telford III SR, Wormser GP. Bartonella spp. transmission by ticks not established.Emerg Infect Dis.2010;16:379-84.
29. Angelakis E, Billeter SA, Breitschwerdt EB, et al. Potential for tick borneBartonelloses. Emerg Infect Dis.2010;16:385-91.
30. Reis1C, Cote1M, Le Rhun D, et al. Vector competence of the tick Ixodes ricinusfor transmission of Bartonella birtlesii. PLoS Negl Trop Dis;5:e1186.
31. Raoult D, Drancourt M, Carta A, Gastaut JA. Bartonella (Rochalimaea) quintanaisolation in patient with chronic adenopathy, lymphopenia, and a cat. Lancet.1994;343:977.
32. Drancourt M, Mainardi JL, Brouqui P, et al. Bartonella (Rochalimaea) quintanaendocarditis in three homeless men. N Engl J Med.1995;332:419–23.
33. La Scola B, Raoult D. Culture of Bartonella quintana and Bartonella henselaefrom human samples: a5-year experience (1993to1998). J Clin Microbiol.1999;37:1899–905.
34. Fournier PE, Mainardi JL, Raoult D. Value of microimmunofluorescence fordiagnosis and follow-up of Bartonella endocarditis. Clin Diagn Lab Immunol.2002;9:795–801.
35. Houpikian P, Raoult D. Western immunoblotting for Bartonella endocarditis. ClinDiagn Lab Immunol.2003;10:95–102.
36. Rolain JM, Brouqui P, Koehler JE, et al. Recommendations for treatment ofhuman infections caused by Bartonella species. Antimicrob Agents Chemother.2004;48:1921–33.
37. Raoult D, Fournier PE, Vandenesch F, et al. Outcome and treatment of Bartonellaendocarditis. Arch Intern Med.2003;163:226–30.
38. Meghari S, Rolain JM, Grau GE, et al. Antiangiogenic effect of erythromycin: anin vitro model of Bartonella quintana infection. J Infect Dis.2006;193:380–6.
39.杨发莲,杨慧,白鹤鸣,于彬彬,张青.不明原因发热病人中五日热巴尔通体感染调查.地方病通报.2007;22:90.
40.杨慧,白鹤鸣,杨发莲,于彬彬.云南巴尔通体感染的血清学调查.中国自然医学杂志.2007;9:277-80.
2. Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of agranulocytotropic Ehrlichia species as the etiologic agent of human disease. J ClinMicrobiol.1994;32:589–95.
3. Buller RS, Arens M, Hmiel SP, et al. Ehrlichia ewingii, a newly recognized agentof human ehrlichiosis. N Eng J Med.1999;341:148–55.
4. Perez M, Rikihisa Y, Wen B. Ehrlichia canis-like agent isolated from a man inVenezuela: antigenic and genetic characterization. J Clin Microbiol.1996;34:2133–9.
5. Perez M, Bodor M, Zhang C, Xiong Q, Rikihisa Y. Human infection withEhrlichia canis accompanied by clinical signs in Venezuela. Ann N Y Acad Sci.2006;1078:110–7.
6. Newton PN, Rolain JM, Rasachak B, et al. Sennetsu neorickettsiosis: a probablefish-borne cause of fever rediscovered in Laos. Am J Trop Med Hyg.2009;81:190-4.
7. Schouls LM, Van De Pol I, Rijpkema SG, Schot CS. Detection and identificationof Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in DutchIxodes ricinus ticks. J Clin Microbiol.1999;37:2215-22.
8. Kawahara M, Rikihisa Y, Isogai E, et al. Ultrastructure and phylogenetic analysisof 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolatedfrom wild rats and found in Ixodes ovatus ticks. Int J Syst Evol Microbiol.2004;54:1837-43.
9. Welinder-Olsson C, Kjellin E, Vaht K, Jacobsson S, Wenneras C. First case ofhuman "Candidatus Neoehrlichia mikurensis" infection in a febrile patient withchronic lymphocytic leukemia. J Clin Microbiol.2010;48:1956-9.
10. von Loewenich FD, Geissdorfer W, Disque C, et al. Detection of "CandidatusNeoehrlichia mikurensis" in two patients with severe febrile illnesses: evidencefor a European sequence variant. J Clin Microbiol.2010;48:2630-5.
11. Fehr JS, Bloemberg GV, Ritter C, et al. Septicemia caused by tick-borne bacterialpathogen Candidatus Neoehrlichia mikurensis. Emerg Infect Dis.2010;16:1127-9.
12. Pekova S, Vydra J, Kabickova H, et al. Candidatus Neoehrlichia mikurensisinfection identified in2hematooncologic patients: benefit of molecular techniquesfor rare pathogen detection. Diagn Microbiol Infect Dis.2011;69:266-70.
13. Wen BH, Cao WC, Pan H. Ehrlichiae and ehrlichial diseases in China. Ann N YAcad Sci.2003;990:45–53.
14. Zhang LJ, Liu Y, Ni D, et a1. Nosocomial transmission of human granulocyticanaplasmosis in China. JAMA.2008;300:2263-70.
15. Cao WC, Zhao QM, Zhang PH, et al. Prevalence of Anaplasma phagocytophilaand Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China. AmJ Trop Med Hyg.2003;68:547-50.
16. Cao WC, Zhan L, De Vlas SJ, et al. Molecular detection of spotted fever groupRickettsia in Dermacentor silvarum from a forest area of northeastern China. JMed Entomol.2008;45:741-4.
17. Zhan L, Cao WC, Jiang JF, et al. Anaplasma phagocytophilum from Rodents andSheep, China. Emerg Infect Dis.2010;16:764-8.
18. Wen B, Jian R, Zhang Y, Chen R. Simultaneous detection of Anaplasmamarginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis bysequence analyses of16S ribosomal DNA in Boophilus microplus ticks from Tibet.J Clin Microbiol.2002;40:3286-90.
19. Parola P, Roux V, Camicas JL, et al. Detection of ehrlichiae in African ticks bypolymerase chain reaction. Trans R Soc Trop Med Hyg.2000;94:707-8.
20. Liz JS, Anderes L, Sumner JW, et al. PCR detection of granulocytic ehrlichiaeinIxodes ricinus ticks and wild small mammals in western Switzerland. J ClinMicrobiol.2000;38:1002–7.
21. Alekseev AN, Dubinina HV, Van De PI, Schouls LM. Identification of Ehrlichiaspp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J ClinMicrobiol.2001;39:2237–42.
22. Pan H, Liu S, Ma Y, Tong S, Sun Y. Ehrlichia-like organism gene found in smallmammals in the suburban district of Guangzhou of China. Ann N Y Acad Sci.2003;990:107-11.
23. Brouqui P, Sanogo YO, Caruso G, Merola F, Raoult D. Candidatus Ehrlichiawalkerii: a new Ehrlichia detected in Ixodes ricinus tick collected fromasymptomatic humans in Northern Italy. Ann N Y Acad Sci.2003;990:134–40.
24. Bakken JS, Dumler JS. Human granulocytic ehrlichiosis. Clin Infect Dis2000;31:554–60.
25. Horowitz HW, Aguero-Rosenfeld ME, McKenna DF, et al. Clinical and laboratoryspectrum of culture-proven human granulocytic ehrlichiosis: comparison withculture-negative cases. Clin Infect Dis.1998;27:1314–7.
26. Standaert SM, Yu T, Scott MA, et al. Primary isolation of Ehrlichia chaffeensisfrom patients with febrile illnesses: clinical and molecular characteristics. J InfectDis.2000;181:1082–8.
27. Olano JP, Masters E, Hogrefe W, Walker DH. Human monocytotropic ehrlichiosis,Missouri. Emerg Infect Dis.2003;9:1579–86.
1. Schouls LM, Van De Pol I, Rijpkema SG, Schot CS. Detection and identificationof Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in DutchIxodes ricinus ticks. J Clin Microbiol.1999;37:2215-22.
2. Kawahara M, Rikihisa Y, Isogai E, et al. Ultrastructure and phylogenetic analysisof 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolatedfrom wild rats and found in Ixodes ovatus ticks. Int J Syst Evol Microbiol.2004;54:1837-43.
3. Welinder-Olsson C, Kjellin E, Vaht K, Jacobsson S, Wenneras C. First case ofhuman "Candidatus Neoehrlichia mikurensis" infection in a febrile patient withchronic lymphocytic leukemia. J Clin Microbiol.2010;48:1956-9.
4. von Loewenich FD, Geissdorfer W, Disque C, et al. Detection of "CandidatusNeoehrlichia mikurensis" in two patients with severe febrile illnesses: evidencefor a European sequence variant. J Clin Microbiol.2010;48:2630-5.
5. Fehr JS, Bloemberg GV, Ritter C, et al. Septicemia caused by tick-borne bacterialpathogen Candidatus Neoehrlichia mikurensis. Emerg Infect Dis.2010;16:1127-9.
6. Pekova S, Vydra J, Kabickova H, et al. Candidatus Neoehrlichia mikurensisinfection identified in2hematooncologic patients: benefit of molecular techniquesfor rare pathogen detection. Diagn Microbiol Infect Dis.2011;69:266-70.
7. Brouqui P, Sanogo YO, Caruso G, Merola F, Raoult D. Candidatus Ehrlichiawalkerii: a new Ehrlichia detected in Ixodes ricinus tick collected fromasymptomatic humans in Northern Italy. Ann N Y Acad Sci.2003;990:134–40.
8. Andersson M, Raberg L. Wild rodents and novel human pathogen CandidatusNeoehrlichia mikurensis, southern Sweden. Emerg Infect Dis.2011;17:1716-18.
9. Fertner ME, Molbak L, Boye Pihl TP, Fomsgaard A, Bodker R. First detection oftick-borne “Candidatus Neoehrlichia mikurensis” in Denmark2011.2012;17:pii=20096.
10. Pan H, Liu S, Ma Y, Tong S, Sun Y. Ehrlichia-like organism gene found in smallmammals in the suburban district of Guangzhou of China. Ann N Y Acad Sci.2003;990:107-11.
11. Rar VA, Livanova NN, Panov VV, et al. Genetic diversity of Anaplasma andEhrlichia in the Asian part of Russia. Ticks Tick Borne Dis.2010;1:57-65.
12. Alekseev AN, Dubinina HV, Van De PI, Schouls LM. Identification of Ehrlichiaspp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J ClinMicrobiol.2001;39:2237–42.
13. Yabsley MJ, Murphy SM, Luttrell MP, et al. Characterization of “CandidatusNeoehrlichia lotoris”(family Anaplasmataceae) from raccoons (Procyon lotor).Int J Syst Evol Microbiol.2008;58:2794–8.
14. Maurer FP, Keller PM, Beuret C, et al. Close geographic association of humanneoehrlichiosis and tick populations carrying “Candidatus Neoehrlichia lotoris” ineastern Switzerland. J Clin Microbiol.2013;51:169-76.
15. Tabara K, Arai S, Kawabuchi T, et al. Molecular survey of Babesia microti,Ehrlichia species and Candidatus Neoehrlichia mikurensis in wild rodents fromShimane Prefecture, Japan. Microbiol Immunol.2007;51:359–67.
16. Dumler JS, Barbet AF, Bekker CPJ, et al. Reorganization of genera in the familiesRickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification ofsome species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichiawith Neorickettsia, descriptions of six new species combinations and designationof Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichiaphagocytophila. Int J Syst Evol Microbiol.2001;51:2145-65.
1. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. ClinMicrobiol Rev.1996;9:273–92.
2. McNee JW, Renshaw A, Brunt EH.“Trench fever”: a relapsing fever occurringwith the British forces in France. BMJ.1916;12:225–34.
3. Byam W, Carroll JH, Churchill JH, et al. Trench fever. Oxford (UK): OxfordUniversity Press;1919.
4. Vinson JW. In vitro cultivation of the rickettsial agent of trench fever. Bull WorldHealth Organ.1966;35:155–64.
5. Brouqui P, Lascola B, Roux V, Raoult D. Chronic Bartonella quintana bacteremiain homeless patients. N Engl J Med.1999;340:184–9.
6. Drancourt M, Mainardi JL, Brouqui P, et al. Bartonella (Rochalimaea) quintanaendocarditis in three homeless men. N Engl J Med.1995;332:419–23.
7. Koehler JE, Quinn FD, Berger TG, LeBoit PE, Tappero JW. Isolation ofRochalimaea species from cutaneous and osseous lesions of bacillaryangiomatosis. N Engl J Med.1992;327:1625–31.
8. Raoult D, Roux V. The body louse as a vector of reemerging human diseases. ClinInfect Dis.1999;29:888–911.
9. Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of Bartonellaquintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, andWolbachia pipientis in cat fleas, France. Emerg Infects Dis.2003;9:338-42.
10. La VD, Tran-Hung L, Aboudharam G, Raoult D, Drancourt M. Bartonellaquintana in domestic cat. Emerg Infect Dis.2005;11:1287-9.
11. Kelly P, Rolain JM, Maggi R, Sontakke S, Keene B. Bartonella quintanaendocarditis in dogs. Emerg Infect Dis.2006;12:1869-72.
12. O'Rourke LG, Pitulle C, Hegarty BC, et al. Bartonella quintana in cynomolgusmonkey (Macaca fascicularis). Emerg Infect Dis.2005;11:1931-4.
13. Huang RT, Liu QY, Li G, et al. Bartonella quintana infections in captive monkeys,China. Emerg Infect Dis.2011;17:1707-9.
14.杨发莲,杨慧,白鹤鸣,于彬彬,张青.不明原因发热病人中五日热巴尔通体感染调查.地方病通报.2007;22:90.
15.杨慧,白鹤鸣,杨发莲,于彬彬.云南巴尔通体感染的血清学调查.中国自然医学杂志.2007;9:277-80.
16. Jensen WA, Fall MZ, Rooney J, Kordick DL, Breitschwerdt EB. Rapididentification and differentiation of Bartonella species using a single-step PCRassay. J Clin Microbiol.2000;38:1717-22.
17. Birtles RJ, Raoult D. Comparison of partial citrate synthase gene (gltA) sequencesfor phylogenetic analysis of Bartonella species. Int J Syst Bacteriol.1996;46:891-7.
18. Roux V, Raoult D. The16S-23S rRNA intergenic spacer region of Bartonella(Rochalimaea) species is longer than usually described in other bacteria. Gene.1995;156:107-11.
19. Roux V, Raoult D. Inter-and intraspecies identification of Bartonella(Rochalimaea) species. J Clin Microbiol.1995;33:1573-9.
20. Pitulle C, Strehse C, Brown JW, Breitschwerdt EB. Investigation of thephylogenetic relationships within the genus Bartonella based on comparativesequence analysis of the rnpB gene,16S rDNA and23S rDNA. Int J Syst EvolMicrobiol.2002;52:2075-80.
21. Ohl ME, Spach DH. Bartonella quintana and Urban Trench Fever. Clin Infect Dis.2000;31:131-5.
22. Guptill L. Bartonellosis. Vet Microbiol,2010;140:347-59.
23. Chomel BB, Boulouis HJ, Breitschwerdt EB, et al. Ecological fitness andstrategies of adaptation of Bartonella species to their hosts and vectors. Vet Res.2009;40:29.
24. Bass JW, Vincent JM, Person DA. The expanding spectrum of Bartonellainfections: I. Bartonellosis and trench fever. Pediatr Infect Dis J.1997;16:2-10.
1. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. ClinMicrobiol Rev.1996;9:273–92.
2. Raoult D, Roux V. The body louse as a vector of reemerging human diseases. ClinInfect Dis.1999;29:888–911.
3. O'Rourke LG, Pitulle C, Hegarty BC, et al. Bartonella quintana in cynomolgusmonkey (Macaca fascicularis). Emerg Infect Dis.2005;11:1931-4.
4. Huang RT, Liu QY, Li G, et al. Bartonella quintana infections in captive monkeys,China. Emerg Infect Dis.2011;17:1707-9.
5. Alsmark CM, Frank AC, Karlberg EO, et al. The louse-borne human pathogenBartonella quintana is a genomic derivative of the zoonotic agent Bartonellahenselae. PNAS.2004;101:9716–21.
6. Spach DH, Kanter AS, Dougherty MJ, et al. Bartonella (Rochalimaea) quintanabacteremia in inner-city patients with chronic alcoholism. N Engl J Med.1995;332:424–8.
7. Rodriguez-Barradas MC, Hamill RJ, Houston ED, et al. Genomic fingerprintingof Bartonella species by repetitive element PCR for distinguishing species andisolates. J Clin Microbiol.1995;33:1089–93.
8. Roux V, Raoult D. Inter-and intraspecies identification of Bartonella(Rochalimaea) species. J Clin Microbiol.1995;33:1573-9.
9. Foucault C, La Scola B, Lindroos H, Andersson SG, Raoult D. Multispacer typingtechnique for sequence-based typing of Bartonella quintana. J Clin Microbiol.2005;43:41–8.
10. Feil EJ, Enright MC. Analyses of clonality and the evolution of bacterialpathogens. Curr Opin Microbiol.2004;7:308–13.
11. Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol.2006;60:561–88.
12. Iredell J, Blanckenberg D, Arvand M, et al. Characterization of the naturalpopulation of Bartonella henselae by multilocus sequence typing. J ClinMicrobiol.2003;41:5071–9.
13. Arvand M, Viezens J. Evaluation of pulsed-field gel electrophoresis andmulti-locus sequence typing for the analysis of clonal relatedness amongBartonella henselae isolates. Int J Med Microbiol.2007;297:255–62.
14. Arvand M, Feil EJ, Giladi M, Boulouis HJ, Viezens J. Multi-locus sequencetyping of Bartonella henselae isolates from three continents reveals hypervirulentand feline-associated clones. PLoS ONE.2007;2:e1346.
15. Arvand M, Raoult D, Feil EJ. Multi-locus sequence typing of a geographically andtemporally diverse sample of the highly clonal human pathogen Bartonellaquintana. PLoS ONE.2010;5:e9765.
16. Rozas J, Rozas R. DnaSP version3: an integrated program for molecularpopulation genetics and molecular evolution analysis. Bioinformatics.1999;15:174-5.
17. Tamura K, Peterson D, Peterson N, et al. MEGA5: Molecular EvolutionaryGenetics Analysis using maximum likelihood, evolutionary distance, andmaximum parsimony methods. Mol Biol Evoln.2011;28:2731–2739.
18. Huson DH, Bryant D. Application of phylogenetic networks in evolutionarystudies. Mol Biol Evol.2006;23:254–267.
19.王中强,邱少富,王勇,孙岩松,宋宏彬.多位点序列分型技术及其研究进展.军事医学.2010;34:76-9.
20. Drancourt M, Tran-Hung L, Courtin J, de Lumley H, Raoult D. Bartonellaquintana in a4000-year-old human tooth. J Infect Dis.2005;191:607-11.
21. Raoult D, Dutour O, Houhamdi L, et al. Evidence for louse-transmitted diseasesin soldiers of Napoleon's Grand Army in Vilnius. J Infect Dis.2006;193:112-20.
22. Chomel BB, Boulouis HJ, Breitschwerdt EB, et al. Ecological fitness andstrategies of adaptation of Bartonella species to their hosts and vectors. Vet Res.2009;40.
23. Engel P, Salzburger W, Liesch M, et al. Parallel evolution of a type IV secretionsystem in radiating lineages of the host-restricted bacterial pathogen Bartonella.PLoS Genetics.2011;7:e1001296.
24. Saenz HL, Engel P, Stoeckli MC, et al. Genomic analysis of Bartonella identifiestype IV secretion systems as host adaptability factors. Nature.2007;39:1469-76.
25. Greger M. The human/animal interface: emergence and resurgence of zoonoticinfectious diseases. Crit Rev Microbiol.2007;33:243-99.
1. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. ClinMicrobiol Rev.1996;9:273–92.
2. McNee JW, Renshaw A, Brunt EH.“Trench fever”: a relapsing fever occurringwith the British forces in France. BMJ.1916;12:225–34.
3. Byam W, Carroll JH, Churchill JH, et al. Trench fever. Oxford (UK): OxfordUniversity Press;1919.
4. Vinson JW. In vitro cultivation of the rickettsial agent of trench fever. Bull WorldHealth Organ.1966;35:155–64.
5. Kostrzewski J. The epidemiology of trench fever. Bull Acad Pol Sci (Med).1949;7:233–63.
6. Raoult D, Roux V. The body louse as a vector of reemerging human diseases. ClinInfect Dis.1999;29:888–911.
7. Brouqui P, Lascola B, Roux V, Raoult D. Chronic Bartonella quintana bacteremiain homeless patients. N Engl J Med.1999;340:184–9.
8. Spach DH, Kanter AS, Dougherty MJ, et al. Bartonella (Rochalimaea) quintanabacteremia in inner-city patients with chronic alcoholism. N Engl J Med.1995;332:424–8.
9. Brenner DJ, O’Connor SP, Winkler HH, Steigerwalt AG. Proposals to unify thegenera Bartonella and Rochalimaea, with descriptions of Bartonella quintanacomb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., andBartonella elizabethae comb. nov., and to remove the family Bartonellaceae fromthe order Rickettsiales. Int J Syst Bacteriol.1993;43:777–86.
10. Birtles RJ, Harrison TG, Saunders NA, Molyneux DH. Proposals to unify thegenera Grahamella and Bartonella, with descriptions of Bartonella talpae comb.nov., Bartonella peromysci comb. nov., and three new species, Bartonellagrahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov. IntJ Syst Bacteriol.1995;45:1–8.
11. Guptill L. Bartonellosis. Vet Microbiol,2010;140:347-59.
12.杨发莲,白鹤鸣,杨慧,张青.巴尔通体分离培养特性观察.微生物学杂志.2006;26:6-9.
13.杨发莲,白鹤鸣,杨慧,董兴齐,宋志忠.巴尔通体I、II代扫描透射电镜观察.中国病原生物学杂志.2007;2:168-169.
14. Alsmark CM, Frank AC, Karlberg EO, et al. The louse-borne human pathogenBartonella quintana is a genomic derivative of the zoonotic agent Bartonellahenselae. PNAS.2004;101:9716–21.
15. Rodriguez-Barradas MC, Hamill RJ, Houston ED, et al. Genomic fingerprintingof Bartonella species by repetitive element PCR for distinguishing species andisolates. J Clin Microbiol.1995;33:1089–93.
16. Roux V, Raoult D. Inter-and intraspecies identification of Bartonella(Rochalimaea) species. J Clin Microbiol.1995;33:1573-9.
17. Foucault C, La Scola B, Lindroos H, Andersson SG, Raoult D. Multispacer typingtechnique for sequence-based typing of Bartonella quintana. J Clin Microbiol.2005;43:41–8.
18. Arvand M, Raoult D, Feil EJ. Multi-locus sequence typing of a geographically andtemporally diverse sample of the highly clonal human pathogen Bartonellaquintana. PLoS ONE.2010;5:e9765.
19. Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of Bartonellaquintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, andWolbachia pipientis in cat fleas, France. Emerg Infects Dis.2003;9:338-42.
20. La VD, Tran-Hung L, Aboudharam G, Raoult D, Drancourt M. Bartonellaquintana in domestic cat. Emerg Infect Dis.2005;11:1287-9.
21. Kelly P, Rolain JM, Maggi R, Sontakke S, Keene B. Bartonella quintanaendocarditis in dogs. Emerg Infect Dis.2006;12:1869-72.
22. O'Rourke LG, Pitulle C, Hegarty BC, et al. Bartonella quintana in cynomolgusmonkey (Macaca fascicularis). Emerg Infect Dis.2005;11:1931-4.
23. Huang RT, Liu QY, Li G, et al. Bartonella quintana infections in captive monkeys,China. Emerg Infect Dis.2011;17:1707-9.
24. Mooser H, Weyer F. Experimental infection of Macacus rhesus with Rickettsiaquintana (trench fever). Proc Soc Exp Biol Med.1953;83:699-701.
25. Zhang P, Chomel BB, Schau MK, et al. A family of variably expressedouter-membrane proteins (Vomp) mediates adhesion and autoaggregation inBartonella quintana. PNAS.2004;101:13630-5.
26. Sasaki T, Poudel SK, Isawa H, et al. First molecular evidence of Bartonellaquintana in Pediculus humanus capitis (Phthiraptera: Pediculidae), collected fromNepalesechildren. J Med Entomol.2006;43:110–2.
27. Bonilla DL, Kabeya H, Henn J, Kramer VL, Kosoy MY. Bartonella quintana inbody lice and head lice from homeless persons, San Francisco, California, USA.Emerg Infect Dis.2009;15:912-5.
28. Telford III SR, Wormser GP. Bartonella spp. transmission by ticks not established.Emerg Infect Dis.2010;16:379-84.
29. Angelakis E, Billeter SA, Breitschwerdt EB, et al. Potential for tick borneBartonelloses. Emerg Infect Dis.2010;16:385-91.
30. Reis1C, Cote1M, Le Rhun D, et al. Vector competence of the tick Ixodes ricinusfor transmission of Bartonella birtlesii. PLoS Negl Trop Dis;5:e1186.
31. Raoult D, Drancourt M, Carta A, Gastaut JA. Bartonella (Rochalimaea) quintanaisolation in patient with chronic adenopathy, lymphopenia, and a cat. Lancet.1994;343:977.
32. Drancourt M, Mainardi JL, Brouqui P, et al. Bartonella (Rochalimaea) quintanaendocarditis in three homeless men. N Engl J Med.1995;332:419–23.
33. La Scola B, Raoult D. Culture of Bartonella quintana and Bartonella henselaefrom human samples: a5-year experience (1993to1998). J Clin Microbiol.1999;37:1899–905.
34. Fournier PE, Mainardi JL, Raoult D. Value of microimmunofluorescence fordiagnosis and follow-up of Bartonella endocarditis. Clin Diagn Lab Immunol.2002;9:795–801.
35. Houpikian P, Raoult D. Western immunoblotting for Bartonella endocarditis. ClinDiagn Lab Immunol.2003;10:95–102.
36. Rolain JM, Brouqui P, Koehler JE, et al. Recommendations for treatment ofhuman infections caused by Bartonella species. Antimicrob Agents Chemother.2004;48:1921–33.
37. Raoult D, Fournier PE, Vandenesch F, et al. Outcome and treatment of Bartonellaendocarditis. Arch Intern Med.2003;163:226–30.
38. Meghari S, Rolain JM, Grau GE, et al. Antiangiogenic effect of erythromycin: anin vitro model of Bartonella quintana infection. J Infect Dis.2006;193:380–6.
39.杨发莲,杨慧,白鹤鸣,于彬彬,张青.不明原因发热病人中五日热巴尔通体感染调查.地方病通报.2007;22:90.
40.杨慧,白鹤鸣,杨发莲,于彬彬.云南巴尔通体感染的血清学调查.中国自然医学杂志.2007;9:277-80.