蚤、蜱中巴尔通体的分离培养及检测鉴定

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

巴尔通体在厦门市鼠形动物间及各区域间的分布

目的掌握厦门地区鼠形动物巴尔通体流行情况,证实厦门市鼠形动物感染巴尔通体菌属,为控制人巴尔通体流行提供参考依据。方法根据厦门市行政区划和地理状况划片,随机抽取3个不同区作为调查点。在不同生境采用笼日法捕鼠,取血,用细胞培养法分离巴尔通体;PCR扩增细菌gltA基因片段（376 bp）,并进行序列分析,寻查本地巴尔通体属种。结果厦门市以褐家鼠为巴尔通体主要宿主动物（51.21%）,其次为臭鼩鼱（29.09%）、黄胸鼠（16.36%）及小家鼠、黑家鼠和黄毛鼠等（3.33%）。3个区共采集鼠形动物血样330份,培养分离出巴尔通体84株,感染率25.45%,其中以臭鼩鼱感染率最高,为32.29%,黄胸鼠为24.07%、褐家鼠为23.08%;各调查点鼠形动物巴尔通体感染率分别为：湖里22.64%,海沧28.68%,同安24.51%,差异无统计学意义（P〉0.05）。查出的巴尔通体分别为B.elizabethae、B.queenslandensis和B.tribocorum,其中B.tribocorum分为A、B两群,B群仅感染臭鼩鼱。结论厦门市鼠形动物巴尔通体感染普遍,且存在宿主多样性和菌株对宿主的选择性。     

浙江省鼠形动物巴尔通体的遗传进化分析

目的分析浙江省鼠形动物中巴尔通体分子遗传进化关系,为巴尔通体人群感染的预防控制提供科学依据。方法用夹夜法在浙江省不同地区、不同季节捕获鼠形动物,无菌操作取鼠肝和脾,用PCR和分离培养检测巴尔通体,对部分阳性产物测序,提交到GenBank,用CLUSTAL W进行匹配,然后用PAUP4.0beta10软件构建系统关系,分析其遗传进化关系。结果我们分别从黑线姬鼠、黄毛鼠、褐家鼠、黑腹绒鼠、社鼠、臭鼩鼱、东方田鼠、黄胸鼠和大林姬鼠中检测到巴尔通体特异DNA片段,浙江首次从黑线姬鼠脾中分离出一株巴尔通体。遗传进化分析显示我们检测到的巴尔通体与Bartonellaratti massiliensis以及对人类有致病性的B.grahamii的遗传关系最近。结论浙江省鼠类中广泛存在巴尔通体感染,而且携带人类致病性巴尔通体,存在人群感染风险。     

广东省鼠形动物携带巴尔通体的调查和基因特征分析

目的 了解巴尔通体在广东省鼠形动物中的流行分布情况及其基因型特征,为巴尔通体的研究和自然疫源性疾病的防控提供科学依据。方法 在广东省惠来县、惠东县、潮安县、罗定县和高州市5个地区进行捕鼠,采集鼠肝脏标本提取总核酸,利用聚合酶连反应(PCR)检测巴尔通体病原,并对其进行分离培养,阳性产物进行测序,并根据序列进行系统进化分析,统计分析巴尔通体的分布情况和特征。结果在广东省5个地区共捕鼠375只,检出巴尔通体阳性标本73份,阳性率为19.47%。不同鼠种(χ²=6.361)、不同地区(χ²=7.778)、不同性别(χ²=0.292)、不同生境间(χ²=0.621),阳性率差异均没有统计学意义(P>0.05)。73份样本中,10份成功分离培养出巴尔通体。系统进化分析表明广东鼠形动物携带的巴尔通体存在6种基因型:Bartonella elizabethae、 B.phoceensis、 B.japonica、 B.henselae、 B.rochalimae、B.tribocorum。结论 巴尔通体在广东省鼠形动物中广泛存在,且基因型呈多样性,其中4种基因型对人类有致病性,对人类健康具有潜在威胁。     

福建沿海臭鼩鼱感染巴尔通体调查研究

目的证实臭鼩鼱感染巴尔通体,了解其分布,分析菌株基因型,为巴尔通体病的防治提供依据。方法采集福建沿海6地市臭鼩鼱血样,培养分离巴尔通体菌株,PCR扩增病原体gltA基因片段进行序列分析,构建生长发育树,并分析各属种的宿主和地区分布。结果臭鼩鼱占本调查鼠形动物的25.33%,与褐家鼠、黄胸鼠构成了室内鼠形动物优势种群。臭鼩鼱血样中分离并证实63份感染巴尔通体,感染率21.43%(63/294),分布在各调查区域内。臭鼩鼱被B.tribocorum A、B种群感染,A、B群的分布也体现了一定的地域特征。结论臭鼩鼱是B.tribocorum A、B种群的保存宿主,且感染率高、分布广,应加强调查研究。     

福建省鼠形动物巴尔通体感染调查

目的了解福建省巴尔通体在鼠形动物中的感染分布状况。方法被检标本于2005年5~9月收集自福建省厦门市所捕捉活鼠,用5%兔心血的脑心浸液琼脂培养基分离巴尔通体,可疑菌落分纯1~4代;以聚合酶链反应(PCR)扩增特异性高的枸橼酸合酶基因(gltA)的379bp片段,以证实为巴尔通体。结果共捕鼠形动物151只,分离到22株巴尔通体,分离率为14.57%。菌株分布于2属2目,其中褐家鼠8.97%,臭鼩鼱20.55%,臭鼩鼱带菌的报告在国内外尚属首次。结论本文报道首次证实巴尔通体在福建省鼠形动物中流行,且感染率高,需对感染分布状况开展进一步的流行病学调查;各种巴尔通体对应于不同的人类疾病,还需采用分子生物学方法作巴尔通体系统发育关系分析。臭鼩鼱在南方占室内鼠形动物的50%左右,组成比例有增大趋势,臭鼩鼱感染巴尔通体的流行病学意义不言而喻。     

福建省鼠类感染巴尔通体调查及序列分析

目的了解福建省鼠类中巴尔通体(Bartonellaspp.)的感染状况和基因特征。方法 2014-2016年采用笼日法在福建省闽东、闽西、闽南、闽北和闽中捕鼠,现场鉴定并记录捕获鼠类的釆集时间、地点、鼠种、性别、鼠龄等资料。采集鼠心脏血,PCR扩增巴尔通体的gltA和16S~23SrRNA基因,阳性PCR产物送测序并进行序列比对分析,构建系统进化树。感染率间的比较采用χ~2检验或Fisher精确检验法。结果调查共布放鼠笼5 917笼次,捕鼠381只,鼠密度为6.44%。巴尔通体感染率为12.34%。家鼠的巴尔通体感染率为10.61%,褐家鼠(Rattus norvebicus)和黄胸鼠(Rattus flavipectus)感染率分别为11.30%和10.00%;野鼠的感染率为13.86%,黄毛鼠(Rattus losea)和针毛鼠(Rattus fulvescens)感染率分别为22.86%和18.00%,野鼠的巴尔通体感染率高于家鼠的感染率,但无统计学意义。从地区分布看,闽西、闽北一带的感染率较高,分别为20.00%和25.33%。闽南一带感染率最低,为0。各地区感染率存在统计学意义(P0.05)。不同性别、鼠龄的巴尔通体感染率差异无统计学意义,而不同的生境下的感染率存在统计学差异(P0.05)。阳性样本测序分析显示,福建省鼠类感染的巴尔通体序列与B.tribocorum、B.elizabethae和B.grahamii序列最接近。结论福建省鼠类存在巴尔通体感染,存在对人群致病的风险。     

江西省部分地区鼠形动物中巴尔通体分布及基因组序列研究

目的 了解江西省鼠形动物中巴尔通体的携带状况和基因型特征,为巴尔通体感染的疾病预防控制提供科学依据.方法 2018-2019年在南城县、广信区、横峰县、广丰区、铜鼓县5个鼠传疾病监测项目点,采集130只鼠形动物的肝、脾组织标本进行巴尔通体分离培养,挑取形态疑似巴尔通体的菌落纯培养后提取核酸,PCR检测巴尔通体属特异性基...     

湖北孝感某地蜱虫及人感染蜱媒病原体现状调查

目的了解驻湖北孝感某部营区蜱虫及蜱媒病原体感染现状,为防治蜱媒病对人群健康危害提供科学依据。方法 2012年对某营区的仓库及训练场开展蜱虫调查,采集营区警犬饲养员及离营区20 km医院发热待查患者血样、警卫犬体表及营区草地上的蜱虫,分别提取其基因组DNA,PCR方法检测分析测定病原体基因分型。结果累计收集患者血110份,将血样混合分组,共7组;警犬饲养员血1份。患者血样检出巴尔通体和肺炎军团菌分别为3组和1组,最大似然估计(maximum likelihood estinate,MLE)感染率分别为27.77%(4/110),8.52‰(1/110);警犬饲养员血液检测到巴尔通体。从警犬身上、营区草地上分别采集蜱虫6只、20只。警卫犬体表蜱虫和营区草地蜱虫均检测到巴尔通体和立克次体。营区警犬饲养员及医院发热待查患者血样与营区警犬体表蜱虫检测到的巴尔通体基因型不同,分别为牛巴尔通体(B.bovis USAMRIID-000002),杆菌巴尔通体(B.birtlesii USAMRIID-000020),伊莉萨白巴尔通体(B.elizabethae USAMRIID-000008 or B.grahamii USAMRIID-000026),巴尔通体变形菌(B.grahamii USAMRIID-000026);而警犬体表蜱虫携带的为巴尔通伯格霍夫亚种(Bartonella vinsonii subsp.berkhoffii)基因型Ⅲ。不同来源的样本检测的巴尔通体基因型不同。结论该调查点蜱虫易见,蜱媒病原体感染率高,应采取蜱虫防制措施。     

剑川自然疫源地的甘油阳性鼠疫菌株在黄胸鼠体内的选择优势

在云南家鼠与野鼠两类鼠疫动物流行之间关系的研究中,一个很重要的问题是:如果剑川自然疫源地中的绒鼠型(甘油阳性)鼠疫菌株侵入当地的主要家栖鼠类——黄胸鼠间发生动物流行时,将发生什么变化?而如果当时黄胸鼠种群中正经历着家鼠鼠疫(甘油阴性株)的动物流行,哪一种能够排挤对方而留存下来?为了探索这个问题,我们进行了云南两型鼠疫杆菌在黄胸鼠体内的选择实验。     

Genetic and ecologic characteristics of Bartonella communities in rodents in southern China

Ecologic and bacteriologic observations of small mammals captured in Yunnan Province in the People's Republic of China indicated that Bartonella infections occurred at a high prevalence among some rodent species. Sequence analyses of the citrate synthase genes of these Bartonella demonstrated that rodents in this region harbored a diverse assemblage of strains. The Bartonella isolates obtained from Apodemus, Eothenomys, and Rattus typically clustered separately by genus of rodent host. Cultures obtained from Rattus rats were genetically related to Bartonella elizabethae, a recognized human pathogen. The finding of Bartonella species in a high proportion of the rodent samples from Yunnan suggests the need to investigate whether these agents might be responsible for cases of febrile illnesses of unknown etiology in southern China and elsewhere in southeastern Asia.     

Prevalence and diversity of Bartonella in rodents of northern Thailand: a comparison with Bartonella in rodents from southern China

We report results of the first study to investigate the distribution and diversity of Bartonella in rodents from Thailand. Whole blood from 195 rodents, representing six species, was tested for the presence of Bartonella species using standard culture techniques. Isolates were obtained from 17 (8.7%) of the samples, and 14 of those isolates represented distinct strains, based upon partial sequencing of the citrate synthase (gltA) gene. Phylogenetic analysis of the isolates and other Bartonella species indicated that five unique isolates from Bandicota indica form a cluster that may represent a new Bartonella species. Two additional isolates from B. indica clustered together, and were nearly identical to an isolate from Apodemus draco collected in southern China. Importantly, a number of the isolates from Thailand rodents are closely related to B. grahamii and B. elizabethae, species which have been associated with human illness.     

Bartonella spp. infection in rodents from different habitats in the Mazury Lake District, Northeast Poland

Four rodent species (Clethrionomys glareolus, Apodemus flavicollis, Microtus arvalis, M. oeconomus) were captured in the period 2004-2006 in the Mazury Lake District, Northeast Poland, to determine the prevalence and genetic diversity of Bartonella species. The presence of bartonellae was assessed using polymerase chain reaction (PCR) with primers CS140f and BhCS1137n, amplifying a fragment of the gltA gene. Bartonella DNA was detected in 313 (30.6%) of 1024 rodents sampled: in 181 C. glareolus, 68 A. flavicollis, 50 M. arvalis, and 14 M. oeconomus, representing prevalence of 31.0%, 42.2%, 32.9%, and 11.1%, respectively. Comparison of the Bartonella gltA gene sequences from 38 isolates revealed six phylogenetic subgroups, out of 15 unique gltA sequences, and therein from one to five genotypic variants with homology of 88.6-99.1%. Six of 13 (46.2%) isolates from C. glareolus were identical to B. grahamii, species associated with human illness. These results have important public health implication, notably in relation to the risk of infection in humans following exposure to rodent bartonellae.     

2004～2005年滇西地区肾综合征出血热调查

目的 为掌握滇西地区肾综合征出血热流行病学特点,提供防治参考,对人间和鼠间疫情进行了调查.方法 收集滇西本病疫情资料,并在该地区采集人血清以及鼠肺脏和血清作汉坦病毒抗原和抗体检查.结果 滇西地区2004和2005年共报告病例41例,占全省同期发病数的40.59%.主要发病地区为祥云、兰坪、古城、永胜、宁蒗、宾川、巍山、弥渡、大理市(县、区),人群隐性感染率为1.81%.2004和2005年在玉龙、古城、永胜、宾川、祥云、大理、澜沧、景东、龙陵、盈江、腾冲、昌宁、宁蒗县和兰坪县(市、区)采集到16种1 825份鼠肺标本,鼠间汉坦病带毒率为2.47%.居民区以褐家鼠和黄胸鼠为优势鼠种,野外以高山姬鼠和大绒鼠为优势种,带毒鼠种为褐家鼠、黄胸鼠、大绒鼠、大足鼠、高山姬鼠和臭鼩鼱.结论 该地区广泛存在以褐家鼠和黄胸鼠为主要宿主动物的家鼠型疫源地,也存在着以高山姬鼠和大绒鼠为主的野鼠型疫源地.滇西疫区正在扩大,应采取以灭家鼠和健康人群接种家鼠型或两型混合疫苗为主的防治措施.     

云南省2001～2002年肾综合征出血热监测研究

目的为掌握云南省肾综合征出血热流行病学特点,提供防治参考,对人间和鼠间疫情进行了监测.方法收集全省本病疫情资料,并在监测县采集人血清以及鼠肺脏和鼠血清作汉坦病毒抗原和抗体检查.结果 2001～2002年全省共报告本病102例,死亡3例,年发病率为0.12/10万,病死率为2.94%.主要发病地区为红河州、昆明市、楚雄州.疫区人群隐性感染率为4.19%.2002年在泸西、寻甸和永胜监测点捕获鼠类9种891只,居民区以褐家鼠和黄胸鼠为优势鼠种,野外以高山姬鼠为优势种;鼠间汉坦病毒带毒率为3.65%,带病毒鼠种为褐家鼠、黄胸鼠、小家鼠和高山姬鼠.2001年在大理市野外捕鼠12种140只,大绒鼠为优势种,带毒鼠为大绒鼠、大足鼠、黄胸鼠、社鼠和短尾鼠句.结论监测区内存在有以褐家鼠和黄胸鼠为主要宿主动物的家鼠型疫源地,也存在着以高山姬鼠和大绒鼠为主的野鼠型疫源地.发病率上升与较高的鼠密度和鼠间感染率有关.应采取以灭家鼠和接种家鼠型或两型混合疫苗为主的防治措施.     

云南省泸西县啮齿动物携带立克次体调查

目的 调查云南省泸西县啮齿动物携带恙虫病东方体、无形体和埃立克体的状况,了解该类病原体在当地自然界中的保存状况和基因特征。方法 用鼠笼和鼠夹在云南省泸西县捕鼠,将捕获的动物种类鉴定后解剖取脾脏,活鼠取血。采用巢式PCR扩增脾脏的恙虫病东方体groEL基因,无形体和埃立克体的16S rRNA基因特异片段;测定PCR扩增阳性产物的DNA序列,对获得序列进行序列比对和系统进化分析。IFA法检测鼠血清中恙虫病东方体IgG抗体。结果 在泸西县共捕获啮齿动物10种225只。其中黄胸鼠36.89%(83/225)、大绒鼠35.11%(79/225)和中华姬鼠13.78%(31/225)为优势鼠种。获得鼠血清85份。鼠脾脏中检测到5株东方体groEL基因阳性标本,带毒鼠种为黄胸鼠2.41%(2/83)和大绒鼠3.80%(3/79)。同源性比较显示,这5株东方体的相似性在99.02%~100%之间,他们分别与GenBank中已知立克次体序列的相似性在98.75%~100%。系统发生树显示,5株OT与来自日本、泰国和中国安徽的菌株位于同一分支。3份16S rRNA阳性标本,其中1份埃立克体阳性,来源于大绒鼠;1份沃尔巴克氏体和1份巴尔通体阳性均来源于黄胸鼠。无形体均为阴性。埃立克体株序列比对显示与来自美国、中国和巴西的埃立克体基因同源性为98.0%~100%,并与分离自美国野外工作者皮肤的伊文氏埃立克体在同一分支。鼠血清恙虫病IgG抗体阳性7份,阳性率8.24%(7/85)。结论 该地区存在以黄胸鼠和大绒鼠为主要宿主的恙虫病自然疫源地。埃立克体、巴尔通体和沃尔巴克氏体在啮齿动物中也存在感染,需注意防控。     

Diversity of blood parasites of genus Bartonella in wild rodents in Mazury Lakes District

This long-term study of genetic diversity and epidemiology of the alpha-proteobacterium Bartonella in wild rodents from forest (Myodes glareolus and Apodemus flavicollis) and abandoned farmland (Microtus arvalis and Mi. oeconomus) was carried out in the years 2007-2009 in the Mazury Lakes District. In total, 1193 rodents were marked and recaptured, and 2226 blood samples were collected. The highest Bartonella prevalence was found in A. flavicollis (43.5%), the lowest in Mi. oeconomus (9.4%), while prevalence in My. glareolus and Mi. arvalis was, respectively, 13.2% and 11.8% (PCR of citrate synthase gltA gene fragment). Prevalence varied according to year and season, as well as sex of rodents. For woodland animals, a rapid decrease of prevalence was observed in late 2008, due to the dilution effect. Multiple (different species/genotypes of Bartonella in successive months) and mixed infections (more than one bacteria genotype in the same blood sample) were also diagnosed. Between 2835 and 4800000 colony forming units (CFU) per ml blood were recorded, with, for B. taylorii, significant differences between isolates from hosts belonging to different host families. Sequence analysis of 147 isolates revealed 37 gltA variants. In all four rodents, B. taylorii was the most prevalent, and could be divided into three main clades. One clade of B. grahamii was present in My. glareolus, A. flavicollis and Mi. arvalis, and both Microtus species were infected with a single clade of B. doshiae. A single isolate of B. birtlesii from A. flavicollis was collected, while two isolates could not be assigned to any known species. Nested clade analysis showed host specificity of 1st step clades (connected with rodent species) and 2nd step clades (connected with rodent family). Analysis was then extended to other housekeeping genes (cell division proteinftsZ, heat shock protein groEl, riboflavin synthase ribC, beta subunit RNA polymerase rpoB) and gene encoding 16S rRNA. Comparison of alleles of these genes in 27 isolates revealed numerous recombinant events, primarily involving groEl and 16S rRNA genes. Moreover, genetic recombination within housekeeping genes was also identified, and one of the unidentified Bartonella isolates was found to involve recombination within gltA between B. grahamii and B. taylorii. Examination of two T4SS pathogenicity genes (virB5 and bepA), revealed a similar pattern of extensive recombination. BepA from 17 isolates showed little diversity, concomitant with its role as an intra-cellular messenger. The virB5 gene (encoding a putative extra-cellular adhesin) from 22 isolates from voles (Arvicolidae) and A. flavicollis was distinctively different in sequence and putative structure, and showed a clear signature of horizontal gene transfer. Moreover, these recombinant events were often identified in the same isolates in which recombination of groEl or 16S rRNA was observed, suggesting that selection for this pathogenicity gene is important in the microevolution of Bartonella within rodents. In particular, Microtus spp. was central in the appearance of novel Bartonella isolates.     

Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella species: an Old World origin for a New World disease?

Bartonella species were isolated from the blood of 63 of 325 Rattus norvegicus and 11 of 92 Rattus rattus from 13 sites in the United States and Portugal. Infection in both Rattus species ranged from 0% (e.g., 0/87) to approximately 60% (e.g., 35/62). A 337-bp fragment of the citrate synthase (gltA) gene amplified by polymerase chain reaction was sequenced from all 74 isolates. Isolates from R. norvegicus were most similar to Bartonella elizabethae, isolated previously from a patient with endocarditis (93%-100% sequence similarity), followed by Bartonella grahamii and other Bartonella species isolated from Old World rodents (Clethrionomys species, Mus musculus, and Rattus species). These data suggest that Rattus species are a reservoir host for pathogenic Bartonella species and are consistent with a hypothesized Old World origin for Bartonella species recovered from Rattus species introduced into the Americas.