用16S rRNA基因序列分析从西藏的微小牛蜱中检出的埃立克体

用埃立克体属特异性套式PCR和DNA序列测定从西藏微小牛蜱检出边缘无形体和埃立克体;用半套式PCR扩得该埃立克体的16S rRNA基因,并测定了它的序列;将该埃立克体的16S rRNA基因序列与其它埃立克体的16S rRNA基因序列进行对比分析,结果证明它的16S rRNA基因与埃立克体属的犬埃立克体群16S rRNA基因相似水平最高(97%～98%);用16S rRNA基因序列做系统发育分析,结果显示该采自西藏的微小牛蜱的埃立克体可能是与查菲埃立克体密切相关的一种新埃立克体.     

荧光定量PCR检测查菲埃立克体

依据查菲埃立克体16SrRNA基因序列设计特异性引物和TaqMan-MGB探针,以克隆的查菲埃立克体16SrRNA基因片段作DNA模板,建立实时荧光定量PCR检测方法。与套式PCR相比较,荧光定量PCR检测的灵敏度是其30倍;用荧光定量PCR检测其他相关立克次体和细菌DNA样本,检出结果为0;对荧光定量PCR检测重复性进行分析,变异系数(CV)批内和批间误差在0·2%~2·0%之间。结果证明本研究建立的荧光定量PCR方法具有种特异性和良好的重复性,可用于检测感染样本中的微量查菲埃立克体DNA。     

人埃立克体的传播途径及其遗传基础

本文从媒介、宿主、传播途径及其遗传学基础对埃立克体传播循环中获得的最新进展进行综述性回顾,为人埃立克体病的预防提供技术支持和基础资料。     

PCR检测蜱中查菲埃立克体DNA及其序列分析

应用从查菲埃立克体１６ＳｒＲＮＡ基因序列高变区构建的特异引物，进行ＰＣＲ，检测蜱标本中病原体ＤＮＡ。结果从云南采集的龟形花蜱、从福建采集的越原血蜱和卵形硬蜱扩增出３８９ｂｐ的特异ＤＮＡ片段。收集龟形花蜱和越原血蜱的特异ＰＣＲ产物，通过与Ｔ载体连接，进行克隆和序列测定。其ＤＮＡ序列与美国查菲埃立克体分离株对应位置相差一个核苷酸，与其它种埃立克体的同源性为８０．７％～９６．１％。这是首次在我国发现埃立克体存在的病原线索，表明在我国南方可能存在人单核细胞埃立克体感染的自然疫源地     

人粒细胞埃立克体病——又一种蜱传病

人粒细胞埃立克体病是由人粒细胞埃立克体(HumanGranulocyticEhrlichia,HGE)引起的一种经蜱传播的自然疫源性疾病。该病是继莱姆病、斑点热及人单核细胞埃立克体病等病之后,于1994年发现的又一种蜱传疾病。研究结果表明,HGE是一种以粒细胞为主要靶细胞的埃立克体。通过测?..     

查菲埃立克体表面抗原P120蛋白基因的克隆与表达

查菲埃立克体 (Ｅｈｒｌｉｃｈｉａｃｈａｆｆｅｅｎｓｉｓ)是人单核细胞埃立克体病 (ｈｕｍａｎｍｏｎｏｎ ｃｙｔｉｃｅｈｒｌｉｃｈｉｏｓｉｓ,ＨＭＥ)的病原体 ,已证实我国存在该病原。血清学分析是目前诊断ＨＭＥ及其流行病学调查的主要方法。但由于该病原体为严格胞内寄生菌 ,难以通过细胞培养制备大量抗原去满足临床ＨＭＥ的血清学诊断和流行病学调查的需要。Ｐ12 0 (相对分子质量为 12 0×10 3蛋白 )为查菲埃立克体主要表面抗原 ,具有种特异性。该蛋白基因包含了多个连续的 2 40ｂｐ重复单位 ,占全基因的 6 0 % ,并均为Ｂ淋巴细胞识…     

我国北方蜱中人粒细胞埃立克体16S rRNA基因的检测

本文应用人粒细胞埃立克体病 ( HGE)的病原体的 1 6S r RNA基因序列的特异引物对采自我国北方地区的一些蜱标本进行扩增 ,首次从内蒙古大兴安岭采集的全沟硬蜱、森林革蜱和嗜群血蜱 ,以及从新疆精河采集的全沟硬蜱和草原革蜱中扩增出该病原体的 1 6S r RNA基因片段。所测出的 967bp序列与美国 HGE株 ( Gen Bank U0 2 52 1 )的同源性为 1 0 0 %     

人类无浆体病的病原学和流行病学研究进展

无浆体病是由无浆体属（Anaplasma）微生物引起的一类反刍动物慢性和急性传染病，其特征为高热、贫血、消瘦、黄疸和胆囊肿大。近年随着立克次体目微生物分类学的变化，原属埃立克体的马埃立克体、嗜吞噬细胞埃立克体和新近发现的引起人粒细胞埃立克体病（HGE）的病原体（HGE-A）三者被重新分类，成为无浆体属中的新种一嗜吞噬细胞无浆体。嗜吞噬细胞无浆体是一种人畜共患病病原体，对人、畜均有较强致病性，因此．人类嗜吞噬细胞无浆体病成为一种新的人类传染病。本文重点总结人类无浆体病的病原学和流行病学方面的一些研究进展。     

中国—尼泊尔边境樟木口岸蜱类携带病原体调查研究

本研究于2012～ 2014年在中国-尼泊尔樟木口岸采集游离蜱和寄生蜱,形态学鉴定后,采用PCR检测蜱标本中伯氏疏螺旋体、埃立克体、立克次体、贝氏柯克斯体、土拉菌和巴通体6种病原体;采用RT-PCR检测森林脑炎、新疆出血热病毒、布尼亚病毒3种病原体.结果在296只蜱中检出伯氏疏螺旋体阳性5个、埃立克体16个、立克次体11个、巴通体16个.本研究初步获得中尼边境樟木口岸蜱媒携带病原情况,为该区域蜱传疾病的防控和研究提供依据.     

贝氏柯克斯体毒力相关基因芯片对其他胞内寄生菌基因组DNA检测结果分析

目的用建立的贝氏柯克斯体毒力相关基因芯片研究贝氏柯克斯体与其致病性相似的专性胞内寄生菌（立克次体、埃立克体、新立克次体）,以及与其密切相关的兼性胞内寄生菌（巴通体和肺炎军团菌）等病原菌的毒力基因的差异。方法贝氏柯克斯体国际标准株-九里株作为本研究参考株,从全基因序列中选取166个毒力及毒力相关基因进行PCR扩增,纯化后的产物制备基因芯片。采用双色荧光标记,待测DNA（即用于分析的各菌株基因组DNA）用Cy5标记,贝氏柯克斯体九里株参照基因组DNA用Cy3标记。芯片杂交后,采用软件GenePixPro4.1,辅助以软件Excel,进行图片处理和数据分析。结果芯片的166个基因片段中有165个存在于所有贝氏柯克斯体属的6个分离株中,另一锚定蛋白（AnkI）基因（CBU1213）仅存在于九里株及Grita株;立克次体属的5株菌株均与CBU1631和CBU1125杂交;埃立克体属的3种菌株与新立克次体属的3种菌株均含有基因CBU1125;东方属的1株菌株具有基因CBU1449,而巴通体属的菌株,军团菌和大肠杆菌与基因芯片均无杂交。结论贝氏柯克斯体种内基因组具有高度保守性,它的专性细胞内寄生菌的毒力相关基因与胞外寄生菌的毒力相关基因有非常显著差异。     

Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet

To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5'-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus EHRLICHIA: Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis.     

Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections

The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.     

Haemolytic activity of the Streptococcus milleri group' and relationship between haemolysis restricted to human red blood cells and pathogenicity in S. intermedius

A collection of 297 clinically documented 'Streptococcus milleri' strains, identified to the genotype level by 16S rRNA gene hydridisation, was screened for haemolysis of human and animal red blood cells. Forty-nine strains (65%) of the S. intermedius genotype displayed haemolysis restricted to human blood; they were named 'exclusive human haemolytic' (EHH) S. intermedius strains. The 26 remaining S. intermedius strains were named S. intermedius non-EHH strains. Quantitative studies on the haemolysis indicated that intermedilysin was the factor involved. The S. intermedius EHH strains represented the S. intermedius phenotype, whereas the S. intermedius non-EHH strains were phenotypically characteristic of S. constellatus. The complete 16S rRNA sequences of the S. intermedius EHH strains exhibited identity with S. intermedius strains ATCC 27335 (= NCDO 2227, NCTC 11324); the 16S rRNA sequences of the S. intermedius non-EHH strains were identical to S. constellatus strain ATCC 27823 (= NCDO 2226, NCTC 11325) except for positions 228 and 229 that carried an S. intermedius sequence signature. The 16S sequence similarities between the non-EHH strains and the S. constellatus and the S. intermedius type strains were 99.5% and 98.6%, respectively. Hybridisations of the complete 16S rRNA genes with oligonucleotide probes indicated a 16S rRNA homogeneity within the S. intermedius EHH and the non-EHH strains respectively. The S. intermedius EHH strains were isolated most frequently from infection- and abscess-related specimens. The present data emphasise the genetic variability within the S. constellatus species and redefine the S. intermedius species as a homogeneous group at the 16S rRNA level.     

Ehrlichia chaffeensis, a new species associated with human ehrlichiosis.

The bacterial 16S rRNA genes from blood samples of two patients with human ehrlichiosis and from an isolate recovered from one of the patients were amplified by using the polymerase chain reaction. The amplimers were then cloned and sequenced. The 16S rRNA gene sequence was also determined for Ehrlichia canis (two strains), E. equi, E. phagocytophila (two strains), and E. sennetsu (two strains). These sequences, along with a previously published 16S rRNA gene sequence of E. risticii, were compared. The 16S rRNA gene sequences were identical for all three sources of the human ehrlichiosis agent. The sequence comparisons indicate that the human ehrlichiosis agent is a new species most closely related to E. canis (98.2%) and more distantly related to other Ehrlichia spp. We propose that this species be named Ehrlichia chaffeensis sp. nov., with the Arkansas strain as the type strain.     

Nested PCR Assay for Detection of Granulocytic Ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

Automated identification of medically important bacteria by 16S rRNA gene sequencing using a novel comprehensive database, 16SpathDB

Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the "best match in 16SpathDB." For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories.     

北京市房山区长角血蜱携带山羊无形体的调查?

为确认我国华北地区广泛分布的长角血蜱是否携带新近发现的山羊无形体Anaplasma capra,2012、2015年5～9月期间,在北京房山地区采集长角血蜱并进行检测。结果共采集游离的长角血蜱311只,其中成蜱95只,若蜱156只,幼蜱60只。提取蜱基因组DNA后对山羊无形体的gltA和16S rRNA两个基因片段进行PCR扩增。结果显示,共有3（3?2％）只成蜱阳性,若蜱和幼蜱中未检测到山羊无形体。遗传进化分析显示,扩增出的gltA和16S rRNA序列和早先报道的牡丹江人感染的A． capra的序列相一致。证实了在我国华北地区的媒介蜱携带该新发现的山羊无形体,该地区为山羊无形体潜在的自然疫源地。加强该地区蜱媒传染病的防控工作具有必要性和紧迫性。     

Detection and differentiation of Vibrio vulnificus in seawater and plankton of a coastal zone of the Mediterranean Sea

Vibrio vulnificus, a human and animal pathogen, is present in low numbers in the Mediterranean Sea. Seawater and plankton samples were collected from a marine coastal zone of the Straits of Messina in the Mediterranean Sea (Italy) in order to investigate V. vulnificus as free-living (>0.2 microm) and associated with small (>64 microm) and large plankton (>200 microm) utilizing cultural and molecular techniques. Characteristic colonies, grown on thiosulfate, citrate, bile salts and sucrose agar plates, were identified using a biochemical protocol system. A PCR assay was used to confirm isolates and to directly detect V. vulnificus in environmental concentrated samples. Specific primers were used to target the structural cytotoxin/hemolysin gene and the variable regions of 16S rRNA species-specific for V. vulnificus. In addition, a tri-primer PCR of 16S rRNA was used for the differentiation of V. vulnificus strains. Direct detection in marine samples was more frequent than isolation of culturable forms. All isolates were assigned to V. vulnificus biotype 1, 16S rRNA type B. These results confirm the low incidence of V. vulnificus in Mediterranean coastal waters. The isolation of cultivable forms is limited to the warmest months. 16S rRNA primers were the most sensitive molecular tool as they allowed detection of V. vulnificus in 79.1% of samples. Due to the low incidence of V. vulnificus in the Mediterranean coastal environment, its detection requires a molecular approach. The occurrence of V. vulnificus as plankton-associated confirms the role of plankton as a potential reservoir for this pathogen.     

Molecular diversity of the Planctomycetes in the human gut microbiota in France and Senegal

Until now, Planctomycetes bacteria were considered as environmental organisms. Nevertheless, some studies detected Planctomycetes DNA from human gut. We therefore explored the human gut Planctomycetes content. Planctomycetes‐specific PCR primers were designed to amplify a 240–bp 16S rRNA gene fragment in human stool specimens from individuals in France and in Senegal and from endocarditis patients receiving antibiotics in France. PCR products were then cloned and sequenced. PCR detection revealed a significantly higher prevalence (1.8% vs 0.4%, p = 0.05) and higher diversity (62 vs 6 phylotypes, p = 0.02) of Planctomycetes 16S rRNA gene in stool specimens collected in Senegal than in France. Also, stool specimens from endocarditis patients exhibited non‐significantly higher prevalence (0.6% vs 0.4%) and the ratio of phylotypes by positive patient (3 vs 1.5) than those collected from untreated French individuals. Gemmata sp. related sequences were found in 6/12 individuals. Planctomycetes organisms are a part of the human digestive tract microbiota. Their diversity varied by environment including the geographical origin of the individual and antibiotics treatment.