附红细胞体的16S rDNA分类学研究及重庆市鸡附红细胞体的分子流行病学调查
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摘要
附红细胞体(Eperythrozoon)寄生于宿主的红细胞表面或游离于血浆、组织液及脑脊液中。可感染山羊、牛、猪、马、绵羊、鼠、犬、蓝狐、鸡等多种动物和甚至人类。但对于附红细胞体的分类,一直存在着很大分歧。由于附红细胞体具有原虫的特点,初始将附红细胞体属于原虫;尔后又发现其具有立克次氏体目的特性,又将它归入立克次体目。近年来,一些国内外学者采用分子生物学技术对附红细胞体的分类进行了研究,发现其16S rDNA与无形体的同源性相对较远,而与支原体目,支原体科的血巴尔通氏体同源性更近,建议将其重新归入支原体科。2005年出版的《伯杰氏系统细菌学手册》也将无形体科的附红细胞体属(Eperythrozoon)和血巴通体属(Haemobartonella)重新归入支原体科的支原体(Mycoplasma)。随着人类基因组测序的完成,后基因组时代的到来,生物信息学在生命科学研究领域得到了广泛的应用。特别是针对原核生物16S rDNA序列的生物信息学分析可作为微生物表型分类的有效补充,用于微生物的系统分类研究。本研究利用网络数据库中丰富的资源,从附红细胞体代表株的16S rDNA序列寻找新的分子标记,对附红细胞体的分类及附红细胞体病的分子诊断进行研究。
从NCBI的GenBank公共数据库下载猪、牛、羊、犬、猫、鼠等动物的附红细胞体和与之相近生物代表株的16S rDNA序列(截至2008年5月),如:血巴尔通氏体及致病性支原体、立克次体,利用生物信息学方法进行同源性分析。结果显示,猪、牛、羊、犬、猫、鼠等动物的附红细胞体与血巴尔通氏体的同源性相对较近,与部分常见致病性支原体形成三个并列的分支,而与立克次体同源性相对较远。
对3株猪附红细胞体(M.suis)及3株牛温氏附红细胞体(M.wenyonii)代表株的16S rDNA序列进行多序列比对分析后,在高变区内选出一段碱基差异较大的片段(约40bp)(后经分析该片段位于16S rDNA高变区V9)。进一步进行二级结构分析后发现,两个种的种间构象有明显区别,M.suis种内几乎无差异,M.wenyonii种内差异较明显,说明在猪、牛附红细胞体16SrDNA的各种间高变区二级结构的异同不完全依赖一级结构的组成,即二级结构相同的序列,其一级结构可能相同也可能不同。(或一级结构的细微差异可能造成二级结构出现差异,也可能不出现差异。)因此,二级结构可能体现出一级结构无法表现的差异。
对几种附红细胞体及近缘种血巴尔通氏体的16S rDNA进行的RFLP模拟分析发现,经限制性酶切图谱分析后,筛选出AluⅠ、DdeⅠ、HaeⅢ、HhaⅠ、HinfⅠ、HpaⅡ(MspⅠ)、RsaⅠ、XbalⅠ等8种内切酶,通过一种或几种酶切带型的组合,基本可用于进行生物的种一级分类单位的比较分析。同时,用8种内切酶对这些种的16S rDNA所进行的模拟酶切(大于100bp),筛选出可区分附红细胞体与血巴尔通氏体的16S rDNA RFLP候选分子标记。
根据NCBI数据库中附红细胞体代表株的16S rDNA序列,设计附红细胞体的具有属特异性的引物,分别从镜检呈附红细胞体阳性的猪血和鸡血中扩增出两条约700bp的目的条带,经克隆测序鉴定为目的条带。通过优化Mg2+浓度、退火温度及循环数等PCR反应条件,建立了附红细胞体的属特异PCR诊断法。优化反应条件后的PCR,经特异性试验、敏感性试验,结果证明该法特异性强,敏感性高,可用于对附红细胞体病的诊断及分子流行病学调查。
本研究根据重庆市各区县鸡的养殖方式及不同地理位置和地形特征,并参考2006年重庆市各区县鸡的养殖情况,选择规模化养鸡的荣昌、大足、永川、江津、合川、璧山、长寿地区及具有典型山地养殖方式武隆万州、云阳、城口、黔江、秀山等等13个区(县)作为采样点,共采集鸡抗凝血2442羽份。经全血压片镜检、姬姆萨染色染镜检、通用引物PCR、特异PCR方法检测的阳性率分别为9.986%、3.398%、0.555%和0。结果表明:重庆市暂时还没有真正意义上鸡感染附红体的病例,镜检法检测的假阳性较高,应以PCR检测结果作为准确诊断的依据。
Eperythrozoon,parasitized on surface of akaryocyte or liberated in blood plasma,tissue and cerebrospinal fluid,often infected animals(such as goats,cattle,pigs,horses,sheep,mice,dogs, foxes and chicken)and even human.There are lots of divergence in the classification of Eperythrozoon,which was considered as a kind of protozoon for its protozoon-like feature initially, then classified it into Rickettsiales and recently was suggested to classify into Mycoplasmatales. Through molecular biology technologies,scientists confirmed that homology of 16S rDNA between Eperythrozoon and Anaplasmataceae was low and a relatively high homology between Eperythrozoon and Mycoplasmatales.,Anaplasmatacea,Eperythrozoon Haemobartonella were classified into Mycoplasmataceae,Mycoplasma again in《Bergey's Manual of Systematic Bacteriology》published in 2005.After completion of Human Genome Sequencing Plan,age of postgenome forthcame with broad utilization of bioinformatics in the field of life science. Bioinformatic analysis based on 16S rDNA would be competent for microbe genealogical classification as an supplement of phenotype classification.In our study,many 16S rDNA sequences of representative strains of Eperythrozoon from NCBI were chose to search novel molecular markers for the exploration of genealogical classification and molecular diagnosis.
Eperythrozoon 16S rDNA sequences(up to may 2008)of animals including pigs,cattle,sheep, dogs and mice and other relative spices(such as Haemobartonella and pathogenic Mycoplasma、Rickettsiales)were downloaded from GeneBank in NCBI and analyzed in homology.The results indicated that Eperythrozoon was not only more close to Haemobartonella than Rickettsia colombiensis but also one of the three apposite lineages in partial common pathogenic mycoplasma.
16S rDNA of three M.suis strains and three M.wenyonii strains were analyzed by multiple sequence alignment.It manifested that there was a kind of segments with considerable differences(about 40 bp)in hypervariable regionlocated V9 of 16S rDNA.After further secondary structure analysis,we found markedly interspecific conformational differences between those two species as well as insignificant intraspecific differences among M.suis and significant intraspecific differences among M.wenyonii.Generally, tendency of interspecific differences of secondary structure in hypervariable regions was inconsistent with that of primary structure for 16S rDNA of Mycoplasma suis, Mycoplasma wenyonii.In other word,sequences with identical secondary structure might be either identical or different at primary structure level.The differences between secondary structures may be not expressed in primary strctures.
After RFLP analysis for 16S rDNA of Eperythrozoon and Haemobartonella,eight restriction enzymes,namely AluⅠ,DdeⅠ,HaeⅢ,HhaⅠ,HinfⅠ,HpaⅡ(MspⅠ),RsaⅠand XbalⅠ,were choosed to analyze specific-class taxonomically unit according to band types. Consequently,candidate molecular markers(over 100bp)distinguishing 16S rDNA of Eperythrozoon and Haemobartonella were screened by simulated restriction enzyme digestion.
Designed genus specific primers according to 16S rDNA of Eperythrozoon from NCBI,two bands about 700 bp were amplified respectively from positive blood of hog and chicken by microscopic examination,and were determined as target strips by cloning and sequencing.Thus genus specific PCR method for Eperythrozoon detection was established through optimizing conditions such as Mg2~+ concentration,anneal temperature and number of cycles.The results indicated that this method had better specificity and sensitivity with specific tests and sensibility tests,and could be used in molecular diagnosis and molecular epidemiology for Eperythrozoon.
According to differences of geography and terrain in Chongqing,breed styles of each county, and also breed information of each district or county in 2006,thirteen counties(Rongchang,Dazu, Yongchuan,Jiangjin,Hechuan,Bishan,Changshou and typical mountain-breed area including Wulong,Wanzhou,Yunyang,Chengkou,Qianjiang,and Xiushan.)were chose to collected blood samples and 2442 samples were obtained.Positive rates of 2442 samples tested by blood squash, Giemsa stain,universal primers and specific primers were 9.986%,3.398%,0.555%and 0, respectively.The results suggested that there was no real case of Eperythrozoon in Chongqing and PCR method was competent to diagnose chiken infected by Eperythrozoon instead of microscopic examination which had high false positive rate.
从NCBI的GenBank公共数据库下载猪、牛、羊、犬、猫、鼠等动物的附红细胞体和与之相近生物代表株的16S rDNA序列(截至2008年5月),如:血巴尔通氏体及致病性支原体、立克次体,利用生物信息学方法进行同源性分析。结果显示,猪、牛、羊、犬、猫、鼠等动物的附红细胞体与血巴尔通氏体的同源性相对较近,与部分常见致病性支原体形成三个并列的分支,而与立克次体同源性相对较远。
对3株猪附红细胞体(M.suis)及3株牛温氏附红细胞体(M.wenyonii)代表株的16S rDNA序列进行多序列比对分析后,在高变区内选出一段碱基差异较大的片段(约40bp)(后经分析该片段位于16S rDNA高变区V9)。进一步进行二级结构分析后发现,两个种的种间构象有明显区别,M.suis种内几乎无差异,M.wenyonii种内差异较明显,说明在猪、牛附红细胞体16SrDNA的各种间高变区二级结构的异同不完全依赖一级结构的组成,即二级结构相同的序列,其一级结构可能相同也可能不同。(或一级结构的细微差异可能造成二级结构出现差异,也可能不出现差异。)因此,二级结构可能体现出一级结构无法表现的差异。
对几种附红细胞体及近缘种血巴尔通氏体的16S rDNA进行的RFLP模拟分析发现,经限制性酶切图谱分析后,筛选出AluⅠ、DdeⅠ、HaeⅢ、HhaⅠ、HinfⅠ、HpaⅡ(MspⅠ)、RsaⅠ、XbalⅠ等8种内切酶,通过一种或几种酶切带型的组合,基本可用于进行生物的种一级分类单位的比较分析。同时,用8种内切酶对这些种的16S rDNA所进行的模拟酶切(大于100bp),筛选出可区分附红细胞体与血巴尔通氏体的16S rDNA RFLP候选分子标记。
根据NCBI数据库中附红细胞体代表株的16S rDNA序列,设计附红细胞体的具有属特异性的引物,分别从镜检呈附红细胞体阳性的猪血和鸡血中扩增出两条约700bp的目的条带,经克隆测序鉴定为目的条带。通过优化Mg2+浓度、退火温度及循环数等PCR反应条件,建立了附红细胞体的属特异PCR诊断法。优化反应条件后的PCR,经特异性试验、敏感性试验,结果证明该法特异性强,敏感性高,可用于对附红细胞体病的诊断及分子流行病学调查。
本研究根据重庆市各区县鸡的养殖方式及不同地理位置和地形特征,并参考2006年重庆市各区县鸡的养殖情况,选择规模化养鸡的荣昌、大足、永川、江津、合川、璧山、长寿地区及具有典型山地养殖方式武隆万州、云阳、城口、黔江、秀山等等13个区(县)作为采样点,共采集鸡抗凝血2442羽份。经全血压片镜检、姬姆萨染色染镜检、通用引物PCR、特异PCR方法检测的阳性率分别为9.986%、3.398%、0.555%和0。结果表明:重庆市暂时还没有真正意义上鸡感染附红体的病例,镜检法检测的假阳性较高,应以PCR检测结果作为准确诊断的依据。
Eperythrozoon,parasitized on surface of akaryocyte or liberated in blood plasma,tissue and cerebrospinal fluid,often infected animals(such as goats,cattle,pigs,horses,sheep,mice,dogs, foxes and chicken)and even human.There are lots of divergence in the classification of Eperythrozoon,which was considered as a kind of protozoon for its protozoon-like feature initially, then classified it into Rickettsiales and recently was suggested to classify into Mycoplasmatales. Through molecular biology technologies,scientists confirmed that homology of 16S rDNA between Eperythrozoon and Anaplasmataceae was low and a relatively high homology between Eperythrozoon and Mycoplasmatales.,Anaplasmatacea,Eperythrozoon Haemobartonella were classified into Mycoplasmataceae,Mycoplasma again in《Bergey's Manual of Systematic Bacteriology》published in 2005.After completion of Human Genome Sequencing Plan,age of postgenome forthcame with broad utilization of bioinformatics in the field of life science. Bioinformatic analysis based on 16S rDNA would be competent for microbe genealogical classification as an supplement of phenotype classification.In our study,many 16S rDNA sequences of representative strains of Eperythrozoon from NCBI were chose to search novel molecular markers for the exploration of genealogical classification and molecular diagnosis.
Eperythrozoon 16S rDNA sequences(up to may 2008)of animals including pigs,cattle,sheep, dogs and mice and other relative spices(such as Haemobartonella and pathogenic Mycoplasma、Rickettsiales)were downloaded from GeneBank in NCBI and analyzed in homology.The results indicated that Eperythrozoon was not only more close to Haemobartonella than Rickettsia colombiensis but also one of the three apposite lineages in partial common pathogenic mycoplasma.
16S rDNA of three M.suis strains and three M.wenyonii strains were analyzed by multiple sequence alignment.It manifested that there was a kind of segments with considerable differences(about 40 bp)in hypervariable regionlocated V9 of 16S rDNA.After further secondary structure analysis,we found markedly interspecific conformational differences between those two species as well as insignificant intraspecific differences among M.suis and significant intraspecific differences among M.wenyonii.Generally, tendency of interspecific differences of secondary structure in hypervariable regions was inconsistent with that of primary structure for 16S rDNA of Mycoplasma suis, Mycoplasma wenyonii.In other word,sequences with identical secondary structure might be either identical or different at primary structure level.The differences between secondary structures may be not expressed in primary strctures.
After RFLP analysis for 16S rDNA of Eperythrozoon and Haemobartonella,eight restriction enzymes,namely AluⅠ,DdeⅠ,HaeⅢ,HhaⅠ,HinfⅠ,HpaⅡ(MspⅠ),RsaⅠand XbalⅠ,were choosed to analyze specific-class taxonomically unit according to band types. Consequently,candidate molecular markers(over 100bp)distinguishing 16S rDNA of Eperythrozoon and Haemobartonella were screened by simulated restriction enzyme digestion.
Designed genus specific primers according to 16S rDNA of Eperythrozoon from NCBI,two bands about 700 bp were amplified respectively from positive blood of hog and chicken by microscopic examination,and were determined as target strips by cloning and sequencing.Thus genus specific PCR method for Eperythrozoon detection was established through optimizing conditions such as Mg2~+ concentration,anneal temperature and number of cycles.The results indicated that this method had better specificity and sensitivity with specific tests and sensibility tests,and could be used in molecular diagnosis and molecular epidemiology for Eperythrozoon.
According to differences of geography and terrain in Chongqing,breed styles of each county, and also breed information of each district or county in 2006,thirteen counties(Rongchang,Dazu, Yongchuan,Jiangjin,Hechuan,Bishan,Changshou and typical mountain-breed area including Wulong,Wanzhou,Yunyang,Chengkou,Qianjiang,and Xiushan.)were chose to collected blood samples and 2442 samples were obtained.Positive rates of 2442 samples tested by blood squash, Giemsa stain,universal primers and specific primers were 9.986%,3.398%,0.555%and 0, respectively.The results suggested that there was no real case of Eperythrozoon in Chongqing and PCR method was competent to diagnose chiken infected by Eperythrozoon instead of microscopic examination which had high false positive rate.
引文
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