基于ITS-1基因序列的隐孢子虫种群分类

本试验以内转录间隔区1(ITS-1)基因作为研究对象,用PCR技术分别对广东(GD)禽源、广东(GD)和安徽(AH)牛源3个隐孢子虫分离株ITS-1基因进行扩增、克隆、序列测定和分析.将扩增序列用DNAStar(4.0)和ClustalX1.81软件与GenBank上登录的参考序列比对,用Phylip3.67构建种群发育进化树,以确定分离株与其他隐孢子虫虫种之间的亲缘关系.通过对ITS-1基因全序列分析,结果表明:禽源隐孢子虫GD分离株为Cryptosporidium baileyi,牛源隐孢子虫GD分离株和AH分离株均为C.andersoni.因此,ITS-1基因可作为隐孢子虫分离株种群分类的遗传标记,从而为隐孢子虫属的种间鉴定以及进一步的分子流行病学调查和分子诊断学研究奠定了基础.     

鸡艾美耳球虫18SrDNA序列与系统进化分析

为了确定鸡艾美耳球虫（Eimeria）不同种以及来自不同地区同种不同株之间的亲缘关系,研究其分类地位,对实验室保藏的柔嫩艾美耳球虫（Etenella）、毒害艾美耳球虫（Eneeatrix）、巨型艾美耳球虫（Emaxima）、堆形艾美耳球虫（Eaaervulina）等4种15株鸡球虫孢子化卵囊的18SrDNA基因进行克隆、测序,并与从GenBank下载的鸡球虫18SrDNA序列一起,使用软件DNAstar 5．0 MegAlign进行系统发育分析。结果显示,4种艾美耳球虫种间同源性在94．6％～99．4％之间,7株柔嫩艾美耳球虫的株间同源性在99．0％-99．9％之间,5株巨型艾美耳球虫的株间同源性在96．9％～99．8％之间。用该4种鸡球虫的18SrDNA序列与GenBank下载的另外4种鸡球虫18SrDNA序列构建系统发育树,显示这8种鸡艾美耳球虫形成2个分支,即堆形艾美耳球虫（EASH）、巨型艾美耳球虫（EMSH）、变位艾美耳球虫（Emivati）、和缓艾美耳球虫（Emitis）、布氏艾美耳球虫（Ebrunetti）、早熟艾美耳球虫（Epraecox）构成1个分支,柔嫩艾美耳球虫（ENSH）、毒害艾美耳球虫（ETAS）构成另1分支。巨型艾美耳球虫、柔嫩艾美耳球虫各株的系统发育树均根据地域关系产生2个分支。柔嫩艾美耳球虫、毒害艾美耳球虫的亲缘关系较近,不同地理区域的同种不同株的亲缘关系相对较远,种间和种内的鉴定结果与普通生物学结果一致。本研究提示18SrDNA基因可用于鸡球虫不同种／株的分类鉴定,为艾美耳球虫分子遗传学鉴定提供了理论基础。     

菲莱氏温扬球虫18S rDNA基因的扩增与克隆分析

菲莱氏温扬球虫是鸭球虫病的重要病原之一,为了寻找种特异性的遗传标记,本研究采集广东省某鸭场的新鲜鸭粪,通过Sheather’s蔗糖漂浮法收集球虫卵囊,经形态学鉴定为菲莱氏温扬球虫;提取该球虫DNA样品,经过PCR扩增,首次获得了该虫的18S rDNA基因部分片段;对该基因进行了克隆和测序,比较了该虫株与其他原虫的亲缘关系。结果显示,对菲莱氏温扬球虫序列与艾美耳科其他球虫关系较近,系统进化树分析属于艾美耳科的另一分支。表明18S rDNA基因在鸭菲莱氏温扬球虫的分类鉴定上是一种有效的分子标记。     

菲菜氏温扬球虫18S rDNA基因的扩增与克隆分析

菲莱氏温扬球虫是鸭球虫病的重要病原之一,为了寻找种特异性的遗传标记,本研究采集广东省某鸭场的新鲜鸭粪,通过Sheather's蔗糖漂浮法收集球虫卵囊,经形态学鉴定为菲莱氏温扬球虫;提取该球虫DNA样品,经过PCR扩增,首次获得了该虫的18S rDNA基因部分片段;对该基因进行了克隆和测序,比较了该虫株与其他原虫的亲缘关系.结果显示,对菲莱氏温扬球虫序列与艾美耳科其他球虫关系较近,系统进化树分析属于艾美耳科的另一分支.表明18S rDNA基因在鸭菲莱氏温扬球虫的分类鉴定上是一种有效的分子标记.     

牛源环孢子虫的发现与分子鉴定

根据GenBank上已发表的环孢子虫序列设计并合成2对引物，利用巢式PCR技术对首次在牛体内发现的形态学特征与人环孢子虫极为相似的牛源环孢子虫的18SrRNA基因进行了扩增，并以KpnⅠ酶对PCR产物进行RFLP分析；扩增出的片段纯化后克隆至pGEM-T Easy载体，对阳性克隆进行序列测定并对测序结果进行了同源性及系统发育分析。结果显示，扩增的18SrRNA基因片段大小为501bp，不含KpnⅠ酶切位点，与对照的艾美尔球虫的酶切图谱明显不同。序列同源性及系统发育分析显示该牛源环孢子虫与环孢子虫同源性最高，系统树中位于同一分支，可以确定其为一种环孢子虫。     

黄艾美耳球虫河北株18SrDNA部分序列测定及系统发育分析

为了进一步确定黄艾美耳球虫河北株虫种,试验根据GenBank中发表的黄艾美耳球虫18SrDNA基因序列设计引物,建立PCR方法对黄艾美耳球虫河北株基因片段扩增、测序,并进行系统发育分析.结果表明:扩增出大小为1 465bp的清晰条带;黄艾美耳球虫河北株18S rDNA序列测定结果与GenBank中的黄艾美耳球虫EF69...     

牛瑟氏泰勒虫吉林株ITS基因的克隆与系统进化分析

本研究根据GenBank上的牛瑟氏泰勒虫内转录间隔区基因(ITS基因)设计合成1对特异性引物,以采自吉林省珲春市某养牛场的血液样本为试验材料,PCR扩增牛瑟氏泰勒虫ITS基因,克隆至pMD-18T Simple载体,进行序列测定,将该基因序列与GenBank上9种已知的泰勒虫相应序列进行比较分析,建立系统进化树。结果表明:牛瑟氏泰勒虫吉林株与美国株亲缘关系较近,其次是水牛泰勒虫,与其他泰勒虫亲缘关系较远。     

以ITS-1+序列鉴定牛源环孢子虫

根据OenBank上已发表的环孢子虫（Cyclospora）rDNA序列设计并合成1对引物，利用PCR技术时首次在牛体内发现的形态学特征与人环孢子虫（Cyclospora cayetanensis）极为相似的牛源环孢子虫的ITS-1＋序列进行了扩增。将PCR扩增出的片段纯化后，克隆至pGEM—TEasy载体后进行序列测定，并利用NCBI在线BLAST程序对测序结果进行了同源性比较和序列分析。分析结果显示，扩增的ITS-1＋大小为865bp的片段，包含18S部分序列（371bp）、ITS-1全序列（385bp）和5．8S部分序列（109bp）。序列同源性分析表明，该牛源环抱子虫为艾美尔科原虫，但不同于目前已知的各种艾美尔科原虫，可能是一种新发现的原虫。     

3株柔嫩艾美耳球虫28SrRNA基因部分序列的扩增与分析

本研究应用PCR技术扩增来自广东的3株柔嫩艾美耳球虫的28S rRNA基因部分序列,并与GenBank^TM登录的柔嫩艾美耳球虫、堆型艾美耳球虫、鼠肉孢子虫和刚地弓形虫虫株的相应序列进行比对分析。试验结果显示,柔嫩艾美耳球虫3个样品均获得1172 bp的28S rRNA基因部分有效序列,不同虫株序列没有差异,与GenBank^TM登录的柔嫩艾美耳球虫相应序列只有一个碱基差异,显示种内序列高度保守,而与堆型艾美耳球虫、鼠肉孢子虫、刚地弓形虫相应的序列存在不同程度的差异。结果表明,28S rRNA基因部分序列可作为研究艾美耳球虫种间及其他顶复门原虫遗传变异的标记。     

河北省奶牛场犊牛安氏隐孢子虫的分离鉴定

本研究从河北省奶牛场有腹泻症状2月龄左右的犊牛粪便中分离卵囊,进行病原分离与虫株鉴定。采集腹泻犊牛的新鲜粪便24份,采用饱和蔗糖溶液漂浮法和抗酸染色法检测隐孢子虫卵囊,观察卵囊形态、大小。提取卵囊基因组DNA,进行18S rRNA基因PCR扩增及琼脂糖凝胶电泳检测。对扩增片段进行序列测定及分析,进一步确定分离虫株隐孢子虫的种类/基因型,根据18S rRNA基因核苷酸序列构建系统发育进化树,确定虫株亲缘关系。结果显示,5份样品检出隐孢子虫卵囊,感染率为20.83%。形态学观察卵囊呈长圆形或椭圆形,大小为(5.0～8.2)μm×(4.2～6.3)μm,平均大小为6.6μm×5.3μm,卵囊指数为1.24,鉴定分离虫株为安氏隐孢子虫。PCR扩增出预期大小为1 188 bp的特异性片段,序列分析和同源性分析结果表明,分离株与安氏隐孢子虫AB089285.2株、AB513856.1株、AY954885.1株的同源性为98.7%～98.8%,进一步表明分离的隐孢子虫虫株为安氏隐孢子虫。在种系进化关系上,分离株与安氏隐孢子虫AB513856.1株亲缘关系最近。本研究为揭示河北省奶牛隐孢子虫病的流行特征,实施有效防制措施提供了科学依据。     

桂林蛇源裂头蚴分离株的分子鉴定及种系发育关系分析

为研究从桂林草花蛇中分离到的裂头蚴的分子分类地位,对其核糖体18 S rDNA、28 S rDNA、ITS全基因及线粒体coxl部分基因进行了克隆和序列分析.从GenBank获取相应基因序列,应用CLUSTAL W2软件进行多重比对分析序列差异,应用MEGA 4.0软件构建种系发育树.结果表明,在基于18 S、28 S...     

First molecular detection of Babesia vogeli in dogs from Brazil

The present work describes the detection and first molecular characterization of Babesia vogeli in dogs, naturally infected in Brazil and even in South America. Microscopic examination of Giemsa-stained peripheral blood smears collected from dogs originating from four different locations in Brazil revealed the presence of large Babesia merozoites and trophozoites (>2.5 microm). DNA was extracted from infected blood samples and PCR amplifications of the 18S rDNA were carried out. As a reference, DNA from an isolate of B. vogeli originated from Egypt was used. PCR products were purified and sequenced. The DNA sequences demonstrated 100% identity among the Brazilian isolates. Comparisons with the 18S rDNA sequence of the B. vogeli isolate from Egypt and with other B. vogeli sequences from Spain, France, Japan, Australia and South Africa confirmed the affiliation of all Brazilian isolates to the species B. vogeli.     

Genotypic characterization and phylogenetic analysis of Cryptosporidium sp. from domestic animals in Brazil

The purpose of the present study was the genetic characterization, sequencing and phylogenetic analysis of 18S rDNA sequences of Cryptosporidium isolates obtained from different animal hosts in Brazil. Fecal samples containing Cryptosporidium oocysts were obtained from chickens, ducks, quails, guinea pigs, dairy calves, dogs and cats. For amplification of 18S rDNA sequences the Secondary-PCR product of the extracted DNA from fecal suspension of each studied animal was utilized. The primary genetic characterization of Cryptosporidium sp. was performed using RFLP with the enzymes SspI and VspI. DNA samples were sequenced and subjected to phylogenetic analysis. The results showed C. baileyi infecting two ducks and one quail and C. melagridis infecting one chicken. The sequences obtained from Cryptosporidium sp. infecting guinea pigs were not identified within groups of known Cryptosporidium species. The isolates found parasitizing cats and one dog were diagnosed as C. felis and C. canis, respectively. One isolate of calf origin was identified as C. parvum. The phylogenetic analysis showed clear distribution of isolates between two Cryptosporidium sp. groups according to their gastric or intestinal parasitism. A great genetic distance was observed between C. felis and C. canis from Brazil when compared to the reference sequences obtained from GenBank. The results obtained during this study constitute the first report of rDNA sequences from C. baileyi, C. meleagridis, C. felis, C. canis and C. parvum isolated in Brazil.     

Phylogenetic relationship of Pasteurella pneumotropica isolates from laboratory rodents based on 16S rDNA sequence

A 1344 bp fragment of the 16S ribosomal DNA (rDNA) sequence was used to determine the genetic relationship of Pasteurella pneumotropica isolates from laboratory rodents. A total of 30 nucleotide sequences of P. pneumotropica, including 24 wild strains, 3 reference strains, and 3 nucleotide sequences deposited in GenBank, were examined for heterogeneity of their 16S rDNA sequences. Phylogenetic analysis based on 16S rDNA sequence discriminated 5 types of branching lineages. Of these 5 types, 3 types had significant associations with mice or rats, and 2 had significant associations with the beta-hemolytic phenotype. These results suggest that 16S rDNA sequencing of P. pneumotropica isolates demonstrates genetic heterogeneity and phylogenetic discrimination in terms of their hemolytic phenotype and host associations.     

黄鳝胃瘤线虫ITS及5.8S rDNA的克隆及序列分析

本研究旨在阐明黄鳝胃瘤线虫湖南分离株的核糖体DNA(rDNA)内转录间隔区(ITS)及5.8 S rDNA序列的遗传变异情况,并用ITS序列重构胃瘤线虫与其它线虫的种群遗传关系.利用聚合酶链反应(PCR)扩增胃瘤线虫rDNA的ITS-1、5.8S及ITS-2片段,将PCR扩增出的片段纯化后克隆至pGEM-T Easy载体,重组质粒通过菌落PCR鉴定后,对阳性菌落进行序列测定并进行序列分析.结果显示所获得的胃瘤线虫ITS及5.8 S rDNA序列总长存在一定差异(922～927 bp),其中包含部分的18S、28 S及全部的ITS-1 (350～351 bp)、5.8S(102 bp)及ITS-2 (340～344 bp)序列.本研究系国内首次报道胃瘤线虫的ITS序列,其结果为黄鳝胃瘤线虫的分类鉴定以及进一步的分子流行病学调查和群体遗传研究奠定了基础.     

家蚕核糖体18S RNA基因的序列分析及分子系统学研究

为研究家蚕18S DNA基因的特点及分子进化,以家蚕丝腺为材料提取家蚕基因组DNA,通过PCR扩增、测序鳞翅目家蚕(Bombyx mori)核糖体小亚基18S RNA基因(18S rDNA)的全序列,将该序列与等翅目、直翅目、衤责翅目、鞘翅目、膜翅目、双翅目、捻翅目、弹尾目、蜉蝣目各一种昆虫的18S rDNA进行了比较。用DNAstav软件分析并进行序列比对,结果表明,昆虫18S rDNA有4段序列较为保守,以18S rDNA比对的第2保守区段构建的分子系统发育树表明鳞翅目和双翅目、膜翅目、鞘翅目进化关系最近,等翅目、直翅目、衤责翅目进化上较为接近,捻翅目昆虫与弹尾目昆虫的亲缘关系较为接近,捻翅目在分类上应是一种古老的昆虫。     

家蚕消化道来源蒙氏肠球菌的鉴定

研究了从家蚕消化道内分离出的4株肠球菌(C1、GC、FD、A20)的生理生化特征和16S rDNA序列,并与肠球菌种特异性探针序列进行了比较。生理生化特征测定结果表明,除A20外,C1、GC和FD与蒙氏肠球菌种的特征基本一致。由16S rDNA序列分析结果可知,分离菌株均与蒙氏肠球菌(Ent.mundtiiAJ301836)有高度同源性,在系统发育树内位于同一分支。分析肠球菌种特异性探针序列,分离菌株的16S rDNA含有与蒙氏肠球菌种特异性探针完全相同的序列。因此认为家蚕消化道内存在蒙氏肠球菌。     

Genetic diversity of Trypanosoma evansi in buffalo based on internal transcribed spacer (ITS) regions

The nucleotide sequences of 18S rDNA and internal transcribed spacer (ITS) regions were used for studying the relationships of Trypanosoma evansi isolate from a buffalo. The sequences were analyzed and compared to 18S rDNA and the ITS regions of the other Trypanosoma spp. Maximum likelihood phylogenetic trees were constructed using Leishmania major as the outgroup. The tree of 18S rDNA indicated that T. evansi (buffalo B18) isolate was closely related to those of Taiwan and T. brucei stock. The ITS tree showed the genetic diversity among 32 clones of T. evansi (B18) within a single host. This data will be useful for epidemiological and dynamic studies for designing the rational control programs of the disease.     

Comparison of partial ribosomal DNA sequences of Babesia gibsoni occurring in Miyazaki prefecture, Japan.

Nucleotide sequences of ribosomal DNA (rDNA) of Babesia (B.) gibsoni occurring in Miyazaki, western Japan, were examined using blood samples obtained from seven dogs suffering from natural canine babesiosis. DNA isolated from these blood samples was subjected to the polymerase chain reaction (PCR). The nucleotide sequences of the PCR products were determined and compared with other rDNA sequences of B. gibsoni isolated from Asia, Europe and U.S.A. Although homology values between our isolates and those isolated from Europe and U.S.A. were both 84.0%, respectively, our isolates were identical to the Asian types. In conclusion, B. gibsoni occurring in Miyazaki was revealed to have the genotype Asia 1 or Asia 2 from a comparison of the partial rDNA sequences.