【作者】 李巍；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2011， 博士

【摘要】 为评估非人灵长动物与人之间微孢子虫病、隐孢子虫病及环孢子虫病相互传播的可能性,本研究分析了来自肯尼亚狒狒的235份粪便样本。首先用巢氏PCR扩增毕氏肠微孢子虫(Enterocytozoon bieneusi)SSU rRNA基因中的转录间隔区(ITS)和隐孢子虫(Cryptosporidium spp.)与环孢子虫(Cyclospora spp.)SSU rRNA基因中的多态区。PCR检测出29(12.3%)个毕氏肠微孢子虫阳性,测序分析显示它们属于10个基因型(包括4个已知基因型A、D、Peru7和Peru11及6个新基因型,新基因型命名为KB-1到KB-6),这10个基因型的核酸序列与参考序列在系统进化分析中形成3个系统进化组。在这10个毕氏肠微孢子虫基因型中,有些曾在人的感染中发现,有些与能感染人的基因型系统进化关系相近。同时,PCR扩增结合测序分析检测出6(2.6%)个人隐孢子虫(Cryptosporidium hominis),亚型分别为IbA9G3、IfA12G2和IiA14。前两个亚型分属Ib与If亚型家族,曾多次在非洲多个国家艾滋病人中发现。Ii家族为本研究首次发现,系统进化分析显示其与人隐孢子虫亚型家族中的Ih、Ia和Ig及兔隐孢子虫(Cryptosporidium cuniculus)亚型家族Va亲缘关系相近。对环孢子虫PCR检测及测序分析显示42只狒狒(17.9%)感染了同一种环孢子虫:Cyclospora papionis。因此,在肯尼亚狒狒中检测出能感染人的毕氏肠微孢子虫基因型和人隐孢子虫亚型,当人与非人灵长类动物接触时可能发生微孢子虫病与隐孢子虫病的相互传播。应用Feng等[1]建立的多位点序列分型(MLST)方法对32个毕氏肠微孢子虫阳性样本(包括3个在ITS位点PCR检测呈阳性但测序混乱的样本)进行多位点序列分型。分别在毕氏肠微孢子虫微卫星(MS1、MS3和MS7)及小卫星(MS4)四个基因多态性位点上扩增这32个样本。PCR扩增结果显示:在MS1位点得到24个阳性、MS3位点得到8个阳性、MS4位点得到15个阳性及MS7位点得到21个阳性。序列分析显示MS1位点多态性包括三核苷酸TGC、TAA和TAC循环及单核苷酸多态性(SNP),共产生10个基因型;MS3位点多态性包括二核苷酸TA循环和SNP,共产生5个基因型;MS4位点多态性包括35 bp核苷酸循环、两个(GGTA和TTTTTTTCTT)核苷酸插入或缺失区和SNP,共产生5个基因型,MS7位点多态性包括三核苷酸TAA循环和SNP,共产生7个基因型。对各个位点核酸序列进行基因差异(gene diversity)分析显示,差异值(Hd)分别为:0.88(MS1)、0.86(MS3)、0.70(MS4)和0.73(MS7)。在章一的研究中,32个样本中的29个在ITS位点得到核酸序列,它们属于10个基因型。系统进化分析显示这10个ITS基因型中的KB-6具有宿主特异性(host-adapted),而基团型A、D、Peru7、Peru11、KB-1、KB-2、KB-3和KB-4具有人兽共患潜力(zoonotic)。本章中,在微卫星及小位星位点得到的系统进化分析结果与在ITS位点上得到的结果相似。此外,具有宿主特异性的ITS基因型KB-6在MS3位点上没有得到扩增,表明这个位点具有区分宿主特异性和人兽共患基因型的潜力。因此,Feng等建立的MLST工具能有效对毕氏肠微孢子虫进行多位点高精度分型,这必将促进对毕氏肠微孢子虫分类学、流行病学和公共卫生学的深入研究。本研究首次运用MLST工具对来自秘鲁艾滋病人72个毕氏肠微孢子虫样本进行群体结构分析。ITS分型已经鉴定这72个样本属于9个ITS基因型:A、D、IV、EbpC、WL11、Peru7、Peru8、Peru10和Peru11。巢氏PCR结合测序分析得到72个样本在4个多态性位点(MS1、MS3、MS4和MS7)的全部序列。基于序列长度多态性及单核苷酸多态性,在72个样本中共鉴定出39个多位点基因型(MLGs)。对全部72个样本进行基于核酸序列的群体遗传结构分析,强烈的基因内连锁不平衡(linkage disequilibrium)和有限的基因重组(genetic recombination)说明毕氏肠微孢子虫具有克隆性群体结构(clonal population stucture)。基于等位基因型分布(allelic profile)的群体遗传学分析显示总群体5个遗传位点(包括ITS位点)间存在强基因间连锁不平衡,对标准群丛指数(ISA)的计算和分析进一步证实克隆群体结构的存在。基于核酸序列和等位基因型分布的系统进化分析显示在总群体中存在两个具有不同遗传结构的系统进化组:组1包括ITS基因型为A的全部样本(基因型A目前只在人的感染中发现);组2包括ITS基因型为人兽共患基因型(主要为基因型D和IV)的样本。对这两个进化组分别进行群体遗传学研究发现组1为流行性群体结构(epidemic population structure),而组2为克隆性群体结构。用STRUCTURE对总群体进行亚群体结构分析显示根据K值的不同存在2-3个亚群体,通过对Wright’s固定指数(FST)的计算和分析进一步证明亚群体的存在。因此,基于ITS、MS1、MS3、MS4和MS7这5个遗传标记位点的群体遗传学研究能清晰描绘出毕氏肠微孢子虫的群体结构,这将促进对人毕氏肠微孢子虫流行病学和传播方式的深入研究。对秘鲁艾滋病人72个毕氏肠微孢子虫样本的群体遗传学研究证实其存在不同的亚群体结构,包括流行性群体结构和克隆性群体结构。为研究毕氏肠微孢子虫地域性群体结构特征,本章对来自尼日利亚和印度艾滋病人的33个样本及来自肯尼亚狒狒的5个样本进行群体遗传学研究。结合来自秘鲁艾滋病人的72个样本,共在总群体110个样本中鉴定出65个多位点基因型。首先对这110个样本进行总群体遗传学分析,强烈的基因内和基因间的连锁不平衡及有限的基因重组说明其存在克隆性群体结构。地域性群体遗传学研究显示毕氏肠微孢子虫在不同地域群体中仍然保持克隆性群体结构。基于核酸序列和等位基因型分布的系统进化分析将总群体110个样本分成两个系统进化组:组1包括ITS基因型为A的全部样本,组2包括ITS基因型为人兽共患基因型(主要为基因型D、PigEBITS7和IV)的样本。用STRUCTURE对总群体进行亚群体结构分析显示根据K值的不同存在2-3个亚群体。然而,无论基于系统进化分析或是群体亚结构分析都没有发现毕氏肠微孢子虫存在地域特异性亚群体的证据。这充分说明毕氏肠微孢子虫经过长期的克隆进化,其稳定的基因型广泛分布于世界不同的研究地域。更多还原

【Abstract】 To assess the potential for transmission of cryptosporidiosis, microsporidiosis, and cyclosporiasis between humans and non-human primates that are in close contact with humans, 235 fecal specimens from captive baboons in Kenya were analyzed for Cryptosporidium spp., Enterocytozoon bieneusi, and Cyclospora spp. by PCR and DNA sequence analysis of the small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region of the rRNA gene, and the SSU rRNA gene, respectively. Cryptosporidium hominis was identified in six (2.6%) of the specimens. DNA sequence analysis of the 60kDa glycoprotein (gp60) gene of five C. hominis specimens revealed the presence of three subtypes in subtype families, Ib, If and a novel subtype family named Ii. E. bieneusi was detected in 29 (12.3%) specimens, belonging to 10 genotypes (four known genotypes: A, D, Peru7 and Peru11; six new genotypes: KB-1 to KB-6) that formed two phylogenetic clusters. All the E. bieneusi genotypes were previously found in humans or were genetically related to those in humans. Only one species of Cyclospora, C. papionis, was identified in 42 (17.9%) specimens. Results of this study indicated that non-human primates in Kenya were infected with human-pathogenic Cryptosporidium genotypes and subtypes and E. bieneusi genotypes. Thus, cross-species transmission of cryptosporidiosis and microsporidiosis is possible between humans and non-human primates that are in close contact with each other.Then, we applied multilocus sequence typing (MLST) tool described by Feng[1] to type 32 E. bineusi-positive specimens (3 with DNA sequencing failure at the ITS locus were included). Using nested PCR, we obtained 24, 8, 15, and 21 positives at loci MS1, MS3, MS4, and MS7, respectively. At the MS1 locus, genotype classification was based on a combination of the number of trinucleotide TGC, TAA and TAC repeats in the microsatellite region and the SNP in the rest of the sequence, revealing the presence of 10 distinct genotypes in 24 specimens with a gene diversity of 0.88. At the MS3 locus, the TA indels and SNP were used to determine the genotypes, resulting in the generation of 5 distinct genotypes in 8 specimens with a gene diversity of 0.86. The MS4 locus has a 35 bp minisatellite repeat region and two regions of insertion-deletion polymorphisms (a 4 bp fragment of GGTA between positions 147 and 150 and a 10 bp fragment of TTTTTTTCTT between positions 358 and 367). Coupled with SNPs outside the repeat region, MS4 yielded a total of 5 genotypes in 15 specimens with a gene diversity of 0.70. The MS7 locus has a microsatellite region with trinucleotide TAA repeats and the SNP in the rest of the sequence. It produced 7 genotypes (Hd = 0.77) in 21 specimens. ITS genotyping have shown that there were 10 ITS genotypes in 29 specimens, including host-adapted and zoonotic genotypes. The absence of PCR amplification at the locus MS3 in most isolates of host-adapted ITS genotypes such as KB-6 implied MS3 had the ability of discriminating the host-adapted from zoonotic E. bieneusi. Phylogenetic analysis of the nucleotide sequences obtained at microsatellite and minisatellite loci produced genetic relationship similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most human-pathogenic E. bieneusi genotypes. Thus, the MLST tool was appropriate for high resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., public health significance of E. bieneusi in animals, and source of human E. bieneusi infections.Genotyping based on DNA sequence analysis of the ITS locus has revealed significant genetic diversity in E. bieneusi. Thus far, the population genetics of E. bieneusi and its significance in microsporidiosis epidemiology have not been examined. To better understand the extent of genetic heterogeneity within E. bieneusi and examine its genetic structure, a MLST study of E. bieneusi in AIDS patients in Lima, Peru was conducted, using 72 specimens previously genotyped as A, D, IV, EbpC, WL11, Peru7, Peru8, Peru10, and Peru11. Together with the ITS, the use of four other genetic markers (MS1, MS3, MS4, and MS7) enabled high resolution genotyping of E. bieneusi. By a combination of sequence length polymorphism and nucleotide substitutions, 39 multilocus genotypes (MLGs) were identified among the 72 specimens. The observation of strong intragenic linkage disequilibria (LD) and detection of limited genetic recombination among loci were highly indicative of an overall clonal population structure of E. bieneusi. Measures of pair-wise intergenic LD and standardized index of association (ISA) based on allelic profile data further supported this conclusion. Both sequence-based and allelic profile-based phylogenetic analyses showed the presence of two genetically isolated groups in the study population, one (group 1) containing isolates of the anthroponotic ITS genotype A, and the other (group 2) containing isolates of multiple ITS genotypes (mainly genotypes D and IV) with zoonotic potential. The measurement of LD and recombination indicated group 2 had a clonal population structure, whereas group 1 had an epidemic population structure. Sub-structuring analysis using STRUCTURE resulted in the formation of two or three sub-populations depending on the setting of K values, and this was verified via measuring Wright’s fixation index (FST) between sub-populations. In conclusion, the MLST analysis of five markers had adequate power of resolution in delineating the population genetic structure of E. bieneusi. The data highlight the power of high resolution MLST in understanding the epidemiology of E. bieneusi.Thereafter, we applied MLST to genotype 105 E. bieneusi specimens isolated from AIDS patients in Peru, Nigeria, and India and five specimens isolated from captive baboons in Kenya using a combination of the ITS marker and four microsatellite and minisatellite markers, of which 72 isolates from Peru have been analyzed before. We determined 65 MLGs in total 110 isolates based on combined sequence length and nucleotide polymorphism. When the isolates of various geographical areas were treated as a single total population, the measures of intragenic and intergenic LD coupled with the limited genetic recombination detected indicated that there was an overall clonal population structure in E. bieneusi. The phylogenetic analysis based on either multilocus gene sequence or allelic profile data as well as the substructural analysis using STRUCTURE all found no evidence to support geographic sub-structuring of the population. When geographic populations (India, Kenya, and Nigeria) were analyzed, the observation of strong and significant LD and detection of limited recombination were highly indicative of a clonal population structure in each population tested. Although there was no evidence of geographic sub-population structure variations, both phylogenetic analysis and substructural analysis revealed the presence of two major genetically isolated groups in total population, one (group 1) contained isolates representing all ITS genotype A. The other (group 2) harbored isolates of multiple ITS genotypes with zoonotic potential. The measurement of LD and recombination indicated group 2 had a clonal population structure, whereas group 1 had an epidemic population structure. The data further confirmed the existence of various genetic sub-populations in E. bieneusi, which should be helpful in determining the transmission patterns of human microsporidiosis.更多还原