新疆地区奶牛三种肠道原虫系统进化分析及隐孢子虫群体遗传学研究

发布时间：2018-01-24 18:27

  本文关键词： 隐孢子虫 十二指肠贾第虫 毕氏肠微孢子虫 种类 基因型 基因亚型 群体遗传结构 奶牛　出处：《河南农业大学》2015年博士论文　论文类型：学位论文

【摘要】：隐孢子虫、十二指肠贾第虫和毕氏肠微孢子虫是重要的人兽共患肠道原虫,世界范围内广泛流行,多通过食物和水进行传播。牛被认为是人类感染该3种机会性原虫的重要传染源,特别是含有病原体的犊牛粪便污染水源和食物是导致人体隐孢子虫病和贾第虫病暴发的主要因素。新近研究发现,微小隐孢子虫IId亚型可能从西亚随牛的驯化史而扩散至其他地理区域,且牛安氏隐孢子虫基因亚型结构在中国可能存在独特地理隔离特征。中国新疆维吾尔族自治区自古为丝绸之路要道,在牛驯化和选育过程中有着独特的地理优势,而有关该地区奶牛3种机会性原虫遗传学特征的研究尚未见报道。为进一步验证上述科学推论,本研究对接近中亚地区的新疆地区奶牛进行了隐孢子虫感染情况调查,采用分子生物学方法对新疆地区奶牛犊牛3种原虫进行了分子流行病学调查、种类/基因型鉴定和种系进化分析研究,并对新疆地区牛源安氏隐孢子虫、新疆和宁夏地区牛源微小隐孢子虫群体遗传学进行了研究。采用饱和蔗糖溶液漂浮法对新疆9个地区1827份奶牛粪便样品中进行检查,隐孢子虫总感染率为4.9%,其中褐牛年龄均为成年牛(450 d),隐孢子虫感染率为2.3%;荷斯坦奶牛隐孢子虫感染率为5.8%,以断奶前犊牛(60 d)感染率最高,为12.2%,断奶后犊牛(61-180 d)、育成牛(181-450 d)和成年牛感染率分别为7.7%、5.4%和2.6%,统计学差异极显著(P0.01)。基于SSU rRNA基因,自新疆15个奶牛场514份奶牛犊牛粪便样品中检测出隐孢子虫阳性样品82份,总感染率为16.0%,高于镜检阳性率(10.1%),断奶前和断奶后犊牛隐孢子虫感染率分别为15.6%和16.2%,无统计学差异(P0.05);安氏隐孢子虫是该地区犊牛优势感染虫种;微小隐孢子虫仅感染断奶前犊牛,在gp60基因位点鉴定出2个基因亚型,IIdA14G1和IIdA15G1均存在人畜共患风险;不同地理分布的芮氏隐孢子虫可能发生适应性序列变异;微小隐孢子虫基因亚型与地理分布存在明显相关性。基于SSU rRNA基因,自新疆15个奶牛场514份奶牛犊牛粪便样品中检测出十二指肠贾第虫阳性样品64份,总感染率为13.4%,断奶后(16.6%)较断奶前(9.7%)犊牛感染十二指肠贾第虫更为常见,统计学差异显著(P0.05);集聚体E是犊牛感染的优势基因型,同时也发现人兽共患集聚体A感染,存在一定人兽共患风险;基于tpi、gdh和bg基因位点,分别鉴定出集聚体E有8个、11个和7个亚型,且形成5个MLG亚群结构,表明十二指肠贾第虫可能存在地理隔离,发生适应性变异。基于ITS基因,自新疆15个奶牛场514份奶牛犊牛粪便样品中检测出毕氏肠微孢子虫阳性样品85份,总感染率为16.5%,断奶前和断奶后犊牛感染率分别为17.7%和15.5%,无统计学差异(P0.05);发现6个基因型,其中BEB4、D、EbpC、I和J为已知人兽共患基因型,鉴定出1个新基因型,命名为NEW1;优势感染基因型为J,显著不同于其他国家和地区;种系进化分析显示,D、EbpC和NEW1基因型在人兽共患组1,I、J和BEB4基因型在牛特异性性组2,均有公共卫生意义。经显微镜形态学鉴定,1827份奶牛粪便样品中70份为安氏隐孢子虫阳性,总感染率为3.8%。基于SSU rRNA基因进行虫种鉴定,70个分离株均为安氏隐孢子虫,与镜检结果一致。其中褐牛安氏隐孢子虫感染率为2.3%;荷斯坦奶牛安氏隐孢子虫感染率为4.3%,以断奶后犊牛感染率最高,为8.4%,断奶前犊牛、育成牛和成年牛安氏隐孢子虫感染率分别为3.0%、5.4%和2.6%,统计学差异极显著(P0.01)。多位点序列分型(MLST)方法在70个分离株中鉴定出8个安氏隐孢子虫亚型结构,以A2,A4,A2,A1为优势亚型结构,3个为新的亚型结构,分别是A4,A5,A2,A1、A4,A5,A4,A1和A7,A4,A2,A1;成年荷斯坦奶牛安氏隐孢子虫亚型结构呈多样化,12个成功分型的分离株存在7个亚型结构。连锁不平衡分析显示,新疆牛源安氏隐孢子虫为流行性群体结构。为进一步了解微小隐孢子虫IId家族群体遗传结构特征,收集新疆和宁夏41个牛源微小隐孢子虫IId亚型分离株进行MLST,在12个基因位点形成4个MLST亚型结构。结合先前中国河南省牛源、山东省马源和河南省鼠源微小隐孢子虫IId亚型分离株的数据,在12个基因位点共形成7个MLST亚型结构,MLST1-6形成一个群,而1个马源亚型结构MLST7单独形成一个群,形成流行性群体遗传结构,表明MLST亚型存在宿主适应性;而中国不同地区牛源微小隐孢子虫IId亚型分离株为克隆性群体结构,基因亚型存在地理隔离现象。综上所述,本研究解析了新疆地区奶牛犊牛隐孢子虫的种类和基因亚型遗传特征,十二指肠贾第虫的集聚体分布和亚型结构,毕氏肠微孢子虫的人兽共患风险;牛源安氏隐孢子虫亚型结构呈多样化并存在地理区域分布特征;中国微小隐孢子虫IId分离株MLST亚型形成流行性群体结构,存在宿主适应性和地理隔离特征,为深入研究隐孢子虫的流行传播提供了参考。
[Abstract]:Cryptosporidium, Giardia duodenalis and Pichia intestinal microsporidia is an important zoonotic intestinal protozoa, widely popular in the world, through the spread of food and water. Cattle is considered to be an important source of infection for humans of the 3 kinds of opportunistic protozoa, especially containing pathogen of calf fecal contamination of food and water are the main factors leading to human cryptosporidiosis and giardiasis outbreaks. Recent studies showed that the Cryptosporidium parvum subtype IId may from the West with cattle domestication history and spread to other geographical areas, and Cryptosporidium andersoni genotype structure in Chinese may have unique geographical isolation characteristics. Chinese Xinjiang Uygur the autonomous region since ancient times for the silk road routes, has a unique geographical advantage in cattle domestication and breeding process, research about the cow 3 kinds of genetic characteristics of opportunistic protozoa have not been reported . to further verify the above scientific inference, this study conducted a survey of Cryptosporidium infection in the central region of Xinjiang area close to the cow, using molecular biology methods for molecular epidemiological investigation of dairy calves in Xinjiang and 3 species of protozoa species / genotype identification and phylogenetic analysis, and the source of Xinjiang cattle area c.andersoni, source of cattle in Xinjiang and Ningxia area of Cryptosporidium parvum population genetics were studied. The examination of 9 areas of Xinjiang 1827 dairy manure samples by saturated sucrose flotation method, the total infection rate of 4.9% hidden spore worm, which age brown cattle were adult cattle (450 d) and the infection rate of Cryptosporidium was 2.3%; the infection rate of Cryptosporidium in Holstein cows was 5.8% in pre weaning calves (60 d) the highest infection rate was 12.2%, after weaning calves (61-180 d), breeding cattle (181-450 d) and adult bovine sense The dyeing rate were 7.7%, 5.4% and 2.6%, significant statistical difference (P0.01). The SSU rRNA gene from Xinjiang based on 15 dairy farms of 514 dairy calves fecal samples to detect Cryptosporidium positive samples of 82 copies, the total infection rate was 16%, higher than the positive rate of examination (10.1%), respectively 15.6% and 16.2% calves of Cryptosporidium infection in weaned before and after weaning, there was no significant difference (P0.05); c.andersoni is the advantage of the parasite infection in calves; Cryptosporidium parvum infection only before weaning calves at the gp60 gene locus identified 2 subtypes, IIdA14G1 and IIdA15G1 exist in human and animal both risk; different distribution of Cryptosporidium muris Rui may occur adaptive sequence variation; there is obvious correlation between Cryptosporidium parvum genotype and geographic distribution. Based on SSU rRNA gene, from Xinjiang 15 dairy farms 514 calves fecal samples were detected in twelve The Giardia intestinalis positive samples of 64 copies, the total infection rate was 13.4%, after weaning (16.6%) than before weaning (9.7%) calves infected with Giardia duodenalis is more common, a statistically significant difference (P0.05); agglomeration E is the dominant genotype in calves, but also found that Amphixenosis from agglomeration of A infection. There are some zoonotic risk; based on TPI, GDH and BG loci were identified, agglomeration of E are 8, 11 and 7 subtypes, and the formation of the 5 subsets of MLG structure, show that Giardia duodenalis may exist in geographic isolation, adaptive variation. Based on the ITS gene, 15 cows from Xinjiang 514 copies of calves fecal samples detected in Pichia intestinal microsporidia positive in 85 samples, the total infection rate was 16.5%, 17.7% and 15.5% respectively in calves weaned before and after weaning, there was no significant difference (P0.05); found 6 genotypes, including BEB4, D, EbpC, and I J is known to man Zoonotic genotypes identified 1 new genes, named NEW1; infection genotype was J, significantly different from other countries and regions; phylogenetic analysis showed that D, EbpC and NEW1 genotypes in Amphixenosis group 1, I, J and BEB4 genotypes in bovine specific group of 2, have public health significance. By microscopic morphological identification, 1827 samples of cow dung samples in 70 samples were c.andersoni positive, the total infection rate was 3.8%. for the identification of SSU rRNA gene based on 70 isolates were c.andersoni, consistent with the microscope. Results of the ANN's hidden brown cattle Cryptosporidium infection rate was 2.3%; the infection rate of Holstein cow Cryptosporidium andersoni was 4.3%, with the highest rate of infection after weaning calves, 8.4% pre weaning calves, heifers and cows c.andersoni infection rates were 3%, 5.4% and 2.6%, significant statistical difference (P0.01). Multilocus sequence type (M LST)鏂规硶鍦，

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