本文關(guān)鍵詞： 隱孢子蟲 十二指腸賈第蟲 畢氏腸微孢子蟲 種類 基因型 基因亞型 群體遺傳結(jié)構(gòu) 奶牛　出處：《河南農(nóng)業(yè)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：隱孢子蟲、十二指腸賈第蟲和畢氏腸微孢子蟲是重要的人獸共患腸道原蟲,世界范圍內(nèi)廣泛流行,多通過食物和水進(jìn)行傳播。牛被認(rèn)為是人類感染該3種機(jī)會(huì)性原蟲的重要傳染源,特別是含有病原體的犢牛糞便污染水源和食物是導(dǎo)致人體隱孢子蟲病和賈第蟲病暴發(fā)的主要因素。新近研究發(fā)現(xiàn),微小隱孢子蟲IId亞型可能從西亞隨牛的馴化史而擴(kuò)散至其他地理區(qū)域,且牛安氏隱孢子蟲基因亞型結(jié)構(gòu)在中國可能存在獨(dú)特地理隔離特征。中國新疆維吾爾族自治區(qū)自古為絲綢之路要道,在牛馴化和選育過程中有著獨(dú)特的地理優(yōu)勢,而有關(guān)該地區(qū)奶牛3種機(jī)會(huì)性原蟲遺傳學(xué)特征的研究尚未見報(bào)道。為進(jìn)一步驗(yàn)證上述科學(xué)推論,本研究對接近中亞地區(qū)的新疆地區(qū)奶牛進(jìn)行了隱孢子蟲感染情況調(diào)查,采用分子生物學(xué)方法對新疆地區(qū)奶牛犢牛3種原蟲進(jìn)行了分子流行病學(xué)調(diào)查、種類/基因型鑒定和種系進(jìn)化分析研究,并對新疆地區(qū)牛源安氏隱孢子蟲、新疆和寧夏地區(qū)牛源微小隱孢子蟲群體遺傳學(xué)進(jìn)行了研究。采用飽和蔗糖溶液漂浮法對新疆9個(gè)地區(qū)1827份奶牛糞便樣品中進(jìn)行檢查,隱孢子蟲總感染率為4.9%,其中褐牛年齡均為成年牛(450 d),隱孢子蟲感染率為2.3%;荷斯坦奶牛隱孢子蟲感染率為5.8%,以斷奶前犢牛(60 d)感染率最高,為12.2%,斷奶后犢牛(61-180 d)、育成牛(181-450 d)和成年牛感染率分別為7.7%、5.4%和2.6%,統(tǒng)計(jì)學(xué)差異極顯著(P0.01)�；赟SU rRNA基因,自新疆15個(gè)奶牛場514份奶牛犢牛糞便樣品中檢測出隱孢子蟲陽性樣品82份,總感染率為16.0%,高于鏡檢陽性率(10.1%),斷奶前和斷奶后犢牛隱孢子蟲感染率分別為15.6%和16.2%,無統(tǒng)計(jì)學(xué)差異(P0.05);安氏隱孢子蟲是該地區(qū)犢牛優(yōu)勢感染蟲種;微小隱孢子蟲僅感染斷奶前犢牛,在gp60基因位點(diǎn)鑒定出2個(gè)基因亞型,IIdA14G1和IIdA15G1均存在人畜共患風(fēng)險(xiǎn);不同地理分布的芮氏隱孢子蟲可能發(fā)生適應(yīng)性序列變異;微小隱孢子蟲基因亞型與地理分布存在明顯相關(guān)性�；赟SU rRNA基因,自新疆15個(gè)奶牛場514份奶牛犢牛糞便樣品中檢測出十二指腸賈第蟲陽性樣品64份,總感染率為13.4%,斷奶后(16.6%)較斷奶前(9.7%)犢牛感染十二指腸賈第蟲更為常見,統(tǒng)計(jì)學(xué)差異顯著(P0.05);集聚體E是犢牛感染的優(yōu)勢基因型,同時(shí)也發(fā)現(xiàn)人獸共患集聚體A感染,存在一定人獸共患風(fēng)險(xiǎn);基于tpi、gdh和bg基因位點(diǎn),分別鑒定出集聚體E有8個(gè)、11個(gè)和7個(gè)亞型,且形成5個(gè)MLG亞群結(jié)構(gòu),表明十二指腸賈第蟲可能存在地理隔離,發(fā)生適應(yīng)性變異。基于ITS基因,自新疆15個(gè)奶牛場514份奶牛犢牛糞便樣品中檢測出畢氏腸微孢子蟲陽性樣品85份,總感染率為16.5%,斷奶前和斷奶后犢牛感染率分別為17.7%和15.5%,無統(tǒng)計(jì)學(xué)差異(P0.05);發(fā)現(xiàn)6個(gè)基因型,其中BEB4、D、EbpC、I和J為已知人獸共患基因型,鑒定出1個(gè)新基因型,命名為NEW1;優(yōu)勢感染基因型為J,顯著不同于其他國家和地區(qū);種系進(jìn)化分析顯示,D、EbpC和NEW1基因型在人獸共患組1,I、J和BEB4基因型在牛特異性性組2,均有公共衛(wèi)生意義。經(jīng)顯微鏡形態(tài)學(xué)鑒定,1827份奶牛糞便樣品中70份為安氏隱孢子蟲陽性,總感染率為3.8%�；赟SU rRNA基因進(jìn)行蟲種鑒定,70個(gè)分離株均為安氏隱孢子蟲,與鏡檢結(jié)果一致。其中褐牛安氏隱孢子蟲感染率為2.3%;荷斯坦奶牛安氏隱孢子蟲感染率為4.3%,以斷奶后犢牛感染率最高,為8.4%,斷奶前犢牛、育成牛和成年牛安氏隱孢子蟲感染率分別為3.0%、5.4%和2.6%,統(tǒng)計(jì)學(xué)差異極顯著(P0.01)。多位點(diǎn)序列分型(MLST)方法在70個(gè)分離株中鑒定出8個(gè)安氏隱孢子蟲亞型結(jié)構(gòu),以A2,A4,A2,A1為優(yōu)勢亞型結(jié)構(gòu),3個(gè)為新的亞型結(jié)構(gòu),分別是A4,A5,A2,A1、A4,A5,A4,A1和A7,A4,A2,A1;成年荷斯坦奶牛安氏隱孢子蟲亞型結(jié)構(gòu)呈多樣化,12個(gè)成功分型的分離株存在7個(gè)亞型結(jié)構(gòu)。連鎖不平衡分析顯示,新疆牛源安氏隱孢子蟲為流行性群體結(jié)構(gòu)。為進(jìn)一步了解微小隱孢子蟲IId家族群體遺傳結(jié)構(gòu)特征,收集新疆和寧夏41個(gè)牛源微小隱孢子蟲IId亞型分離株進(jìn)行MLST,在12個(gè)基因位點(diǎn)形成4個(gè)MLST亞型結(jié)構(gòu)。結(jié)合先前中國河南省牛源、山東省馬源和河南省鼠源微小隱孢子蟲IId亞型分離株的數(shù)據(jù),在12個(gè)基因位點(diǎn)共形成7個(gè)MLST亞型結(jié)構(gòu),MLST1-6形成一個(gè)群,而1個(gè)馬源亞型結(jié)構(gòu)MLST7單獨(dú)形成一個(gè)群,形成流行性群體遺傳結(jié)構(gòu),表明MLST亞型存在宿主適應(yīng)性;而中國不同地區(qū)牛源微小隱孢子蟲IId亞型分離株為克隆性群體結(jié)構(gòu),基因亞型存在地理隔離現(xiàn)象。綜上所述,本研究解析了新疆地區(qū)奶牛犢牛隱孢子蟲的種類和基因亞型遺傳特征,十二指腸賈第蟲的集聚體分布和亞型結(jié)構(gòu),畢氏腸微孢子蟲的人獸共患風(fēng)險(xiǎn);牛源安氏隱孢子蟲亞型結(jié)構(gòu)呈多樣化并存在地理區(qū)域分布特征;中國微小隱孢子蟲IId分離株MLST亞型形成流行性群體結(jié)構(gòu),存在宿主適應(yīng)性和地理隔離特征,為深入研究隱孢子蟲的流行傳播提供了參考。
[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)鏂規(guī)硶鍦，

本文編號(hào)：1460709