林芝藏猪消化道寄生虫调查及蛔虫、细颈囊尾蚴线粒体基因组学研究

发布时间：2018-06-28 00:17

本文选题：藏猪 + 蛔虫　；参考：《华中农业大学》2017年博士论文

【摘要】：作为一种以草为主的地方特色品种,藏猪因对高海拔地区严寒、缺氧等极端气候具有极好的适应能力而成为青藏高原牧民主要的食物来源和经济来源,其作用不可或缺。由于采取自由放牧的形式,粗放的管理模式,畜群结构不合理以及藏猪品种的退化,再加上薄弱的疾病防控意识,因此近年来藏猪的各种疾病,尤其是寄生虫疾病的发病率逐年升高。其中最为常见的寄生虫是蛔虫和细颈囊尾蚴,一旦发生感染,严重影响藏猪的生长发育和生产性能,不但给藏猪产业带来巨大的经济损失,同时也会提高人类感染人畜共患寄生虫疾病的风险。故本研究在西藏藏猪消化道寄生虫病流行情况,蛔虫和细颈囊尾蚴的分离和鉴定以及线粒体基因组学等方面展开调查和研究,结果如下:1.西藏林芝地区藏猪消化道寄生虫流行情况调查采用寄生虫学完全剖检法、粪便虫卵检查法,112头屠宰藏猪、73份粪便进行寄生虫病流行情况调查。结果显示:共检测出寄生虫11种,隶属于5门、6纲、9目、10科、10属,其中线虫5种,绦虫蚴2种,原虫2种,吸虫1种,棘头虫1种。优势虫种为细颈囊尾蚴(42.9%)、野猪后圆线虫(38.4%)、棘球蚴(33.0%)、蛔虫(30.4%)、肝片吸虫(26.8%)、食道口线虫(18.8%)、毛首线虫(15.2%)、球虫(15.1%)、结肠小袋纤毛虫(6.8%)、蛭形巨吻棘头虫(5.6%)、六翼泡首线虫(2.7%)。调查结果表明,林芝地区藏猪胃肠道寄生虫感染普遍,感染率较高。2.藏猪蛔虫nad1、cox1和cox2基因扩增及序列分析本试验以屠宰藏猪体内分离的蛔虫为研究对象,运用PCR方法,以不同引物分别扩增nad1、cox1和cox2三个基因序列,扩增后的片段纯化后克隆至pGEM?-T Easy载体,转化,并对扩增后阳性产物进行测序和序列分析,以鉴定藏猪蛔虫的种类。结果显示:藏猪蛔虫的nad1、cox1和cox2序列均与猪蛔虫(登录号:X54253.1,猪蛔虫)相似性为99%。调查结果表明,此次分离的蛔虫属于猪蛔虫。本研究是首次通过藏猪蛔虫三个线粒体基因片段nad1、cox1、cox2进行分离、鉴定和分子标记。3.藏猪细颈囊尾蚴病流行病学调查及cox2基因扩增及序列分析本研究以西藏林芝地区屠宰藏猪体内分离出来的细颈囊尾蚴为研究对象,用ELISA和PCR方法,首次扩增藏猪细颈囊尾蚴的cox2基因序列,并对扩增后阳性产物进行测序和序列分析,以鉴定藏猪蛔虫的种类。结果显示:藏猪细颈囊尾蚴的血清抗体阳性率平均为43.93%(2014:42.86%;2015:45.35%),其中公猪和母猪的阳性率分别为43.39%,44.56%,不同年龄段的藏猪细颈囊尾蚴的阳性率范围为30.20%~63.79%,结果显示:本次分离到细颈囊尾蚴株与甘肃、青海和四川分离株具有很大的相似性。本研究是首次通过对藏猪细颈囊尾蚴进行流行病学调查和cox2基因进行分离和鉴定,旨在为西藏地区藏猪细颈囊尾蚴的防治提供流行病学和分子生物学数据。4.藏猪蛔虫、细颈囊尾蚴线粒体基因组序列测定与分析本试验对藏猪蛔虫、细颈囊尾蚴的线粒体DNA进行提取、建库和测序、功能注释和生物信息学分析。结果表明:猪蛔虫线粒体基因组大小为14128 bp,编码基因12个(cox1-3,nad1-6,nad4L,atp6,cytb)、rRNA 2个(rrnL、rrnS)、t RNA 35个;细颈囊尾蚴线粒体基因组大小为13607 bp,编码基因12个(cox1-3,nad1-6,nad4L,atp6,cytb)、rRNA2个(rrnL,rrnS)、t RNA 22个。猪蛔虫、细颈囊尾蚴线粒体基因碱基A、T含量明显高于C、G含量,分别为71.74%和70.90%。猪蛔虫、细颈囊尾蚴线粒体基因密码子分别为4385(不含终止密码子304个)和4194个(不含终止密码子341个)。两者密码子种类均为63个,TTT使用最多,分别为15.51%和10.44%;蛔虫最少的为CAA(0.05%)、CGC(0.05%),细颈囊尾蚴最少的为CGC(0.05%)。均翻译20种氨基酸,猪蛔虫苯丙氨酸、亮氨酸、缬氨酸比例最高,为17.13%、16.03%、10.86%;细颈囊尾蚴为亮氨酸、苯丙氨酸、缬氨酸比例最高,为16.48%、11.76%、11.06%。猪蛔虫氨基酸比列最低的为谷氨酰胺(0.73%)、组氨酸(0.62%);细颈囊尾蚴氨基酸比列最低的为谷氨酰胺(1.29%)、色氨酸(1.67%)、丙氨酸(1.81%)。进化分析表明:藏猪蛔虫与人蛔虫(NC-016198.1)同源性高达99%;与X54253.1同源性高达99%;与中国湛江分离虫体(登录号为HQ704901.1)同源性为98%。该结果与之前报道的人蛔虫与猪蛔虫高度同源结果一致,进一步证实了二者可能为同一种虫体。藏猪细颈囊尾蚴与参考的甘肃分离株(登录号:GQ228819.1)同源性均高达99%。综上所述,本研究通过对西藏藏猪消化道寄生虫流行病学调查以及2种常见寄生虫(蛔虫、细颈囊尾蚴)进行线粒体基因组学研究,较为全面了解藏猪常见消化道寄生虫的流行情况,并通过对其进行分离鉴定及线粒体基因组测序,这对于了解藏猪寄生虫的分离鉴定、进化分析,种内遗传变异情况,乃至藏猪寄生虫的防控具有重大的意义。
[Abstract]:As a local characteristic variety with grass mainly, the Tibetan Pig has become the main food source and economic source for the herdsmen of the Qinghai Tibet Plateau because of its excellent adaptability to the extreme cold and anoxic extreme climate in high altitude areas, and its function is indispensable. In recent years, the incidence of various diseases in Tibetan pigs, especially parasitic diseases, is increasing year by year. The most common parasites are Ascaris Ascaris and cysticercus cellulosae, which seriously affect the growth and production performance of Tibetan pigs, not only to the Tibetan Pig Industry, but also to the pig industry. Huge economic losses will also increase the risk of human zoonosis parasitic diseases. Therefore, this study investigates and studies the epidemic of parasitic diseases in the digestive tract of the Tibetan pigs in Tibet, the isolation and identification of Ascaris and cercariae and the mitochondrial genomics. The results are as follows: 1. the digestive tract of the Tibetan pigs in Linzhi, Tibet Parasitology epidemiological survey using parasitology complete examination, fecal egg examination, 112 slaughtered hogs and 73 faeces for parasitic diseases. The results showed that 11 species of parasites were detected in 5 doors, 6 classes, 9 orders, 10 families and 2 species, 2 species of tapeworms, 2 species of protozoa, 1 species of acanthosis and 1 species of echinohead. Insect species are cysticercus cellulosae (42.9%), hydatidis (38.4%), hydatid (33%), Ascaris (30.4%), Fasciola hepatica (26.8%), esophagus nematode (18.8%), nematode (15.2%), coccidia (15.1%), small colonic ciliates (6.8%), leech giant spinous echinohead (5.6%), and six winged nematode (2.7%). The results of the investigation showed that the gastrointestinal parasitism of Tibetan pigs in Linzhi area was parasitized. The infection rate is common, the infection rate is high.2. hid NaD1, cox1 and COX2 gene amplification and sequence analysis. This experiment takes the Ascaris Ascaris isolated in the slaughtered pig as the research object. The PCR method is used to amplify three gene sequences of NaD1, cox1 and COX2 with different primers. After the amplification, the fragment is cloned to pGEM? -T Easy carrier. The positive products were sequenced and sequenced to identify the species of Ascaris Ascaris. The results showed that the NaD1, cox1 and COX2 sequences of Ascaris Ascaris were all similar to that of Ascaris Ascaris (X54253.1, Ascaris Ascaris). The results showed that the Ascaris belonged to Ascaris Ascaris. This study was the first three mitochondrial bases of Ascaris Ascaris. NaD1, cox1, COX2 were isolated, identified and labeled by molecular marker.3., the epidemiological investigation of cysticercosis in porcine cysticercosis and the amplification and sequence analysis of COX2 gene were studied. The COX2 gene of the cysticercus cercariae was amplified by ELISA and PCR method for the study of the cysticercus isolated from the slaughtered and Tibetan pigs in Linzhi, Tibet. Sequence analysis and sequence analysis of positive products were carried out to identify the species of Ascaris Ascaris. The results showed that the positive rate of serum antibody was 43.93% (2014:42.86%; 2015:45.35%), and the positive rate of boar and sow was 43.39%, 44.56%, and the positive rate of Cysticercus cellulosae in different age groups. The range was 30.20%~63.79%. The results showed that the isolated strains of Cysticercus cellulosae were very similar to Gansu, Qinghai and Sichuan isolates. This study was the first time to isolate and identify the COX2 gene by epidemiological investigation of the cysticercus cercariae and to provide the epidemic prevention and control of Cysticercus cercariae in Tibet. Disease and molecular biology data.4. hid Ascaris Ascaris, mitochondrial genome sequencing of Cysticercus cercariae and analysis of mitochondrial DNA from Ascaris Ascaris and cysticercus cercariae were extracted, constructed and sequenced, functional annotation and bioinformatics analysis. The results showed that the size of the mitochondrial genome of Ascaris Ascaris was 14128 BP and 12 (co X1-3, nad1-6, nad4L, Atp6, cytb), rRNA 2 (rrnL, rrnS) and t RNA 35. The size of the mitochondrial genome of Cysticercus cercariae is 13607 BP, and 12 encoded genes. The codon of the mitochondrial gene of Ascaris Ascaris and cercariae is 4385 (304 without terminating codon) and 4194 (without terminating codon 341). Both codon is 63, TTT is used most, 15.51% and 10.44% respectively. The Ascaris is CAA (0.05%), CGC (0.05%), and the cercariae at least CGC (0.05%). Both translates 20 ammonia. The ratio of phenylalanine, leucine and valine in Ascaris Ascaris is the highest, 17.13%, 16.03%, 10.86%. The proportion of Cysticercus is leucine, phenylalanine and valine is the highest, 16.48%, 11.76%. The lowest amino acid of Ascaris 11.06%. is glutamine (0.73%), histidine (0.62%), and the lowest amino acid in Cysticercus cercariae is glutamine. (1.29%), tryptophan (1.67%) and alanine (1.81%). The phylogenetic analysis showed that the homology of Ascaris Ascaris (NC-016198.1) was up to 99%, and the homology of X54253.1 was 99%, and the homology of the Chinese Zhanjiang isolate (login number HQ704901.1) was 98%., which was consistent with the high homologous results of Ascaris Ascaris and Ascaris Ascaris reported before. The two species may be the same type of insect. The homology of the porcine cysticercus and the reference Gansu isolate (login number: GQ228819.1) are all up to 99%.. The study on the mitochondrial genomics of the parasites of the digestive tract of the Tibetan pigs in Tibet and the 2 common parasites (Ascaris Ascaris, cysticercus cercariae) are studied in this study. It is of great significance for understanding the isolation, identification, evolution analysis, intraspecific genetic variation and even the prevention and control of parasites in Tibetan pigs, through the isolation and identification of the common digestive tract parasites in Tibetan pigs and the sequencing of the mitochondrial genome.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.28

【参考文献】