一种貉僵化症病因、流行病学调查及比较转录组、蛋白组研究

发布时间：2018-01-20 03:02

本文关键词： 貉僵化症毕氏肠微孢子虫附红细胞体比较转录组比较蛋白组　出处：《吉林农业大学》2017年博士论文　论文类型：学位论文

【摘要】：貉子是我国广泛养殖的珍贵的毛皮动物之一,近几年出现的貉僵化症,又称僵貉病,严重影响养殖貉经济效益。本试验首先以河北某地的典型僵化症貉为研究对象,从僵貉临床症状、血液生理生化指标、感染源确诊和疾病治疗方面对貉僵化病产生的影响及影响程度进行了探索性的初步研究,并调查了我国养貉主产区微孢子虫、附红细胞体和僵化病的流行情况。然后利用高通量测序技术对僵貉与正常貉的肝脏、脾脏进行比较转录组、蛋白组研究,取得了以下发现:1、针对河北某地的僵化貉样品,利用常规病理剖检、胶体金试纸条、PCR、细菌分离培养、动物回归和血常规和血液生化检测,并利用相应药物治疗,排除冠状病毒、犬瘟热病毒、细小病毒、水貂阿留申病毒、貉蓝狐源阿留申病毒和遗传原因,综合确诊该场为以附红细胞体为主由附红细胞体和毕氏肠微孢子虫混合感染导致僵化。2、利用巢氏PCR调查我国养貉主产区发现貉毕氏肠微孢子虫总阳性率为22.30%,感染率最高的地区为河北省阳性率为40.74%,公貉阳性率为27.10%,母貉阳性率为17.33%;DNA测序结果发现鉴定了六个不同的ITS区,包括四个已知的基因型(D,CHN-DC1,NCF2和CHN-F1)和两个新的基因型(NCR1和NCR2),系统发育分析显示所有六种基因型属于组1-人兽共患病组,表明貉是人类毕氏肠微孢子虫感染的潜在来源,应注意貉毕氏微孢子对人类公共健康的威胁。3、采用血液滴片检测方法检测了我国养貉主产区貉血液样品,发现附红细胞体阳性率为62.29%,感染率最高的地区为河北省阳性率为68.64%,母貉和公貉附红细胞体感染率分别为61.16%和63.38%。应引起我们对附红细胞体大面积感染引起的免疫抑制问题关注。4、调查统计7省区38个貉养殖场发现貉僵化症总体阳性率为4.71%,各省区之间差异不显著,不同省区内各个养殖场之间差异明显,发生率高的比低的场子相差18.4倍,僵化症发生率与管理水平呈相反趋势,管理好的场子貉僵化症发生率低。5、应用比较转录组学技术对僵化症貉和正常貉的肝脏和脾脏2个器官的14个样品进行研究,共得到1,571,229个转录本和290568个unigenes,通过与Nr数据库进行比对注释,发现貉与狗相似性最高达到61.62%,符合已知分类学地位。僵貉脾脏相对于正常貉的转录组有1842个基因上调和876个基因下调,僵貉肝脏相对于正常貉有1392个基因表达上调和604个基因表达下调,其相关基因可以作为僵化诊断治疗及预防的候选基因。6、对僵貉脾脏比较转录组Pathway富集分析,发现共涉及六大分类42个通路,聚类发现,僵貉与正常貉脾脏差异基因Pathway富集显著性最高的为代谢通路和PI3K/Akt信号通路。对僵貉肝脏比较转录组Pathway富集分析,发现共涉及六大分类41个通路。聚类发现,僵貉与正常貉肝脏差异基因Pathway富集显著性最高的为环境信息中的加工信号转导途径PI3K-Akt信号通路、代谢通路、ECM受体相互作用通路。7、对僵化貉和正常貉的脾脏进行比较蛋白质组学研究,共鉴定出2,469个蛋白,其中1,484个蛋白可以被定量,僵化貉脾脏中相对于正常貉正常貉有23个蛋白表达上调,33个蛋白表达下调。上调差异主要是细胞骨架成分涉及生物粘附和细胞粘附过程中的酶活性调节等。下调差异蛋白主要是MHC蛋白复合物涉及免疫系统、免疫反应等功能。KEGG分析发现上调差异基因共涉及25个通路,下调差异基因共涉及2个通路。8、对僵化貉和正常貉的肝脏进行比较蛋白质组学研究,总共鉴定出有2,025个蛋白,其中有1,282个蛋白可以被精确定量,僵化貉肝脏中相对于正常貉有93个蛋白表达上调,119个蛋白表达下调。上调差异基因共涉及1个通路,下调差异基因共涉及16个通路。在僵化貉与正常貉差异蛋白分子功能富集分析排名前三位的是氧化还原酶活性、铁离子结合、血红素结合,与机体感染附红细胞体后内呼吸和氧气运输相关功能分子关系较大。
[Abstract]:Raccoon is one of the precious fur animal extensively cultivated in China, in recent years the raccoon rigid disease, also known as stiff raccoon disease, seriously affect the economic benefits. The raccoon dog breeding first in a typical Hebei province sclerosis raccoon as the research object, from the dead raccoon clinical symptoms, blood physiological and biochemical indexes, the to explore the preliminary study of the effect on treatment of infection disease produces rigid raccoon source diagnosis and disease and the influence, and the survey of the raccoon dog producing microsporidia, Eperythrozoon and epidemic situation of rigid disease. Then use high-throughput sequencing of normal liver and stiff raccoon raccoon, spleen comparative transcriptome, proteome research, made the following findings: 1 in Hebei, a rigid raccoon sample, using routine pathological examination, colloidal gold strip, PCR, bacterial culture, animal regression and blood routine and blood biochemical detection, and use The corresponding drug treatment, exclusion of coronavirus, canine distemper virus, parvovirus, mink Aleutian disease virus, blue fox raccoon source Aleutian disease virus and genetic causes, comprehensive diagnosis of the field to Eperythrozoon dominated by Eperythrozoon and Pichia intestinal microsporidia infection leads to mixed rigid.2, China's main producing areas of the raccoon dog survey found raccoon dog intestinal microsporidia Pichia total positive rate was 22.30% by nested PCR, the highest infection rate in the area for the positive rate of Hebei province was 40.74%, the positive rate of male raccoon was 27.10%, the positive rate of mother raccoon was 17.33%; DNA were found in six different ITS regions were identified, including four known genotypes (D, CHN-DC1, NCF2 and CHN-F1) and two new genotypes (NCR1 and NCR2), phylogenetic analysis showed that all six genotypes belonging to the group of 1- Amphixenosis group, that is the human intestinal raccoon Pichia microsporidia infection potential sources, should pay attention to the raccoon dog Bi Shiwei The spores of human public health threat detection.3, China's main producing areas of the raccoon dog with blood dripping raccoon blood sample detection method, it was found that the positive rate of Eperythrozoon infection rate was 62.29%, the highest positive rate of Hebei province is 68.64%, and female raccoon dog male raccoon dog Eperythrozoon infection rate was 61.16% and 63.38%. should cause us to a large area of Eperythrozoon infection induced immunosuppression on.4, investigation and statistics of 7 provinces in 38 raccoon farms that raccoon sclerosis total positive rate was 4.71%, there was no significant difference between different provinces, different provinces between the various farms significantly higher incidence than low a difference of 18.4 times, the incidence rate of sclerosis and management level had the opposite trend, the incidence rate of low.5 manage place raccoon sclerosis, using comparative transcriptome of raccoon dog and raccoon normal sclerosis of the liver and spleen of 2 organs 14 The sample of a total of 1571229 transcripts and 290568 unigenes, comparing notes with the Nr database, and found that the highest similarity of raccoon dog reached 61.62%, consistent with the known taxonomic status. Compared with the normal group of stiff raccoon spleen transcription of 1842 genes of the raccoon and 876 genes were down regulated compared with normal liver, stiff raccoon raccoon dog there are 1392 up-regulated genes and 604 downregulated genes, the gene can be used as candidate genes for.6 diagnosis and treatment and prevention of the rigid and stiff of raccoon comparative transcriptome Pathway enrichment analysis were found, involving a total of six classification 42 paths, clustering found stiff raccoon and normal raccoon dog Pathway gene significantly enriched spleen the highest metabolic pathway and PI3K/Akt pathway. The liver transcriptome stiff raccoon Pathway enrichment analysis found, involving a total of six classification 41 pathways. Clustering, stiff and normal raccoon dog Raccoon dog liver Pathway gene significantly enriched the highest processing of environmental information in the signal transduction pathway of PI3K-Akt signaling pathway, metabolic pathway, ECM receptor interaction pathway.7, the rigid and normal spleen raccoon raccoon comparative proteomics study, 2469 proteins were identified, of which 1484 proteins can be quantitative, rigid Raccoon in spleen compared with normal raccoon normal raccoon expression of 23 proteins, 33 proteins expression down. Upregulation of differences are mainly components of the cytoskeleton involved enzyme activity process of biological adhesion and cell adhesion in the regulation. The difference is mainly MHC down-regulation of protein protein complexes involved in the immune system, immune response function.KEGG analysis found that 25 pathways involving a total of up-regulated differential gene expression, downregulation of genes involving a total of 2 path.8 of normal liver and rigid raccoon raccoon comparative proteomics study, total discrimination Out of 2025 proteins, including 1282 proteins can be accurately quantified relative to normal liver in rigid raccoon raccoon expression of 93 protein, 119 protein down regulated. 1 pathways involving upregulation of different genes, 16 down regulated pathways involving genes. The rigid differential proteins and normal raccoon raccoon dog the functional enrichment analysis of the top three is the oxidoreductase activity, ferric ion binding, heme binding, and respiratory infection of Eperythrozoon in oxygen transport function of molecular relationship is larger.

【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.92

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