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Analyzing factors involved in genetic variation of porcine response to Salmonella.

机译：分析猪对沙门氏菌反应的遗传变异中涉及的因素。

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摘要

The purpose of the research presented in this dissertation was to contribute to the understanding of factors underlying disease resistance in swine by investigating genetic variation associated with swine response to Salmonella. The main objectives of the research were: 1) using integrated global gene expression and bioinformatics analysis approach to discover candidate genes with single nucleotide polymorphisms (SNPs) that can be associated with Salmonella-related phenotypes and potentially affect Salmonella shedding and/or the carrier status in swine; 2) analyzing expression of a set of genes regulated by IFN-gamma, a cytokine whose serum levels are correlated with Salmonella shedding in swine, to determine potential biomarkers for assessing the quality of swine immune response that could be used to predict the shedding status of swine.;To identify SNPs in functional candidate genes to test for associations with swine response to Salmonella, integrative gene selection approach was employed. Recent global gene expression analysis of the porcine response to Salmonella combined with literature information on important candidate genes as well as sequence alignment-based predictions of SNPs resulted in successful identification of SNPs in functional candidate genes. The selected SNPs were genotyped by Sequenom technology using four porcine populations that total 750 swine. Genotyping and statistical association analysis resulted in discovery of several novel SNPs in 12 functional candidate genes associated with Salmonella-related phenotypes, such as tissue colonization and/or fecal Salmonella shedding. The associated SNPs were identified in such genes as: TAP1, GNG3, CCR1, CD163, AMT, ACP2, VCL, CCT7, HP, EMP1, NCF2 and PGD. Thus, the SNPs analysis study suggest several potential candidate genes to be further evaluated as markers for selection of swine with improved immune function and reduced Salmonella shedding. Additionally, the novel genetic associations provide useful information for the pig industry regarding strategies for selecting swine with improved disease resistance.;One of the major immune response mediators, IFN-gamma, has been shown to be important in swine response to Salmonella by transcriptomic and pathway analysis. Thus, this research focused on a postulated role of IFN-gamma-regulated genes in the differential swine response to Salmonella. Whole blood transcriptome analysis revealed many differentially expressed genes between pigs classified as persistent (PS) or low (LS) Salmonella shedders. Pathway analysis of differentially expressed genes showed that IFN-gamma-responsive genes represent the largest response network in the PS pigs. Using gene expression data and literature evidence, 15 candidate genes representing different aspects of the IFN-gamma-regulated response were selected. qPCR analysis confirmed in vivo patterns of the 15 genes. Further investigations verified that differential patterns observed in vivo in PS and LS pigs could be recapitulated in vitro. Whole blood from 3 healthy pigs was stimulated in vitro with 1, 10 or 100 ng/ml IFN-gamma with or without S. Typhimurium endotoxin (STE). The qPCR analysis revealed that the in vitro response to IFN-gamma alone for five genes (CXCL10, IL10, MMP8, PSMB9, TMEM176) was dose-dependent. Simultaneous stimulation of whole blood with STE and IFN-gamma induced IFN-gamma dose-dependent expression for these genes plus CASP4, CYBA, IRF1, JAK2, NCF1, SOD2, and TAP1. Cluster analysis revealed that in vivo gene expression patterns across all genes in PS swine clustered most closely with patterns in whole blood stimulated with the two highest IFN-gamma levels plus STE. This research indicates that quantitative differences in IFN-gamma levels explains the expression of most tested genes, and that the IFN-gamma regulon is a source of genes whose expression levels two days post-infection can predict shedding outcomes. Such genes can now be further evaluated as candidates for development of predictive assays for shedding outcome in swine. (Abstract shortened by UMI.)
机译：本论文的研究目的是通过研究与猪对沙门氏菌反应有关的遗传变异，为了解猪的抗病性奠定基础。该研究的主要目的是：1）使用整合的全球基因表达和生物信息学分析方法来发现具有单核苷酸多态性（SNP）的候选基因，这些候选基因可能与沙门氏菌相关的表型有关，并可能影响沙门氏菌的脱落和/或携带者的状况猪2）分析由IFN-γ调节的一组基因的表达，IFN-γ是一种细胞因子，其血清水平与猪沙门氏菌脱落有关，以确定潜在的生物标志物以评估猪免疫反应的质量，可用于预测猪的脱落状态为了鉴定功能候选基因中的SNP以测试与猪对沙门氏菌反应的相关性，采用了综合基因选择方法。猪对沙门氏菌反应的最新全球基因表达分析，结合重要候选基因的文献信息，以及基于序列比对的SNP预测，成功鉴定了功能候选基因中的SNP。通过Sequenom技术使用总共750头猪的四个猪群对选定的SNP进行基因分型。基因分型和统计关联分析导致在与沙门氏菌相关表型相关的12个功能候选基因中发现了几个新的SNP，例如组织定植和/或粪便沙门氏菌脱落。在诸如TAP1，GNG3，CCR1，CD163，AMT，ACP2，VCL，CCT7，HP，EMP1，NCF2和PGD的基因中鉴定了相关的SNP。因此，SNPs分析研究表明需要进一步评估几个潜在的候选基因，作为选择具有改善的免疫功能和减少沙门氏菌脱落的猪的标记。此外，新的遗传学关联为猪业提供了有关选择具有更好抗病性的猪的策略的有用信息。；主要的免疫反应介质之一，IFN-γ，已被证明在通过转录组和转录组对沙门氏菌的猪反应中很重要。途径分析。因此，这项研究集中于IFN-γ调节基因在猪对沙门氏菌的差异反应中的假定作用。全血转录组分析显示，猪之间存在许多差异表达的基因，这些基因被分类为持久性（PS）或低（LS）沙门氏菌脱落菌。差异表达基因的途径分析表明，IFN-γ反应基因代表PS猪中最大的反应网络。利用基因表达数据和文献证据，选择了15个代表IFN-γ调节反应不同方面的候选基因。 qPCR分析证实了15个基因的体内模式。进一步的研究证实，在PS和LS猪体内观察到的差异模式可以在体外概括。用1、10或100 ng / mlIFN​​-γ在有或没有鼠伤寒沙门氏菌内毒素（STE）的情况下体外刺激3只健康猪的全血。 qPCR分析显示五个基因（CXCL10，IL10，MMP8，PSMB9，TMEM176）对单独的IFN-γ的体外反应是剂量依赖性的。这些基因加上CASP4，CYBA，IRF1，JAK2，NCF1，SOD2和TAP1的STE和IFN-γ同时刺激全血诱导了IFN-γ剂量依赖性表达。聚类分析表明，PS猪中所有基因的体内基因表达模式与以两种最高的IFN-γ水平加STE刺激的全血中的模式最接近。这项研究表明，IFN-γ水平的定量差异可以解释大多数测试基因的表达，并且IFN-γregulon是基因的来源，其感染后两天的表达水平可以预测脱落的结果。现在可以进一步评估此类基因，作为开发用于预测猪排泄结果的预测分析的候选基因。 （摘要由UMI缩短。）

著录项

  • 作者

    Uthe, Jolita Janutenaite.;

  • 作者单位

    Iowa State University.;

  • 授予单位 Iowa State University.;
  • 学科 Biology Genetics.;Health Sciences Immunology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 179 p.
  • 总页数 179
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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