Distinct Peripheral Blood RNA Responses to Salmonella in Pigs Differing in Salmonella Shedding Levels: Intersection of IFNG, TLR and miRNA Pathways

Transcriptomic analysis of the response to bacterial pathogens has been reported for several species, yet few studies have investigated the transcriptional differences in whole blood in subjects that differ in their disease response phenotypes. Salmonella species infect many vertebrate species, and pigs colonized with Salmonella enterica serovar Typhimurium (ST) are usually asymptomatic, making detection of these Salmonella-carrier pigs difficult. The variable fecal shedding of Salmonella is an important cause of foodborne illness and zoonotic disease. To investigate gene pathways and biomarkers associated with the variance in Salmonella shedding following experimental inoculation, we initiated the first analysis of the whole blood transcriptional response induced by Salmonella. A population of pigs (n = 40) was inoculated with ST and peripheral blood and fecal Salmonella counts were collected between 2 and 20 days post-inoculation (dpi). Two groups of pigs with either low shedding (LS) or persistent shedding (PS) phenotypes were identified. Global transcriptional changes in response to ST inoculation were identified by Affymetrix Genechip® analysis of peripheral blood RNA at day 0 and 2 dpi. ST inoculation triggered substantial gene expression changes in the pigs and there was differential expression of many genes between LS and PS pigs. Analysis of the differential profiles of gene expression within and between PS and LS phenotypic classes identified distinct regulatory pathways mediated by IFN-γ, TNF, NF-κB, or one of several miRNAs. We confirmed the activation of two regulatory factors, SPI1 and CEBPB, and demonstrated that expression of miR-155 was decreased specifically in the PS animals. These data provide insight into specific pathways associated with extremes in Salmonella fecal shedding that can be targeted for further exploration on why some animals develop a carrier state. This knowledge can also be used to develop rational manipulations of genetics, pharmaceuticals, nutrition or husbandry methods to decrease Salmonella colonization, shedding and spread.

目前已有多项针对不同物种对细菌病原体响应的转录组学分析（Transcriptomic analysis）研究被报道，但针对疾病响应表型存在差异的试验猪，其全血转录组差异的相关研究仍较为匮乏。沙门氏菌属（Salmonella）可感染多种脊椎动物，而感染鼠伤寒沙门氏菌（Salmonella enterica serovar Typhimurium, ST）的猪通常无明显临床症状，使得这类沙门氏菌带菌猪的检测难度较大。沙门氏菌不定量的粪便排毒是引发食源性疾病（foodborne illness）与人畜共患病（zoonotic disease）的重要诱因。为探究实验接种沙门氏菌后，与排毒量差异相关的基因通路与生物标志物（biomarker），本研究首次开展了沙门氏菌诱导的猪全血转录响应分析。研究共纳入40头试验猪，经ST接种后，于接种后2至20天（days post-inoculation, dpi）采集外周血样本并进行粪便沙门氏菌计数。根据检测结果，将试验猪分为低排毒（LS）与持续排毒（PS）两个表型组。通过Affymetrix Genechip® 芯片分析接种第0天与第2天的外周血RNA，鉴定ST接种诱导的全转录组表达变化。结果显示，ST接种可引发猪体内显著的基因表达改变，且低排毒与持续排毒组猪之间存在大量差异表达基因（differentially expressed genes）。对两个表型组内及组间的基因表达差异谱进行分析，鉴定出由干扰素-γ（IFN-γ）、肿瘤坏死因子（TNF）、核因子κB（NF-κB）或多种微小RNA（miRNA）介导的特异性调控通路。本研究验证了SPI1与CEBPB两个调控因子（regulatory factors）的激活状态，并证实miR-155的表达在持续排毒组动物中特异性下调。本研究数据揭示了与沙门氏菌粪便排毒极端表型相关的特定通路，可为进一步解析部分动物发展为带菌状态的机制提供重要线索；同时，该研究结果也可用于指导通过遗传学手段、药物干预、营养调控或饲养管理方法的合理优化，以减少沙门氏菌在猪群中的定植、排毒与传播。