Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance

Background: Gastrointestinal nematodes are a serious cause of morbidity and mortality in grazing ruminants. The major ovine defence mechanism is acquired immunity, which develops over time in response to infection. Nematode resistance varies both within and between breeds and is moderately heritable (h ~ 0.3). A detailed understanding of the genes and mechanisms involved in protective immunity, and the factors that regulate this response, is required to aid future breeding strategies as well as the development of effective and sustainable nematode control methods. The aim of this study was to compare the abomasal lymph node transcriptome of resistant and susceptible lambs in order to determine biological processes differentially expressed between resistant and susceptible lambs. Results: Scottish Blackface lambs, with divergent phenotypes for resistance, were challenged with 30,000 Teladorsagia circumcincta larvae (L3), and abomasal lymph node recovered at 7 and 14 days post-infection (dpi). High-throughput sequencing of abomasal lymph node cDNA was used to quantitatively sample the transcriptome with an average of 32 million reads per sample. A total of 194 and 144 genes were differentially expressed between resistant and susceptible lambs at 7 and 14 dpi respectively. Differentially expressed networks and biological processes were identified using Ingenuity Pathway Analysis. Resistant animals appear to generate a more rapid immune response as at 7 dpi processes relating to homing of lymphocytes, leukocyte migration and migration of antigen presenting cells were up-regulated. In susceptible animals this response appears to be delayed until approximately 14 dpi. Twenty-four Single Nucleotide Polymorphims (SNP), within 11 differentially expressed genes were tested for association with gastrointestinal nematode resistance in the Scottish Blackface lambs. Four SNPs in two genes (SLC30A2, and ALB) were suggestively associated with faecal egg count. Conclusions: A large number of genes were differentially expressed in the abomasal lymph node of resistant and susceptible lambs responding to gastrointestinal nematode challenge. Resistant Scottish Blackface lambs appear to generate a more rapid immune response to T. circumcincta. In susceptible lambs this response appears to be delayed until approximately 14 days post infection. SNP in two differentially expressed genes were suggestively associated with faecal egg count indicating that differentially expressed genes can be considered as candidate loci for mediating nematode resistance. Overall design: Scottish Blackface lambs, selected for divergent phenotypes for nematode resistance, were challenged with 30,000 Teladorsagia circumcincta larvae (L3) and abomasal lymph node recovered at 7 and 14 days post-infection for transcriptome sequencing.

背景：胃肠道线虫（gastrointestinal nematodes）是放牧反刍动物发病与死亡的重要诱因。绵羊的核心防御机制为获得性免疫（acquired immunity），该免疫会随感染进程逐步建立。线虫抗性在品种内部与品种之间均存在差异，且具有中等遗传力（h~0.3）。解析保护性免疫相关的基因、调控机制及应答调控因素，可为后续育种策略制定与高效可持续的线虫防控手段开发提供理论支撑。本研究旨在对比抗性与易感羔羊的皱胃淋巴结（abomasal lymph node）转录组（transcriptome），以明确抗性与易感羔羊间存在差异表达的生物学过程。
结果：选取具有极端线虫抗性表型的苏格兰黑面羔羊，以30000枚环状奥斯特线虫（Teladorsagia circumcincta）L3幼虫进行攻毒感染，分别于感染后7天和14天（dpi，days post-infection）采集皱胃淋巴结样本。通过高通量测序（high-throughput sequencing）对皱胃淋巴结cDNA进行转录组定量分析，每个样本平均获得3200万条reads。在感染后7天和14天时，抗性与易感羔羊间分别有194个和144个基因存在差异表达。利用Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）对差异表达的调控网络及生物学过程进行注释。结果显示，抗性个体可产生更为快速的免疫应答：感染后7天时，淋巴细胞归巢、白细胞迁移及抗原呈递细胞迁移相关通路均被上调；而易感个体的免疫应答则延迟至感染后14天左右。对11个差异表达基因内的24个单核苷酸多态性（Single Nucleotide Polymorphisms, SNP，原文存在拼写笔误，应为Polymorphisms）位点进行关联分析，以探究其与苏格兰黑面羔羊胃肠道线虫抗性的相关性。结果发现，2个基因（SLC30A2与ALB）内的4个SNP位点与粪便虫卵计数（faecal egg count）呈提示性关联。
结论：在响应胃肠道线虫攻毒的抗性与易感羔羊的皱胃淋巴结中，存在大量差异表达基因。抗性苏格兰黑面羔羊可对环状奥斯特线虫感染产生更为快速的免疫应答，而易感羔羊的免疫应答则延迟至感染后14天左右。2个差异表达基因内的SNP位点与粪便虫卵计数呈提示性关联，表明差异表达基因可作为介导线虫抗性的候选基因位点。
整体实验设计：选取具有极端线虫抗性表型的苏格兰黑面羔羊，以30000枚环状奥斯特线虫L3幼虫进行攻毒感染，分别于感染后7天和14天采集皱胃淋巴结样本，用于转录组测序。