Kutyrev Ivan, Cleveland Beth, Leeds Timothy, Wiens Gregory D
National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, USDA, 11861 Leetown Rd, Kearneysville, WV 25430, USA; Institute of General and Experimental Biology, Siberian Branch of Russian Academy of Sciences, Sakhyanovoi St., 6, 670047 Ulan-Ude, Russia.
National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, USDA, 11861 Leetown Rd, Kearneysville, WV 25430, USA.
Fish Shellfish Immunol. 2016 Nov;58:542-553. doi: 10.1016/j.fsi.2016.09.053. Epub 2016 Oct 12.
Flavobacterium psychrophilum (Fp) is the causative agent of bacterial cold water disease (BCWD) which causes appreciable economic losses in rainbow trout aquaculture. We previously reported development of a genetic line, designated ARS-Fp-R that exhibits higher survival relative to a susceptible line, designated ARS-Fp-S, following either laboratory or natural on-farm challenge. The objectives of this study were to determine the temporal kinetics of gene expression between experimentally-challenged ARS-Fp-R and ARS-Fp-S fish and the correlation between gene expression and pathogen load. We developed a GeXP multiplex RT-PCR assay to simultaneously examine expression of immune-relevant genes, concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. Spleen tissue was sampled at 6 h, 24 h, 48 h and 144 h post-challenge and pathogen load quantified by qPCR. Transcript abundance of cytokine genes tnfa1, tnfa2, tnfa3, il1b1, il1b2, il11a; acute phase response genes saa and drtp1; and putative cytokine receptors il1r1-like-b, il1r2, tnfrsf1a, tnfrsf9, tnfrsf1a-like-b increased following challenge while the transcript abundance of il1r-like-1 and tnfrsf1a-like-a decreased compared to PBS-injected line-matched control fish. Principal component analysis identified transcript levels of genes il1r-like-1 and tnfrsf1a-like-a as exhibiting differential expression between genetic lines. In summary, Fp i.p. injection challenge elicited a proinflammatory cytokine gene expression response in the spleen, with ARS-Fp-R line fish exhibiting modestly higher basal expression levels of several putative cytokine receptors. This study furthers the understanding of the immune response following Fp challenge and differences in gene expression associated with selective breeding for disease resistance.
嗜冷黄杆菌（Fp）是细菌性冷水病（BCWD）的病原体，该病在虹鳟鱼养殖中造成了可观的经济损失。我们之前报道了一个名为ARS-Fp-R的遗传品系的培育，在实验室或农场自然感染后，该品系相对于一个名为ARS-Fp-S的易感品系表现出更高的存活率。本研究的目的是确定实验感染后的ARS-Fp-R和ARS-Fp-S鱼之间基因表达随时间的动力学变化以及基因表达与病原体载量之间的相关性。我们开发了一种GeXP多重RT-PCR检测方法，以同时检测免疫相关基因的表达，重点关注肿瘤坏死因子和白细胞介素-1配体/受体系统以及急性期反应基因。在感染后6小时、24小时、48小时和144小时采集脾脏组织，并通过qPCR对病原体载量进行定量。细胞因子基因tnfa1、tnfa2、tnfa3、il1b1、il1b2、il11a；急性期反应基因saa和drtp1；以及假定的细胞因子受体il1r1-like-b、il1r2、tnfrsf1a、tnfrsf9、tnfrsf1a-like-b的转录本丰度在感染后增加，而与注射PBS的品系匹配对照鱼相比，il1r-like-1和tnfrsf1a-like-a的转录本丰度降低。主成分分析确定基因il1r-like-1和tnfrsf1a-like-a的转录水平在不同遗传品系之间表现出差异表达。总之，腹腔注射Fp感染引发了脾脏中促炎细胞因子基因表达反应，ARS-Fp-R品系的鱼表现出几种假定细胞因子受体的基础表达水平略高。本研究进一步加深了对Fp感染后免疫反应以及与抗病性选择性育种相关的基因表达差异的理解。
