鸭肠炎病毒感染鸭脾脏的代谢组学分析

Metabolomic analysis of the spleen of ducks infected with duck enteritis virus

为探究鸭肠炎病毒(DEV)感染后雏鸭脾脏差异代谢物的变化,本研究选取41日龄健康绿壳蛋鸭,随机分为实验组(TD)和对照组(CK),实验组鸭经腿部肌肉接种DEV病毒液(0.2 m L/只,3.16×10^(9)TCID_(50)/0.1 m L),对照组注射等体积灭菌生理盐水。于接种后66 h、90 h和114 h时剖杀各组鸭,无菌采集各组鸭脾脏组织样品采用PCR检测病原核酸,观察各组鸭各剖检病变,制备各组鸭脾脏组织切片观察其病理学变化。结果显示,TD组鸭脾脏样品经PCR扩增出DEV特异性DNA片段,而CK组未扩增到该条带;剖检病变观察可见TD组鸭脾脏肿大,呈斑驳状,被膜下有出血点;脾脏组织病变观察可见脾窦内充血,淋巴细胞数量减少,而CK组鸭脾脏无明显病变,确定DEV感染鸭模型建立。采用液相色谱-质谱联用(LC-MS)技术检测实验组和对照组鸭脾脏的代谢组学,筛选并分析各组鸭潜在的差异代谢物及相关信号通路。结果显示,与CK组相比,TD组鸭脾脏在感染66 h、90 h和114 h时的差异代谢物质分别有38、64、71种,主要为花生四烯酸乙醇胺、γ-亚麻酸、吲哚-3-乙酸、二十二碳六烯酸、赖氨酸、前列腺素e3、马L-色氨酸、L-苯丙氨酸等;参与的代谢通路主要是色氨酸代谢、苯丙氨酸代谢、组氨酸代谢、酪氨酸代谢、氨基酸的生物合成、细胞色素P450对外源物质的代谢、氨酰t RNA的生物合成等,其中以花生四烯酸乙醇胺和色氨酸代谢为主的差异代谢物和代谢通路与鸭的免疫应答和炎症反应有关。上述结果表明,花生四烯酸乙醇胺和色氨酸可作为DEV感染鸭的生物标志物,DEV感染可能是通过色氨酸代谢通路引起鸭的炎症反应和免疫应答。本实验为进一步研究DEV的致病机制提供参考。

In order to explore the metabolic alterations in spleen of ducks infected with duck enteritis virus(DEV),41-day-old healthy green shell ducks were selected and randomly divided into test(TD)group and control(CK)group.The experimental group was inoculated with DEV venom(0.2mL/duck,3.16×10^(9)TCID_(50)/0.1mL)via leg muscle.The CK group was inoculated with the same amount of sterile saline.The ducks were euthanized at 66 hours,90 hours and 114 hours after inoculation.The spleen samples from each group were aseptically collected for pathogen nucleic acid detection and tissue lesion observation.After determining the successful infection of DEV,liquid chromatography-mass spectrometry(LC-MS)was employed to analyze the metabolomic profiles of the spleens from both the TD and CK groups.Potential differential metabolites and related signaling pathways were screened and analyzed.The results demonstrated successful amplification of DEV-specific DNA fragments in the spleens of the TD group,while no such DNA fragments were detected in the CK group.The spleens from the TD group exhibited enlargement with a mottled appearance and subcapsular hemorrhagic spots.Histological analysis revealed hyperemia in the splenic sinus and reduced lymphocyte count.In contrast,the spleens of the CK group showed no significant pathological changes.Comparative metabolomic analysis revealed 38,64,and 71 distinct metabolic substances at 66 hours,90 hours,and 114 hours post-inoculation,respectively,in the spleens of ducks from the TD group.These metabolites primarily included arachidonic acid ethanolamine,gamma-linolenic acid,indole-3-acetic acid,docosahexaenoic acid,lysine,prostaglandin e3,L-tryptophan,L-phenylalanine,etc.The main metabolic pathways involved were tryptophan metabolism,phenylalanine metabolism,histidine metabolism,tyrosine metabolism,amino acid biosynthesis,cytochrome P450 metabolism of foreign substances,aminoacyl tRNA biosynthesis.Notably among them are differential metabolites such as arachidonic acid ethanolamine and tryptophan metabolism which are closely associated with immune response and inflammation.The findings suggest that ethanolamine arachidonic acid and tryptophan can serve as sensitive biomarkers for ducks infected with DEV,indicating that DEV infection may induce inflammation and immune response in ducks through the tryptophan metabolic pathway.These results provide a valuable reference for further investigation into the pathogenesis of DEV.