鸭肠炎病毒与A型产气荚膜梭菌共感染鸭回肠代谢组学分析

Metabolomic Analysis of Ileum Co-infected with Duck Enteritis Virus and Clostridium perfringens Type A in Ducks

【目的】探究鸭肠炎病毒(DEV)和A型产气荚膜梭菌(CpA)共感染的致病机制。【方法】研究选取37日龄健康非免疫雏鸭,随机分为对照组与共感染组。共感染组鸭于每天早晚各进行1次灌胃,每次灌CpA菌液8 mL(1×108 CFU/mL),连续5 d,首次灌胃72 h后于腿部肌肉接种DEV-GZ株病毒液(0.2 mL/只,TCID 50=3.16×10-9 TCID 50/0.1 mL),对照组接种灭菌生理盐水0.2 mL,观察鸭临床病变及回肠剖检病变,采用细菌分离鉴定和PCR等方法检测DEV和CpA共感染模型是否成功建立。采集感染90 h对照组与共感染组回肠,采用液相色谱-质谱联用(LC-MS)技术对对照组及共感染组感染90 h鸭回肠进行代谢组学检测,筛选并分析潜在的差异代谢物及相关信号通路。【结果】经检测确定成功构建了鸭DEV和CpA共感染模型。对照组与共感染组鸭回肠差异代谢物质共有36种,在正离子模式下共有16种差异代谢物质,主要为2′-脱氧肌苷、腐胺、8(z),11(z),14(z)-二十碳三烯酸、胞苷和1-油酰基外消旋甘油,在负离子模式下共有20种差异代谢物质,主要为油酸、花生四烯酸、11(z),14(z)-二十二碳二烯酸、二十二碳三烯酸和亚油酸;差异代谢物主要富集的代谢通路为10条,主要为色氨酸代谢、嘧啶代谢、嘌呤代谢等。【结论】11(z),14(z)-二十二碳二烯酸、二十二碳三烯酸和花生四烯酸可成为DEV和CpA共感染鸭回肠的敏感生物标记物,提示DEV与CpA共感染加剧雏鸭回肠炎症,色氨酸代谢通路提示DEV与CpA共感染鸭免疫力变化存在联系。本研究为阐明DEV与CpA感染的致病机制奠定基础。

【Objective】The purpose of this study was to investigate the pathogenic mechanism of Duck enteritis virus(DEV)and Clostridium perfringens type A(CpA)co-infection.【Method】Healthy non-immune ducklings at 37 days of age were randomly divided into control and co-infected groups.The co-infected group were gavaged once a day in the morning and evening,with 8 mL of CpA bacterial solution(1×108 CFU/mL)for 5 consecutive days,and after 72 h of initial gavage,the DEV-GZ strain virus solution(0.2 mL,TCID 50=3.16×10-9 TCID 50/0.1 mL)was inoculated into the leg muscles,and control group was inoculated with sterilized saline 0.2 mL.The clinical lesions and ileal dissection lesions of the ducks were observed.Bacterial isolation and identification and PCR were used to detect whether the co-infection model was successfully established by DEV and CpA.The ileum of control and co-infected ducks was collected at 90 h of infection,and liquid chromatography-mass spectrometry(LC-MS)was used for metabolomic analysis of the ileum of control and co-infected ducks at 90 h of infection to screen and analyze potential differential metabolites and related signaling pathways.【Result】A duck DEV and CpA co-infection model was successfully constructed after testing.A total of 36 different metabolites were found in the ileum of ducks in control and co-infected groups.16 different metabolites were found in the positive ionization mode,mainly including 2′-deoxyinosine,putrescine,8(z),11(z),14(z)-eicosatrienoic acid,cytidine,and 1-oleoyl racemic glycerol,and 20 different metabolites were found in the negative ionization mode,mainly including oleic acid,arachidonic acid,11(z),14(z)-docosa-2-dienoic acid,docosa-2-trienoic acid and linoleic acid.The main metabolic pathways enriched in differential metabolites were 10,mainly tryptophan metabolism,pyrimidine metabolism,purine metabolism and so on.【Conclusion】11(z),14(z)-docosadienoic acid,docosatrienoic acid,and arachidonic acid could be sensitive biomarkers in the ileum of ducks co-infected with DEV and CpA,suggesting that the co-infection of DEV and CpA exacerbated the inflammation of the ileum in ducklings,and that tryptophan metabolism pathway suggested a link between the changes in the immunity in ducks co-infected with DEV and CpA.This study laid a foundation for elucidating the pathogenic mechanism of DEV and CpA infection.