腹泻犊牛粪便菌群及粪便代谢物的变化特征分析

Characteristics of Microbiota and Metabolites in Feces of Calves with Diarrhea

【目的】揭示腹泻犊牛粪便菌群和粪便代谢物的变化特征。【方法】根据粪便评分和临床症状,本研究将30日龄内哺乳犊牛分为健康组(CK)和腹泻组(D),应用16S rRNA扩增子测序技术对CK和D组犊牛粪便菌群进行比较分析。应用液相色谱-质谱联用法,对犊牛粪便进行非靶向代谢组分析,筛选差异代谢物并对其进行KEGG通路富集分析,对粪便菌群和粪便差异代谢物进行Spearman相关性分析。【结果】与CK组相比,D组犊牛粪便菌群OTU数量和Chao1指数极显著升高(P<0.01),Shannon和Simpson指数极显著降低(P<0.01)。D组粪便中梭杆菌门、变形菌门、酸杆菌门和绿弯菌门,以及埃希-志贺属、梭杆菌属、梭菌属等9个菌属相对丰度极显著或显著升高(P<0.01或P<0.05),而放线菌门、厚壁菌门和广古菌门,以及粪杆菌属、罕见小球菌属、厄氏菌属、双歧杆菌属等11个菌属极显著或显著降低(P<0.01;P<0.05)。梭杆菌门、变形菌门、梭杆菌属、埃希-志贺菌属、死亡梭杆菌、大肠埃希菌是腹泻犊牛粪便生物标志物。从CK和D组犊牛粪便中共筛选到54个差异代谢物,其中PC(16∶1(9Z)/16∶1(9Z))富集到甘油磷脂代谢途径,与梭杆菌属极显著正相关(P<0.01),与柯林斯菌属、巨型球菌属、厄氏菌属显著或极显著负相关(P<0.05;P<0.01)。脱硫生物素富集到生物素代谢途径,与梭杆菌属显著负相关(P<0.05),与罕见小球菌属、巨型球菌属、罗尔斯通菌属显著正相关(P<0.05)。二氢生物蝶呤和二氢新蝶呤三磷酸富集到叶酸生物合成途径,与梭杆菌属、消化链球菌属极显著或显著负相关(P<0.01;P<0.05),与罕见小球菌属、巨型球菌属、厄氏菌属、双歧杆菌属、普雷沃菌属、柯林斯菌属、粪杆菌属、副拟杆菌属极显著或显著正相关(P<0.01;P<0.05)。【结论】腹泻犊牛粪便菌群丰富度、多样性、物种组成及粪便代谢物组成均发生了显著变化,犊牛肠道甘油磷脂代谢、生物素和叶酸生物合成受到影响,肠道菌群的紊乱和粪便代谢物的显著变化紧密相关。

【Objective】The objective of this study was to reveal the characteristics of fecal microbiota and metabolites of calves with diarrhea.【Method】According to the fecal score and clinical symptoms,the suckling calves within 30 days of age were divided into healthy group(CK)and diarrhea group(D).The fecal microbiota of calves in CK and D groups were analyzed using 16S rRNA amplification sequencing technology.The non-targeted metabolome analysis was performed to screen differential metabolites in the feces of CK and D groups through LC-MS,and the differential metabolites were conducted for KEGG pathway enrichment analysis.The Spearman correlation analysis was performed on fecal microbiota and differential metabolites in feces.【Result】Compared with CK group,the OTUs and Chao1 indexes of fecal microbiota in D group were extremely significantly increased(P<0.01),and the Shannon and Simpson indexes were extremely significantly decreased(P<0.01).The relative abundances of Fusobacteriota,Proteobacteria,Acidobacteriota,Chloroflexi and 9 bacteria genera that including Escherichia-Shigella,Fusobacterium and Clostridium_sensu_stricto_1,etc.were extremely significantly or significantly increased in D group(P<0.01 or P<0.05),while the relative abundances of Actinobacteriota,Firmicutes,Euryarchaeota and 11 bacteria genera that including Faecalibacterium,Subdoligranulum,Olsenella and Bifidobacterium,etc.were extremely significantly or significantly decreased in D group(P<0.01 or P<0.05).Fusobacteriota,Proteobacteria,Fusobacterium,Escherich-Shigella,Fusobacterium mortiferium and Escherichia coli were biomarkers in the feces of calves with diarrhea.A total of 54 different metabolites were screened from the feces of CK and D groups.PC(16∶1(9Z)/16∶1(9Z))was enriched into the glycerophospholipid metabolism pathway,which was significantly positively correlated with Fusobacterium(P<0.01)and significantly or extremely significantly positively correlated with Collinsella,Megasphaera and Olsenella(P<0.05 or P<0.01).Dethiobiotin was enriched into the biotin metabolic pathway.It was significantly negatively correlated with Fusobacterium(P<0.01),while significantly positively correlated with Subdoligranulum,Megasphaera and Rolstonia(P<0.05).Dihydrobiopterin and dihydroneopterin triphosphate enriched into the folate biosynthesis pathway were extremely significantly or significantly negatively correlated with Fusobacterium and Peptestreptococcus(P<0.01 or P<0.05),which extremely significantly or significantly positively correlated with Subdoligranulum,Megasphaera,Olsenella,Bifidobacterium,Prevotella,Collinsella,Faecalibacterium,and Parabacteroides(P<0.01 or P<0.05).【Conclusion】The richness,diversity,species composition of fecal microbiota and the composition of fecal metabolites were significantly changed in calves with diarrhea.Theglycerophospholipid metabolism,biotin metabolism and folate biosynthesis were affected in the gut of calves with diarrhea.The dysbiosis of intestinal microbiota of calves was closely related to the significant changes of fecal metabolites.