Effect Mechanism Of Pathogenic E. Coli O1 On Intestinal Flora,Immunity And Metabolism Of Newborn Calves

Calf diarrhea induces intestinal flora imbalance,barrier dysfunction,immune imbalance and metabolic disorder.The purpose of this study was to explore the effects and mechanisms of pathogenic E.coli O1 on the health of newborn calves from the perspectives of intestinal barrier,flora diversity and composition,immune function,short-chain fatty acids（SCFAs）and plasma metabolism.In this experiment,calf diarrhea model was established by early artificial intervention of intestinal flora colonization,and the effects and mechanisms of diarrhea caused by pathogenic E.coli O1 infection on intestinal barrier,diversity and composition of colon flora,immune function,diversity and composition of rectal flora,SCFAs metabolism of rectal contents and plasma metabolites of newborn calves were studied,and the pathogenic mechanism involved in inflammatory reaction induced by E.coli was explored to provide theoretical reference for the prevention and treatment of diarrhea of E.coli in calves.The following experiments were carried out in this study:Experiment 1:To study the effects of pathogenic E.coli O1 on the intestinal barrier and the diversity and composition of colonic flora in newborn calves.A total of 64newborn calves（40-43 kg）were selected and divided into normal group（NG）and test group（TG）,with 32 calves in each group.Calves in the TG were given the suspension of pathogenic E.coli O1(2.5×10¹¹CFU/m L,100 m L)to establish diarrhea model.The calves in the NG were natural healthy calves,which were given the same dose of normal saline orally.The experimental period was 30 days,and the calves feeding and management of the two groups remained the same.HE staining was used to observe pathological sections of calf colon tissue,ELISA and real-time fluorescence quantification（RT-QCPR）were used to detect intestinal permeability in calf serum and colon tissue,and 16S r RNA high-throughput sequencing technique was used to analyze the diversity and composition of calves colon flora.The results of showed that pathogenic E.coli O1 had a significant effect on the incidence of diarrhea in calves in the TG.The intestinal villi of calves in the TG broke off,indicating that the intestinal villi structure was damaged due to inflammation.The intestinal villi of calves in the NG were arranged neatly and tightly,and the intestinal mucosa was intact.Pathogenic E.coli O1 infection will increase the contents of bovine diamine oxidase（DAO）and bovine endotoxin（ET）in serum,decrease the contents of intestinal trefoil factor（ITF）and tight junction proteins Occludin,Claudin-1and ZO-1 in colon tissue,and down-regulate the m RNA expression levels of Occludin,Claudin-1 and ZO-1 in colon tissue,which will increase the intestinal permeability and impair the intestinal barrier function.In addition,pathogenic E.coli O1 infection has a significant impact on the diversity and composition of colon flora.At the phylum level,the abundance of Proteobacteria increased,while the abundance of Firmicutes and Bacteroides decreased.At the genus level,the abundance of E.coli-Shigella in the TG increased significantly（P<0.05）,while the abundance of Bacteroides,Butyricicoccus,Rikenellaceae-RC9-gut-group,Blautia and Lactobacillus decreased significantly（P<0.05）.Experiment 2:To study the effect mechanism of pathogenic E.coli O1 on immune function of newborn calves.The immune function related indexes in calves serum and tissues were detected by ELISA and RT-q PCR.The results showed that calves infected with pathogenic E.coli O1 increased the concentrations of ACTH,GH and GC in serum,decreased the concentrations of T₃and T₄,and made the endocrine hormone level disorder.Pathogenic E.coli O1 infection increased the levels of pro-inflammatory cytokines IL-6 in calves serum and colon tissue,decreased the levels of anti-inflammatory cytokines IL-10and IL-4,broke the balance between Th1 and Th2 cells,inhibited Th2 cells from secreting anti-inflammatory factors,and reduced the immune function of the body.Pathogenic E.coli O1 infection increased the expression levels of TLR4,My D88 and NF-κB-p65m RNA in the colon tissue of newborn calves,which led to an increase in the release of downstream pro-inflammatory cytokines,and an increase in the expression levels of TNF-α,IL-2 and IL-6 m RNA in the colon tissue of calves,which induced inflammatory reaction and reduced immune function.Experiment 3:To study the effect mechanism of pathogenic E.coli O1 on rectal flora composition and SCFAs metabolism of newborn calves.16S r RNA high-throughput sequencing,gas chromatography-mass spectrometry and RT-q PCR were used to analyze and detect the composition of rectal flora and SCFAs metabolism in calves.The results showed that the infection of pathogenic E.coli O1 in newborn calves significantly changed the diversity and composition of rectal flora.At the phylum level,the abundance of Proteobacteria in the TG increased,while the abundance of Firmicutes decreased.At the genus level,the abundance of E.coli-Shigella in the rectal flora of calves in the TG increased significantly（P<0.05）,while the abundance of Lactobacillus decreased.Compared with the NG,the contents of acetic acid,propionic acid and butyric acid in calves rectal contents decreased significantly（P<0.05）,and the expression levels of SCFAs receptor GPR41 and GPR43 m RNA decreased significantly（P<0.05）.Through the correlation analysis between bacteria with significantly different abundance and SCFAs content,it was found that propionic acid and butyric acid were negatively correlated with E.coli-Shigella.The above results showed that the rectal flora of calves in the TG was unbalanced,the beneficial bacteria decreased,the harmful bacteria increased,the number of bacteria producing SCFAs decreased,and the metabolic pathway of SCFAs was damaged.Experiment 4:To study the influence mechanism of pathogenic E.coli O1 on plasma differential metabolites and metabolic pathways of newborn calves.According to the previous experimental results,24 experimental animals were selected and divided into normal group（NG）and test group（TG）,with 12 calves in each group.The feeding and management of calves in the two groups were consistent.The plasma metabolites of healthy calves in the NG and diarrhea calves in the TG were detected by LC-MS/MS technology,and the differential metabolites and metabolic pathways were screened out.In addition,the biomarkers involved in the metabolic pathway of diarrhea calves and healthy calves were analyzed.The results showed that the infection of pathogenic E.coli O1 in newborn calves had a significant effect on plasma metabolites.A total of 67 different metabolites were screened out,and 34 metabolites were down-regulated in the TG.In addition,the enriched metabolic pathways are glutamine and glutamic acid metabolism,arginine biosynthesis and arginine and proline metabolism respectively.Generally speaking,the altered metabolic pathway mainly involves amino acid biosynthesis and metabolism.To sum up,the disorder of these markers and metabolic pathways is closely related to the occurrence and development of calves diarrhea.Targeted regulation of metabolic pathways involved in these biomarkers is helpful to the diagnosis,treatment and mechanism research of calves diarrhea.