Effect of Early Pathogenic Escherichia coli Infection on the Intestinal Barrier and Immune Function in Newborn Calves

Abstract

We studied the effect of early pathogenic Escherichia coli infection on newborn calves' intestinal barrier and immune function. A total of 64 newborn Holstein male calves (40-43 kg) were divided into two groups: normal (NG) and test (TG), each with 32 heads. At the beginning of the experiment, the TG calves were orally administered pathogenic E. coli O1 (2.5 × 10¹¹ CFU/mL, 100 mL) to establish a calf diarrhea model. In contrast, the NG calves were given the same amount of normal saline. During the 30 d trial period, the feeding and management of the two groups remained constant. Enzyme-linked immunosorbent assay, quantification PCR, and high-throughput 16S rRNA sequencing technology were used to detect indicators related to the intestinal barrier and immune function in the calf serum and tissues. Pathogenic E. coli O1 had a significant effect on calf diarrhea in the TG; it increased the bovine diamine oxidase (P < 0.05) and endotoxin levels in the serum and decreased (P < 0.05) the intestinal trefoil factor (P < 0.05), Occludin, Claudin-1, and Zonula Occludens 1 (ZO-1) levels in the colon tissue, as well as downregulated the mRNA expression of Occludin, Claudin-1,and ZO-1 in the colon mucosa, leading to increased intestinal permeability and impaired intestinal barrier function. Additionally, pathogenic E. coli had a significant impact on the diversity of colonic microbial flora, increasing the relative abundance of Proteobacteria at the phylum level and decreasing the levels of Firmicutes and Bacteroides. At the genus level, the relative abundance of Escherichia and Shigella in the TG increased significantly (P < 0.05), whereas that of Bacteroides, Butyricicoccus, Rikenellaceae_RC9_gut_group, Blautia, and Lactobacillus was significantly decreased (P < 0.05). In addition, the level of IL-6 in the serum of the TG calves was significantly increased (P < 0.05), whereas the IL-4 and IL-10 levels were significantly decreased (P < 0.05), compared to those in the NG calves. Thus, pathogenic E. coli induced diarrhea early in life disrupts intestinal barrier and impairs immune function in calves.

Keywords: calf; colonic microflora; immune function; intestinal barrier; pathogenic Escherichia coli O1.

Figure 1

Representative H&E staining microscopic images of colon tissues in different groups. (A) NG, normal group, (B) TG, test group.

Figure 2

Effect of pathogenic Escherichia coli O1 on tight junction protein mRNA expression in the colon of newborn calves. (A–C) show the expression levels of Claudin-1, Occludin, and ZO-1, respectively. Compared with the normal group (NG), different letters show significant differences (P < 0.05). The same letter indicates no significant difference (P > 0.05).

Figure 3

Abundance of colonic mucosal flora based on OTU level. (A) Rarefaction curve, (B) rank-abundance curves, and (C) Venn diagram. 1 = 12 h; 2 = 24 h; 3 = 36 h; 4 = 48 h; 5 = 72 h; 6 = 5 d; 7 = 10 d; 8 = 30 d; N= normal group (NG); T= test group (TG).

Figure 4

Analysis of alpha-diversity index of colon contents in calves. (A) Sobs index, (B) Ace index, (C) Chao1 index, (D) Shannon index, (E) Simpson index, and (F) Coverage index. *0.01<P<=0.05, **0.001<P<=0.01, ***P<=0.001.

Figure 5

Abundance of colonic mucosa flora at phylum level (%). 1 = 12 h; 2 = 24 h; 3 = 36 h; 4 = 48 h; 5 = 72 h; 6 = 5 d; 7 = 10 d; 8 = 30 d; N = normal group (NG); T = test group (TG).

Figure 6

Abundance significance of dominant flora in supraportal colonic mucosa at the phylum level (%). (A) Proteobacteria, (B) Firmicutes, and (C) Bacteroidetes abundance. *0.01< P <=0.05, **0.001 < P <=0.01, ***P <=0.001.

Figure 7

Abundance significance of dominant flora in supraportal colonic mucosa at the genus level(%). (A) Escherichia-Shigella, (B) Bacteroides, (C) Butyricicoccus, (D) Rikenellaceae_RC9_gut_group, (E) Blautia, and (F) Lactobacillus.*0.01< P <=0.05, **0.001< P <=0.01, ***P <=0.001.

Figure 8

LDA discrimination in calf colon. (A) Length of the histogram represents the influence of significantly different species, and different colors represent different samples, (B) Circle from inside to outside indicates the level of phylogeny from genus to phylum.