Arula-7 powder improves diarrhea and intestinal epithelial tight junction function associated with its regulation of intestinal flora in calves infected with pathogenic Escherichia coli O1

Abstract

Background: The effects of Arula-7 powder (ASP) on diarrhea and intestinal barrier function associated with its regulation of intestinal microflora in calves infected with pathogenic Escherichia coli O₁ (E. coli O₁) were studied.

Method: Twenty Holstein calves were randomly divided into four treatment groups: normal control (NC), model control (MC), 0.5 mg/kg ciprofloxacin (CIP) and 2.50 g/kg ASP groups.

Results: ASP inhibited the relative abundance of Proteobacteria, Selenomonadales, and Enterobacteriales, and increased the relative abundance of Lactobacillus, Faecalibacterium, and Alloprevotella. Moreover, we demonstrated for the first time that the ASP and CIP promoted weight gain, reduced the diarrhea rate (P < 0.05), and enhanced antioxidant capacity (P < 0.05) due to the increase in average daily gain (ADG), total protein (TP), and albumin (ALB). In addition, ASP and CIP increased the expression of Zunola occludens-1 (ZO-1), Occludin, and Claudin-1 in the ileum (P < 0.05), and improved immunity due to increase levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-γ (IFN-γ), immunoglobulin A (IgA), and immunoglobulin G (IgG) in the serum, strengthened CD4⁺T levels in the ileal mucosa and reducing CD8⁺T and CD11c⁺T (P < 0.05).

Conclusion: Hence, The intestinal microbiota environment formed by early intervention of ASP powder has a protective effect on the intestinal mucosal function of calves infected with pathogenic E. coli. Video Abstract.

Keywords: Arula-7 powder; Calf; Diarrhea; Intestinal barrier function; Intestinal flora; Pathogenic Escherichia coli O1.

Fig. 1

Effect of ASP on the composition of cecal and colonic contents microbiota in calves infected with pathogenic E. coli O₁. Graph A ~ C represents the OTUs at different taxonomical levels in the contents of the cecal: phylum, Order, and Genus. Graph D ~ F represents the OTUs at different taxonomical levels in the contents of the colonic: phylum, Order, and Genus. Red Latin indicates a significant increase (P < 0.05), and blue indicates a significant decrease (P < 0.05)

Fig. 2

Effect of Arula-7 powder on the composition of rectal microbiota in calves infected with pathogenic E. coli O₁. A Graph represents the OTUs at different taxonomical levels: phylum (n = 5). B Graph represents the OTUs at different taxonomical levels: order (n = 5). C The change in the relative abundance of phylum Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria. D The ratio of Firmicutes/Bacteroidetes was significantly low in 2.50 g/kg ASP group. E The change in the relative abundance of genus Faecalibacterium, Fusobacterium, Alloprevotella, Megamonas, and Bacteroides. Metastats analysis was applied to identify the significantly differentially abundant phylum and genera among groups. Different letters above the bars denotes significantly differentially abundant genera among groups. (P < 0.05). n = 5

Fig. 3

Effect of ASP on blood biochemical indexes of calves with E. coli diarrhea. A BUN, blood urea nitrogen. B AST, aspartate aminotransferase. C TP, serum total protein. D ALB, albumin. E ALP, alkaline phosphatase. F Glu, glutamic. n = 5. In the same histogram, the values with the same letter superscripts mean no significant difference (P > 0.05), while with different letter superscripts mean significant difference (P < 0.05)

Fig. 4

Effects of ASP on histopathological changes of jejunum and ileum with H&E staining (original magnification of 100 ×). A Stroke-physiological saline solution + NC group, B E. coli O₁ + MC group, C E. coli O₁ + 0.5 mg/kg CIP group, D E. coli O₁ + 2.50 g/kg ASP group

Fig. 5

Intestinal tight junction proteins in pathogenic E. coli O₁ induced diarrhea calf with ASP and CIP for 7 days (400 ×). Immunofluorescence staining of (A) ZO-1, (B) Occludin, and (C) Claudin-1 in the ileum. D, E, F Each field of ZO-1, Occludin, and Claudin-1 from every group was quantified by using ImageJ. Scale bar = 20 μm; red, ZO-1, Occludin, and Claudin-1; blue, 4,6-diamidino-2-phenylindole counterstaining of the nuclei. Control group was denoted as CG; NC, Normal control group; MC, E. coli group; CIP, Ciprofloxacin group; ASP, Arula-7 powder group. ZO-1, Occludin and Claudin-1

Fig. 6

Intestinal lymphocyte surface molecular proteins in pathogenic E. coli O₁ induced diarrhea calf with ASP and CIP for 7 days (400 ×). Immunofluorescence staining of A CD4⁺, B CD8⁺, and C CD11c⁺ in the ileum. D, E, F Each field of CD4⁺, CD8⁺, and CD11c⁺ from every group was quantified by using Image J. G The ratio of CD4⁺/CD8⁺T was significantly high in ASP group. Scale bar = 20 μm; red, CD4⁺, CD8⁺, and CD11c⁺; blue, 4,6-diamidino-2-phenylindole counterstaining of the nuclei. Control group was denoted as CG; NC, Normal control group; MC, E. coli group; CIP, Ciprofloxacin group; ASP, Arula-7 powder group

Fig. 7

Effects of ASP treatment on calf immune markers. The levels of A serum IgA, B serum IgG, C serum SIgA, D serum IL-4, E serum IL-2, F serum IFN-γ, G serum IL-6, H serum TNF-α