| Summary: | Calf diarrhea caused by pathogenic <i>Escherichia coli</i> is a major cause of death in calves, with a mortality rate of over 50%. It is crucial to understand the pathogenesis and development of calf diarrhea for its prevention and treatment. We aimed to study the effect of pathogenic <i>E. coli</i> on the flora composition, function, and short-chain fatty acid (SCFA) content of calf feces using a calf diarrhea model. Sixty-four newborn Holstein calves (40–43 kg) were divided into a normal group (NG; <i>n</i> = 32) and a test group (TG; <i>n</i> = 32). At the beginning of the experiment, the TG were orally administered pathogenic <i>E. coli</i> O1 (2.5 × 10<sup>11</sup> CFU/mL, 100 mL) to establish a calf diarrhea model, and the NG were orally administered the same amount of physiological saline solution. The calves of the two groups were subjected to the same feeding and management. Fresh feces samples were collected at different time points and subjected to 16S rRNA high-throughput sequencing and gas chromatography–mass spectrometry to determine the fecal microbial composition and SCFA content. Pathogenic <i>E. coli</i> O1 significantly altered microbiotas composition in the feces of calves, increasing the relative abundance of Proteobacteria and decreasing that of Firmicutes. It also led to a significant increase in the relative abundance of <i>Escherichia-Shigella</i> and a decrease in <i>Lactobacillus</i>, as well as significantly decreased SCFA content. Therefore, we postulate that pathogenic <i>E. coli</i> induces calf diarrhea by causing intestinal florae imbalance and reducing the content of SCFA.
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