| Summary: | Several different pathogens, including <i>Escherichia coli</i>, are strongly associated with calf diarrhoea. The population diversity of intestinal <i>E. coli</i> within each diarrhetic calf and between diarrhetic calves is not well understood. In the present study, 391 faecal samples were obtained during 2023–2024 from Danish dairy calves with diarrhoea. Semi-quantified growth estimates of <i>E. coli</i> after culturing did not reflect the diarrhetic grade nor whether <i>E. coli</i> was the only pathogen observed in the sample. From each sample, five isolates were subjected to multiple-locus variable-number tandem repeat analysis (MLVA) and revealed that 70% of faecal samples contained more than one type of <i>E. coli.</i> Genotyping, sequence typing and in silico serotyping showed a large diversity of <i>E. coli</i> between faecal samples. Surprisingly, isolates with a genotype representing mixed features of Diffusely adhering <i>E. coli</i>/Extraintestinal pathogenic <i>E. coli</i> were found in 25% of the isolates, while the classic Enterotoxigenic <i>E. coli</i> genotype was only observed in 5% of the isolates, and only 4% of the faecal samples were positive for <i>E. coli</i> F5 (K99) fimbriae, as determined by PCR. In conclusion, a diverse population of (non-F5) <i>E. coli</i> is associated with diarrhoea in calves. High genomic diversity of <i>E. coli</i> within samples needs to be considered when selecting only one isolate for antimicrobial resistance profiling and vaccination measurements.
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