# Diversity of Escherichia coli from Faecal Samples of Danish Calves with Diarrhoea

## TL;DR

This study shows that Danish calves with diarrhoea are infected by a wide variety of E. coli strains, not just the traditional F5 type, which changes how we diagnose and treat the disease.

## Contribution

The study reveals a shift in the dominant E. coli pathotype causing calf diarrhoea in Denmark and highlights the need for updated diagnostic and vaccination strategies.

## Key findings

- Most faecal samples from calves with diarrhoea contained multiple E. coli types, showing high diversity.
- Only 4% of samples had the classic F5 fimbriae, while many carried genes from other pathotypes like DAEC and ExPEC.
- High genomic diversity within samples suggests that selecting a single isolate may miss important pathogenic variants.

## Abstract

Calf diarrhoea is a major health and welfare problem in dairy farming, often linked to infections with Escherichia coli (E. coli). Traditionally, strains carrying the F5 (K99) fimbriae have been considered the main cause of E. coli-associated diarrhoea. However, recent studies, including ours, show that this is no longer the case. In this study, we examined 391 faecal samples from Danish calves with diarrhoea and found that E. coli was present in most cases, but with a remarkable diversity of types both within individual calves and between calves. More than two-thirds of the samples contained multiple E. coli types. Surprisingly, only 4% of samples contained the classic F5 fimbriae, while a large proportion carried genes associated with other pathotypes, such as diffusely adherent E. coli (DAEC) and extraintestinal pathogenic E. coli (ExPEC). These findings suggest that calf diarrhoea in Denmark is now linked to a wide range of E. coli types rather than one dominant pathotype. This shift has important implications for diagnosis, treatment, and prevention, as selecting only one isolate from a sample may overlook relevant diversity, and vaccines targeting only F5-positive strains may no longer be effective.

Several different pathogens, including Escherichia coli, are strongly associated with calf diarrhoea. The population diversity of intestinal E. coli 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 E. coli after culturing did not reflect the diarrhetic grade nor whether E. coli 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 E. coli. Genotyping, sequence typing and in silico serotyping showed a large diversity of E. coli between faecal samples. Surprisingly, isolates with a genotype representing mixed features of Diffusely adhering E. coli/Extraintestinal pathogenic E. coli were found in 25% of the isolates, while the classic Enterotoxigenic E. coli genotype was only observed in 5% of the isolates, and only 4% of the faecal samples were positive for E. coli F5 (K99) fimbriae, as determined by PCR. In conclusion, a diverse population of (non-F5) E. coli is associated with diarrhoea in calves. High genomic diversity of E. coli within samples needs to be considered when selecting only one isolate for antimicrobial resistance profiling and vaccination measurements.