Gut microbiota in diarrheic and healthy calves: comparative analysis of 16S rRNA gene full-length sequences

Neonatal calf diarrhea (NCD) is a leading cause of morbidity and mortality in the dairy industry. Although multiple pathogens have been identified, widespread alterations in gut microbiota composition associated with diarrhea remain poorly understood. This study aimed to characterize the fecal microbiota and potential metabolic pathways in diarrheic and healthy neonatal calves using 16S rRNA gene full-length sequencing. A total of 40 fecal samples (20 normal and 20 diarrheic) were collected, and 16S rRNA gene full-length sequencing was performed. Alpha diversity measures (Chao1, Shannon, and Simpson indices) were significantly reduced in diarrheic calves (p < 0.01), indicating marked dysbiosis. Principal coordinate analyses, a beta diversity method used to assess difference in microbial composition between groups, revealed distinct clustering of diarrheic versus healthy samples based on both Bray–Curtis and Jensen–Shannon divergences, underscoring a major compositional shift. Taxonomic profiling showed elevated levels of opportunistic/pathogenic bacteria (e.g., Escherichia coli, Salmonella spp., Klebsiella spp.) in diarrheic calves, while beneficial taxa (e.g., Faecalibacterium) were depleted. Functional predictions indicated increased carbohydrate metabolism, notably the N-acetylneuraminate degradation, Entner–Doudoroff, and hexitol fermentation pathways, in diarrheic calves (all adjusted p < 0.05). These findings highlight multifaceted dysbiosis in NCD, including both taxonomic imbalances and heightened carbohydrate metabolic pathways. Identifying key bacterial species and metabolic signatures may facilitate the development of diagnostic biomarkers and targeted microbiome-based therapies. Conclusively, the findings emphasize the prospective efficacy of interventions aimed at restoring microbial diversity and metabolic balance to mitigate illness severity and enhance calf health.