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RESEARCH ARTICLE | 06 Jul 2026

Comparative metagenomic and untargeted metabolomic analyses reveal gut microbiota and metabolite alterations associated with diarrhea in neonatal Holstein calves in Anhui, China

Xing Zhang, Hui Liu, Chaoyue He, Huiyang Chen, Huilin Zhang, and Falei Li Show more
VETERINARY WORLD | Article No. 6 | pg no. 2763-2776 | Vol. 19, Issue 7 | DOI: 10.14202/vetworld.2026.2763-2776
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ABSTRACT

                      
Background and Aim: Neonatal calf diarrhea remains a major cause of morbidity, mortality, and economic losses in the dairy industry. Although alterations in gut microbial communities have been implicated in calf diarrhea, the interactions between intestinal microbiota and metabolites in neonatal Holstein calves remain incompletely understood. This study aimed to characterize gut microbiome and metabolomic alterations associated with diarrhea and to explore the relationships between differential microorganisms and metabolites in neonatal Holstein calves from Anhui, China.
Materials and Methods: Fecal samples were collected from four diarrheic and four healthy female Holstein calves younger than 2 weeks of age. Shotgun metagenomic sequencing was performed using the DNBSEQ-T7 platform, and untargeted liquid chromatography–tandem mass spectrometry metabolomics was used to characterize fecal metabolites. Multivariate analyses, biomarker identification, pathway enrichment, and correlation analyses were conducted to investigate associations between microbial taxa and metabolites.
Results: Distinct microbial and metabolic profiles were observed between healthy and diarrheic calves. Analysis of similarities confirmed significant differences in microbial composition between groups (R = 0.7917, p = 0.025). Forty microbial biomarkers were identified, with <em>Campylobacter</em><em>jejuni</em>, <em>Campylobacter coli</em>, and <em>Bacillus cereus</em> showing increased abundance in diarrheic calves, whereas beneficial taxa such as <em>Faecalibacterium</em><em></em><em>prausnitzii</em> were enriched in healthy calves. Metabolomic analysis identified 377 differential metabolites, including 130 upregulated and 247 downregulated compounds in diarrheic calves. Pathway analysis indicated that D-glutamine and D-glutamate metabolism was the most affected pathway, together with alanine, aspartate, and glutamate metabolism, thiamine metabolism, taurine and hypotaurine metabolism, and cysteine and methionine metabolism. Five key metabolites, glutamate, α-ketoglutaric acid, thiamine, 3-sulfinoalanine, and S-adenosyl-L-homocysteine, were strongly associated with differentially abundant microorganisms. Correlation analyses demonstrated significant microbe–metabolite interactions, suggesting that dysbiosis contributes to metabolic disturbances during diarrhea.
Conclusion: Neonatal calf diarrhea was associated with pronounced alterations in gut microbial communities and fecal metabolites. The enrichment of opportunistic pathogens and disruption of amino acid-related metabolic pathways highlight potential biomarkers and mechanistic links underlying gut dysbiosis. These findings provide novel insights into microbiota–metabolite interactions and may facilitate the development of targeted strategies to improve calf health and reduce economic losses in dairy production.
                      
Keywords: alpha-ketoglutaric acid, calf diarrhea, gut microbiota, metagenomics, metabolomics, neonatal Holstein calves, precision livestock management, Campylobacter jejuni