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Neonatal calf diarrhea (NCD) remains a significant contributor to calf mortality within the first 3 weeks of life, prompting widespread antibiotic use with associated concerns about antimicrobial resistance and disruption of the calf gut microbiota. Recent research exploring NCD treatments targeting gut microbiota dysbiosis has highlighted probiotic supplementation as a promising and safe strategy for gut homeostasis. However, varying treatment outcomes across studies suggest the need for efficient treatment options. In this study, we evaluated the potential of probiotics Limosilactobacillus reuteri, formally known as Lactobacillus reuteri, isolated from healthy neonatal calves to treat NCD. Through in silico whole genome analysis and in vitro assays, we identified nine L. reuteri strains, which were then administered to calves with NCD. Calves treated with L. reuteri strains shed healthy feces and demonstrated restored gut microbiota and normal animal behavior. Leveraging a machine learning model, we evaluated microbiota profiles and identified bacterial taxa associated with calf gut health that were elevated by L. reuteri administration. These findings represent a crucial advancement towards sustainable antibiotic alternatives for managing NCD, contributing significantly to global efforts in mitigating antimicrobial resistance and promoting overall animal health and welfare.

Calf diarrhea is one of the most concerning challenges facing both the dairy and beef cattle industry. Maintaining healthy gut microbiota is essential for preventing gastrointestinal disorders. Here, we observed significantly less bacterial richness in the abnormal feces with watery or hemorrhagic morphology compared to the normal solid feces. The normal solid feces showed high relative abundances of Osllospiraceae, Christensenellaceae, Barnesiella, and Lactobacillus, while the abnormal feces contained more bacterial taxa of Negativicutes, Tyzzerella, Parasutterella, Veillonella, Fusobacterium, and Campylobacter. Healthy calves had extensive bacterial-bacterial correlations, with negative correlation between Lactobacillus and potential diarrheagenic Escherichia coli-Shigella, but not in the abnormal feces. We isolated Lactobacillus species (L. reuteri, L. johnsonii, L. amylovorus, and L. animalis), with L. reuteri being the most abundant, from the healthy gut microbiota. Isolated Lactobacillus strains inhibited pathogenic strains including E. coli K88 and Salmonella Typhimurium. These findings indicate the importance of a diverse gut microbiota in newborn calf’s health and provide multiple potential probiotics that suppress pathogen colonization in the gastrointestinal tract to prevent calf diarrhea.