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(1) Background: We aimed to isolate and identify potential next-generation probiotics (NGP) by investigating the interrelationships between gastrointestinal microbiota and diarrhea in preruminant Holstein calves. (2) Material and methods: Twenty preruminant Holstein calves were divided into healthy and diarrheic groups after the combination outcomes of veterinary diagnosis and fecal scores. The fecal microbiome, plasma cytokines, plasma immunoglobulin (Ig) G and haptoglobin were analyzed. The potential probiotic bacteria were identified by comparing the microbiota difference between healthy and diarrheic calves and correlation analysis with fecal scores and inflammatory markers. The identified bacteria were also isolated for further evaluation for antimicrobial activities and immunoregulatory effects. (3) Results: Microbiota analysis suggested that Ruminococcaceae_UCG_014, Bifidobacterium and Pseudoflavonifractor positively correlated with bovine IgG and negatively correlated with fecal score; inflammatory factors, bovine HP, and IL-8 were classified as beneficial bacteria contributing to the health of the calves. The alternation of gut microbial composition also induced changes in the functional gene enrichment of gut microbiota in calves. The gathering of microbiomic data strongly indicated the possible beneficial effects of Bifidobacterium longum subsp. longum, expected to develop as NGP. After isolation and evaluation of the potential functionality in vitro, two specific bifidobacterial strains demonstrated antimicrobial activities and immunoregulatory effects. (4) Conclusions: The results provide a new probiotic searching approach for preventing gastrointestinal disorders in preruminant calves. Further animal study is necessary to verify the results.

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Development of Next-Generation Probiotics by Investigating the Interrelationships between Gastrointestinal Microbiota and Diarrhea in Preruminant Holstein Calves