Abstract

Background. As a highly reproductive meat sheep breed in China, the Hu sheep is an important economic group in ruminant animal breeding. However, research on their intestinal microbiome under the background of diarrhea and antibiotic treatment remains relatively limited.

Methods. This study investigated the intestinal microbiota of Hu sheep lambs in the preliminary stage of diarrhea (group DM), the late recovery stage of diarrhea (group DL), and the healthy stage (group H). Diseased individuals (groups DM and DL) were treated with a combination of Shuanghuanglian, Cefazolin, Lincomycin, and Dexamethasone (0.2 mL dosage). To characterize the intestinal microbiota, fecal samples were collected from all groups, and metagenomic sequencing was performed. Using metagenomic binning tools and co-assembly methods, we reconstructed 482 high-quality non-redundant metagenome assembled genomes (MAGs).

Results. Among these MAGs, 70% belong to the phyla Firmicutes, Bacteroidetes, and Proteobacteria, highly consistent with the typical structure of intestinal microbiota in ruminants. Functional annotation revealed that the genes encoding carbohydrate-active enzymes (CAZymes) are more abundant in Bacillota and Bacteroidota, which supports the degradation and energy metabolism functions of Hu sheep on fibrous feed. During the preliminary stage of diarrhea, the virulence genes carried by symbiotic bacteria such as Lachnospiraceae, Acutalibacteraceae, and Bacteroidaceae were enriched. Although diarrhea symptoms alleviated during the late recovery stage of diarrhea, the combined use of multiple antibiotics led to the continuous enrichment of antibiotic resistance genes (ARGs) related to lincosamides and cephalosporins. The average abundance of cephalosporin-related ARGs in group DL was significantly higher than that in group DM and H, indicating a risk of residual ARGs. Microbial diversity analysis showed that there was no significant overall difference in MAGs between the group DM and H, but both groups showed significant differences compared to the group DL, suggesting that antibiotic-driven clearance of sensitive bacteria is the core driving force. Moreover, our study shows that the abundance of the zoonotic pathogens Barnesiella and Campylobacter significantly increased in the diarrhea group (p<0.05), and they carry 382 and 567 virulence genes, respectively. Their pathogenicity is regulated by the dynamic changes in the host intestinal microbiota.

Conclusions. This study not only expands the genomic database of ruminant intestinal microorganisms but also provides a key theoretical basis for formulating intestinal microecological regulation strategies and optimizing diarrhea treatment regimens for Hu sheep.