Integrated 16S rRNA and Metagenomic Analysis of Pulmonary Microbiota in Sheep with Pneumonia

Sheep are a major livestock species in China, yet pneumonia-related mortality poses a significant obstacle to intensive farming. In this study, 115 sheep lung samples were collected and classified into different pneumonia severity groups based on lung lesion scoring. Subsequently, this study employed 16S rRNA sequencing to systematically investigate the structure and diversity of the pulmonary microbiota in sheep, including alpha diversity, beta diversity, and LEfSe analyses. Metagenomic techniques were also applied to analyze the abundance of metabolic pathways, exploring the associations between functional gene differences and pneumonia severity, as well as putative antibiotic resistance genes, virulence factors, and the species contributions of functional genes in severe pneumonia cases. Microbial richness and diversity were significantly higher in the severe pneumonia group than in the healthy/mild lesion group (p < 0.05). While the dominant microbial structures were similar across the groups, notable differences were observed in the abundance of respiratory disease-associated genera, with Pasteurella, Mannheimia, Mycoplasma, Bibersteinia, and Moraxella identified as significantly enriched in severe cases. Moreover, several genera originating from the gut and oral cavity were also associated with pneumonia, suggesting a potential gut-lung axis. Carbohydrate metabolism was the most prevalent pathway in all groups, whereas amino acid metabolism was significantly enriched in the severe pneumonia group. Putative antibiotic resistance genes were differentially enriched; the severe pneumonia group showed significant enrichment of genes conferring resistance to aminoglycosides, tetracyclines, and polymyxins. Virulence factor analysis identified nutritional/metabolic factors and adhesion as the predominant virulence mechanisms. Species contribution analysis further revealed that Mannheimia, Mycoplasma, Pasteurella, and Moraxella were the predominant species associated with functional gene enrichment. In conclusion, the current study reveals associations between changes in the pulmonary microbiota structure and function and the severity of pneumonia in sheep, aiming to provide a foundation for future hypothesis-driven research on the role of the pulmonary microbiota in pneumonia progression.