Subclinical disease elevates zoonotic risks of antibiotic resistance and virulence factor genes through gut microbial network in dairy cow production system.

Antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) have become a global public health threat. As one of the main types of livestock, dairy cows under intensive farming practices pose a greater risk as the gastrointestinal tract of subclinically diseased cows serves as a "silent" gene reservoir. However, the distribution, transmission, and impacts of ARGs and VFGs in the gastrointestinal tract of subclinically diseased cows on human health and their own metabolism remain unclear. In this study, a nested case-control study was conducted within a prospective cohort of 211 periparturient dairy cows, collecting a total of 58 samples of rumen fluid, feces, and blood from subclinical ketosis and healthy cows. The results demonstrated that compared to healthy cows, the gastrointestinal microbiome of diseased cows contained a greater number of ARGs and VFGs exhibiting significantly increased abundance (ARGs in rumen: 30 vs 10, ARGs in feces: 66 vs 42; VFGs in rumen: 6 vs 1, VFGs in feces: 30 vs 10). Meanwhile, analysis of mobile genetic elements (MGEs) and MetaCompare2 database further indicated that the gastrointestinal microbiome of diseased cows poses a greater potential risk to human health. Additionally, specific ARGs/VFGs (e.g., FosM1) were found to be involved in the pathological process of ketosis of cow and were significantly associated with endogenous pathways such as bile acid metabolism. This highlights the important role of the ARGs-VFGs-CAZymes (Carbohydrate-active enzymes) co-occurrence network in host metabolic diseases. Our study highlights the human health risks of subclinically diseased cows as a reservoir of resistance genes, providing a theoretical basis and practical direction for improving the "One Health" prevention and control strategy.