摘要Background An imbalance in the rumen microbiota caused by high-concentrate diets(HCD)is a significant endog-enous trigger of mastitis.However,the underlying mechanisms remain largely unknown.Microbial extracellular vesicles(mEVs)are critical mediators of microbe-host communication.However,the role of mEVs in rumen microbi-ota-mediated mastitis has not yet been reported.In this study,we used an HCD-induced rumen microbiota dysbiosis model to investigate the role of mEVs-derived from rumen microbiota in the pathogenesis of mastitis.Results Our results indicate that HCD leads to mastitis and systemic inflammation.Meanwhile,HCD-fed goats exhib-ited substantial rumen microbiota dysbiosis and the disruption of the rumen barrier.Transplanting rumen microbiota from HCD goats into mice induced both mastitis and systemic inflammation in the recipients.Specifically,HCD increases the production of mEVs carrying microbial DNA,which can translocate across the compromised rumen bar-rier to the mammary gland,triggering a mammary inflammatory response via activation of the cGAS-STING-NF-κB/NLRP3 pathway.Furthermore,treating mice with mEVs isolated from the rumen fluid of HCD goats directly induced mastitis,whereas depletion of microbial DNA attenuated mEVs-induced mastitis.Conclusion Our findings suggest that HCD induces rumen microbiota dysbiosis and impairs rumen barrier function.This dysfunction leads to an increase in microbial DNA-containing mEVs,which subsequently leak into the mammary gland.Once there,these mEVs activate the cGAS-STING-NF-κB/NLRP3 signaling pathway,ultimately inducing mastitis.This study provides a new perspective on the"rumen microbiota-mammary gland axis"and enhances the understand-ing of the pathogenesis of mastitis.