The Correlation And Mechanism Between Rumen Microbiota Disturbance And Mastitis In Dairy Cows

Mastitis,one of the most serious diseases of dairy cows,especially of high-yield cows,has brought not only the enormous economic loss in the dairy industry,but also the great threat to the human health.In clinical practice,mastitis,especially subclinical mastitis,has been found to occur in cows at a high frequency during the early and peak lactation period,which is closely associated with the disorder of the ruminal microbiome caused by the increased feeding of concentrated feed.In addition,studies have shown that the rumen flora disorder induced by over-feeding with high-concentration diet promoted the excessive growth and reproduction of conditional pathogenic bacteria,leading to the continuous release of lipopolysaccharide(LPS)into the blood,and inducing systemic low-level inflammation.When cows lactated after delivery,the increased blood flowing to the mammary glands may cause LPS to accumulate in mammary glands with circulation and induce the occurrence of mastitis in cows.Furthermore,our previous studies have shown that perfusion of LPS to mammary gland destroyed the blood-milk barrier and caused a large number of neutrophil to be recruited to the mammary gland.Therefore,we speculated that the increased incidence of mastitis in the early and peak lactation of dairy cows is directly related to ruminal flora disorders,and the following theoretical assumptions are proposed: changes in factors such as feeding and environment will lead to rumen microbiota imbalance,leading to multiply of harmful bacteria and continuous release of LPS into blood,resulting in a systemic flow-grade inflammation.When cows experience lactation stress,more LPS enter the mammary gland with increased blood flowing to the mammary gland.The increased LPS in the mammary gland damages the blood-milk barrier and lowers the threshold for inflammatory cells to cross the barrier,which manifests as subclinical mastitis characterized by excess somatic cell count(SCC)in milk.When pathogenic factors such as pathogenic bacteria in the external environment act on the mammary glands,inducing more inflammatory cells into the mammary gland and increasing the severity to mastitis.In order to verity this theoretical hypothesis,we carried out the following experiments:Firstly,the mice were randomly divided into three groups,including untreated group(normal mice),gut microbiota-dysbiosis group(combinatorial antibiotics was administered to the mice via their drinking water to disrupt intestinal microbiota),and fecal microbiome transplantation group(fecal from healthy mice were transplanted to antibiotics treatment mice),then the three groups mice were infected with S.aureus to induce mastitis.The effects of gut microbiota on mastitis was investigated by examining the histopathological changes,inflammatory cytokines production,MPO activity,and blood-milk barrier permeability in the mammary gland tissues of different group mice.The results showed that structure of the mammary gland was changed,the inflammatory cytokines TNF-α,IL-1β,and IL-6 and MPO activity were significantly increased,as well as the blood-milk barrier and intestinal barrier were damaged in gut microbiota-dysbiosis mice compared to the untreated mice.However,these changes were significantly alleviated in fecal transplant group mice.In addition,these changes became more pronounced when infected with different concentrations of S.aureus.These results suggested that gut microbiota dysbiosis increased the permeability of blood-milk barrier,induced the inflammatory response of mammary gland,and increased the severity to S.aureus infection.Secondly,subacute rumen acidosis(SARA),a typical animal model of rumen microbiota disorder,was induced by feeding a high-concentration diet(ratio of fine to coarse: 7:3)to dairy cows.The concentration of LPS in rumen and blood,the levels of PMN,albumin,AST,inflammatory cytokines TNF-α,IL-1β,IL-6,IL-17,and SAA in blood were significantly increased compared to the control cows.These results suggested that the systemic low-grade inflammation was induced by LPS derived from rumen during cows suffering SARA.Moreover,the expression of tight junction protein of blood-milk barrier was reduced,the SCC and inflammatory cytokines TNF-α,IL-1β,IL-6,and SAA in both milk and mammary glands were significantly increased in SARA compared to control cows.These results suggested that SARA increased the permeability of blood-milk barrier and induced mastitis in dairy cows.In addition,the levels of LPS in lacteal vein,milk and mammary gland of SARA cows was significantly increased,and the continuous LPS infusion induced mammary gland pathological changes,increased the inflammatory cytokines TNF-α,IL-1β,IL-6 production,MPO activity,and the blood-milk barrier permeability.These results indicated that SARA of cows leads to rumen-derived LPS entering the mammary gland through blood circulation,damaging the blood-milk barrier,and inducing inflammation of the mammary gland in cows.Furthermore,16 S rRNA gene sequence analysis was used to investigate the relationship of mastitis and the changes of microbiota community in rumen fluid,milk,feces,and blood.The results showed that although rumen fluid,milk,feces,and blood harbored distinct microbiota,the community of bacterial flora in the rumen and milk of cows suffering from SARA are more similar to the rumen and milk and mainly manifested as the relative abundance of Stenotrophomonas significantly increased.Venn diagrams showed that core genera shared by rumen fluid,milk and blood of SARA cows accounted for the proportion of rumen fluid,milk,and blood were significantly increased compared to the control cows.This suggested that some bacteria may migrate among the rumen fluid,milk,and blood after cow suffered SARA.Further research by LEfSe analysis showed that Stenotrophomonas were enriched in rumen fluid,milk and feces,but no bacteria enriched in the blood of SARA cows,and we did not isolate Stenotrophomonas from the blood.Furthermore,the mice were gavaged by Stenotrophomonas maltophilia(S.maltophilia,theonly species of Stenotrophomonas)to explore the relationship between Stenotrophomonas and mastitis.The results showed that treatment of S.maltophilia significantly increased the pathological damage,and inflammatory cytokines TNF-α,IL-1β and IL-6production in mammary gland compared to the control mice.The above results indicated that SARA increased the abundance of Stenotrophomonas in rumen,which was one of the endogenous factors to induce mastitis in cows.Finally,to evaluate the effect of SARA on bovine mastitis,we tested the milk composition,and the load of S.aureus in milk of control and SARA cows after infection with S.aureus.The results showed that infusion of S.aureus resulted in decreased milk fat,lactose,and dry matter content,while a significant increase in milk protein and SCC,and SARA cows showed a more obvious above changes compared to control cows after the S.aureus infusion.These results suggested cows suffering from SARA may reduce the clearance ability of the mammary gland to S.aureus infection.In present study,we have demonstrated that there was an important relationship between rumen and mammary glands of dairy cows,and this association played an important role in the development of mastitis.The correlation mechanism as follows: LPS originating from the rumen may be translocated to the mammary gland by the bloodstream and then induce mastitis in cows with imbalance rumen microbiota induced by SARA.In addition,SARA-induced abundance of Stenotrophomonas increase in the rumen is also an important endogenous factor to induce mastitis in cows,but the specific mechanism is still unclear.In addition,SARA damages the ability of the mammary gland to clear out pathogens,thus increased severity of mastitis.Our results not only provide a basis of novel therapeutic strategies for treatment of cow mastitis,but also have important reference significance to the prevention and treatment of other infectious diseases.