| Bovine mastitis is an inflammatory reaction of abnormal metabolic and physiological state in bovine mammary gland tissue due to damage of mammary gland tissue or infection of pathogenic microorganisms.It is also a disease that leads to significant economic losses in dairy industry.As an important infectious pathogen,bovine mastitis caused by Streptococcus agalactiae is usually chronic and does not show obvious clinical symptoms,and causes persistent infection of bovine mammary gland tissues.Pyroptosis,as a newly discovered programmed death of inflammatory cells,can be triggered by a variety of stimuli,but it is mainly induced by intracellular bacteria.However,whether Streptococcus agalactiae can induce pyroptosis in mammary epithelial cells has not been reported.The purpose of this study was to deeply understand the prevalence and molecular typing of Streptococcus agalactiae in201 large farms in 20 provinces,municipalities and autonomous regions of China,and to explore whether Streptococcus agalactiae can induce pyroptosis of mammary cells,so as to provide a preliminary theoretical basis for further exploring the molecular mechanism of host cells to resist Streptococcus agalactiae infection.And then to provide scientific basis for the prevention and treatment of bovine mastitis.The samples were collected for bacterial isolation and culture,and the suspected Streptococci strains were isolated by Gram staining,and the suspected Streptococci strains were incubated on a plate containing 5 % sheep blood for culture.The DNA of the suspected strains was extracted and identified by 16 Sr RNA gene sequencing sequencing.The molecular typing of the isolates was studied by multi-sequence locus typing(MSLT).The dominant ST strains were selected for subsequent experiments.Mouse mammary epithelial cells(HC11)were infected with different multiplicity of infection(MOI = 50: 1,40: 1,30: 1,20: 1,10:),and cell viability and LDH release were observed at different infection times(3h,6h,9h).The appropriate MOI was selected to infect mouse mammary epithelial cells,and the enrichment of signal pathways related to pyroptosis was analyzed by transcriptomics.The results of transcriptome analysis were verified by Western blot and ELISA,and whether Streptococcus agalactiae induced pyroptosis in mouse mammary epithelial cells was explored.In this study,140 strains of Streptococcus agalactiae were isolated from4041 samples and Shandong Province was a high incidence area of Streptococcus agalactiae.MLST analysis showed that there were only two major clonal complexes(CC)in these populations,namely CC103 and CC67.ST103 was the dominant ST,accounting for 35.7 % of the 140 isolates.In addition,13 new STs were found.The ST1421 strain was selected to construct a mouse mammary epithelial cell infection model and transcriptional sequencing,and the differential gene expression between the experimental and control groups was obvious after the infection of cells without Streptococcus lactis,and the differential gene expression between the experimental groups was not obvious.The results of GO gene enrichment analysis showed that biological processes accounted for the largest proportion.The results of KEGG enrichment analysis of differentially expressed genes showed that the differential genes of NF-κB and Nod-like receptors were significantly expressed.By analyzing the NOD-like receptor signaling pathway at 6h and 9h after infection,it was found that the related genes of NF-κB signaling pathway were significantly up-regulated,and the genes of inflammasome(NLRP3)were also significantly up-regulated in the whole signaling pathway.After verifying the results of transcription,it was found that Streptococcus agalactiae infection of mouse mammary epithelial cells can induce pyroptosis and through the classical pathway mediated by Caspase-1,and NLRP3 is activated through the classical NF-κB signaling pathway and is significantly timedependent. |
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