Abstract: 【Objective】 This study was aimed to isolate exosomes from bovine mammary epithelial cell (BMEC) infected with Staphylococcus aureus (S.aureus),and investigate the effects of MOI(S.aureus:BMEC) and time post infection on the total concentration of exosomes.Then,the exosomes induced by S.aureus were also isolated and identified.【Method】 BMEC were infected with two levels of S.aureus (MOI=1 and MOI=10) for 3 h,and further cultured for 9,12,and 24 h after the infection.While,a blank control group with no infection of S.aureus was also set up simultaneously.The damage of the ultrastructure of BMEC caused by S.aureus infection was observed by scanning electron microscope.The exosomes in the supernatant of S.aureus-infected BMEC were then isolated by ultracentrifugation.The morphology,particle size and marker proteins of the exosomes were analyzed by transmission electron microscopy,nanoparticle tracking analysis,and Western blotting,respectively.The total amount of exosomes at different MOI and post-infection time was evaluated by detecting the total protein concentration of exosomes.Consequently,the optimal conditions for S.aureus in inducing the release of exosomes from BMEC were determined.【Result】 Scanning electron microscope observation showed that S.aureus infection induced shedding of microvilli,and destruction of cytoskeleton of the BMEC.Exosomes were successfully isolated and purified from the supernatant of S.aureus-infected BMEC by ultracentrifugation.Transmission electron microscopy showed that the purified exosomes were spherical vesicles with a lipid bilayer membrane,and a diameter of 30-150 nm. The morphology of the exosomes in each group was uniform.The total protein concentration of exosomes induced by S.aureus.with MOI=10 was higher than that at MOI=1 at 9,12 and 24 h after the infection respectively, and it was the highest at 12 h after infection.Furthermore,nanoparticle tracking analysis detected the average diameter of exosomes induced by S.aureus was about 116 nm.Western blotting results showed that the exosome protein markers CD9,CD81,and TSG101 were positively expressed.【Conclusion】 The results indicated that the exosomes were successfully isolated in this study by inspecting their morphological and molecular biological characteristics.The releasing of the exosomes derived from BMEC could be induced by S.aureus.And the highest protein content in the exosomes derived from the BMEC was observed after treated by S.aureus for 3 h and cells cultured exosome-freely for 12 h with MOI=10.

Key words: dairy cows; Staphylococcus aureus; mammary epithelial cell; exosomes