| Bovine mastitis is a hazards greater disease in the livestock industry and foodsafety, it produces a wide variety of problems in the dairy farm, Staphylococcusaureus(S. aureus) is one of important pathogen of mastitis.For a long time, theeffective treatment of this disease is based on the use of antibiotics, but in recent yearsdue to dependence and irrational use of antibiotics leads to drug resistance ofpathogens and drug residues, which brought great difficulties to effectively treatmastitis. Our previous acquired S. aureus which has a broad spectrum, high crackingactivity of the phage lyase LysGH15and reveals its three-dimensional structure andmolecular mechanism of action. In the early basis, this study through the use of phageLysGH15rapid ability to kill S. aureus, the model of mastitis caused by S.aureustreatment study to explore the use of the lyase treat mastitis in dairy cows potential.First, optimized expression conditions and purification conditions of S. aureusphage lysin LysGH15, showed that at20℃low temperature induced6h can obtainmaximum expression, eventually obtain a higher purity lysin LysGH15after150mMimidazole of eluention buffer and concentrating measured concentration7.18μg/μl.Determination of LysGH15in vitro activity by plate lysis assay and turbidityreduction assays, shows that concentration of1.0μg/μl of LysGH15with ATCC49525strain LB solid medium plate to form a clear inhibition zone after12h at37℃and theOD600dropped from1.0to0.4with10min in turbidity experiments. By the results ofthe formation of inhibition zone and turbidity reduction to observed Lysin significantbactericidal activity in vitro.It established mouse model of mastitis by use of S. aureus ATCC49525whichpurchased from American Type Culture Collection. we will twenty-four lactatingfemale rats randomly divided into seven groups,include control group, PBS group andinfected group, each group of three lactating female rats. The control group have notreatment, PBS group of mice only the fourth and fifth mammary gland infusion PBS, infection group of mice only the fourth and fifth mammary gland infusion bacteria of105cfu/50μl meanwhile infection after8h,12h,24h,48h,72h were sacrificed todetermine the best time. By visual observation of clinical symptoms, pathologicalanatomy; making biopsy and analyzed pathological changes; ELISA assay factorTNF-α levels in breast tissue cells. Infection group compared with the negativecontrol group and PBS group,the results showed that the mice mammary glandobvious clinical symptoms, redness, swelling, heat, pain, pathological changes with S.aureus infection after48h; mammary gland tissue structural have changed, mammarygland have lot disintegrated, inflammatory cell infiltration,interstitial have wided andblurred, Inflammatory reaction; the level of pro-inflammatory cytokines TNF-αsignificantly increased.These results show the mouse model of mastitis have beenestablished in success.Application of S. aureus phage lysin LysGH15treatment mouse model ofmastitis.Infection after one hour given a low dose lysin (10μL), the results show thatcolonies within mammary gland is significantly decreased, inflammatory factorsTNF-α and IL-6within mammary gland also significantly reduced, infiltration ofinflammatory factors within acinar reduce and can relieve inflammatory responseeffectively. However, Infection after eight hours given a high dose lysin (50μL) canalso play a significant effect. In summary use of S. aureus bacteriophage lysinenzymes LysGH15treatment of mice mastitis models with good results,and laid thefoundation for the follow-up dairy cow mastitis of prevention and cure. |
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