Large Scale Preparation Method Of S.aureus Phage JS25 For Preventing And Controlling Mastitis And Its Application Safety Evaluation

Staphylococcus aureus(S.aureus)is a kind of pathogens that can cause a variety of severe local or systemically infection.It is also one of the significant pathogens of cow mastitis.Long-term use of antibiotics will lead to multi-drug resistance of S.aureus,and cause great difficulties for the treatment of various diseases.Therefore,it is extremely urgent to search for a safe and effective treatment of various types of infection which caused by multi-drug-resistant S.aureus.The rise of phage research provides possible treatments of multi-drug-resistant S.aureus.Our study isolated and obtained the bacteriophage JS25 of S.aureus,which owned broad parasitifer and could effectively kill the isolated pathogen of cow mastitis.Based on the previous studies,in order to realize the industrial production and application of phage.This paper illustrates the large-scale preparation technology of bacteriophage JS25 and its safety evaluation.Experiment one:The large-scale preparation technology of phage JS25 of S.aureus was studied.We chose 20 mL liquid value-added system to select the optimal culture medium,culture time,multiplicity of infection,multiplication temperature and rotation speed of the fermentation system of phage JS25.Finally,the selected optimum culture medium was 0.5 × TSB-YE,the culture time was 8-10h,the infection plural MOI values was 1×10-4,the temperature was 37 ?,the rotation speed was 180 rpm.Using the same conditions on the fermentation tank culture will increase 30 times of the previous amplification efficiency of phage JS25.The phage after fermentation and the amplification phage obtained from the laboratory declared that there were no significant differences in the electron microscopy figures and biological characteristics,which provided the theoretical basis for the mass production and application of phage.Experiment two:We studied the storage conditions and application effects of large scale production of phage.Eventually,we confirmed the optimal storage temperature was 4 ?,and the killing rate of scale preparation of the environmental disinfectants on S.aureus,Klebsiella pneumoniae and Escherichia coli was 90%or more.Experiment three:We use the mice as model to evaluate the application security of JS25 in the large scale production of phage.In the experiment,the mice were inoculated with phage JS25,and the acute toxicity,specific immune toxicity and non-specific immune toxicity of mice were determined.Test methods ICR mice were divided into control group,JS25 in TSB and JS25 in PBS.We studied the effects of JS25 in TSB and JS25 in PBS on the percentage of neutrophils and lymphocytes in mice,cytokines,and phagocytosis of white blood cells.The specific immune group was immunized once every 14 days with 200 ?L Staphylococcus aureus phage JS25(1010 pfu/mL).The changes of IgM and IgG in mice were measured every 7 days.The results showed that Staphylococcus aureus phage JS25 had no acute toxicity,After the injection of phage 4h,the percentages of lymphocytes in the JS25 in TSB and JS25 in PBS group of mice were significantly decreased(P<0.05).The percentage of neutrophil increased significantly(P<0.05),after 24 hours,JS25 in PBS group of lymphocyte percentage and neutrophil percentage were returned to the same level with the control group,but JS25 in TSB group remained significantly different(P<0.05).The results showed that JS25 in PBS group of phage displayed no abnormal cytokine production.The results of specific immune group showed that IgM and IgG increased rapidly in the short time after injection,but IgM declined after 7 days and remained at a low level,while IgG continued to increase as the number of immune times increased.Therefore,the phage will not affect the immune system in mice,but the continuous use of phage to cause the continuous increase of IgG and accelerate the elimination of phage in the body.