Study On Isolation And Identification Of Probiotic Lactic Acid Bacteria And Its Application To Prevent And Control Endometritis In Dairy Cow

Endometritis is common diseases in dairy cows during postparturition period,causing by bacterial infection uterine.It is mainly treated with antibiotics in clinical practice,which is likely to cause antibiotic residues and increase resistant strains.Therefore,non antibiotic therapy has become a research hotspot.As one of the non antibiotic therapies,Lactobacillus has attracted people’s attention.However,there are few reports on the prevention and treatment of the endometritis by Lactobacillus.In this paper lactobacillus was isolated from the uterus of healthy dairy cows 40-60 days postpartum.The potential of Lactobacillus in the prevention and treatment of endometritis was evaluated by the bacteriostasis,acid and H₂O₂ production,detection of drug resistance and drug resistance genes,safety,effectiveness.The results are as follows:1.Isolation and identification of Lactobacillus,test of bacteriostasis adhesion.Eight strains of Lactobacillus were isolated by MRS solid medium,Gram staining,biochemical identification and 16S rDNA identification.The results showed that Lactobacillus plantarum,Lactobacillus paracasei and Lactobacillus rhamnosus had good bacteriostasis.The adhesion test showed that Lactobacillus paracasei had a better ability to adhere to endometrial epithelial cells.Therefore,these 3 strains of Lactobacillus were selected for subsequent experiments.2.Safety and prebiotic evaluation of Lactobacillus.It was found that the three strains had good acid-producing ability,and the pH reached below 4.0 after fermentation for 24 hours.None of the 3strains Lactobacillus had the ability to produce H₂O₂.The drug sensitivity test showed that the three strains were all sensitive to the seven commonly antibiotics,and could significantly inhibit the growth of E.coli,S.aureus,and S.agalactiae.Partial resistance genes of lactic acid bacteria were detected by specific PCR.The results showed that sulfonamide resistance genes sul-2 and sul-3,aminoglycoside resistance genes aphA-1 and aphA-2 andβ-lactam antibiotic resistance gene bla-IMP wrer detected by PCR.The results of rat safety test showed that 0.1 mL 1×10¹⁰ CFU/mL could cause slight inflammation of rat uterus.The results showed that the three strains could not cause pathological damage to the experimental animals and could be used in clinical trials.3.Optimization of Lactobacillus culture medium.The medium was optimized by changing concentration of the main carbon source and nitrogen source in the MRS basic medium.The results show that the optimized medium can increase the growth of Lactobacillus paracasei and Lactobacillus rhamnosus,but the effect on Lactobacillus plantarum is not obvious.The orthogonal test was used to screen the best concentration of freeze-dried embedding agent.The results showed that the three strains freeze-dried protective agent were frozen at-50℃under vacuum condition for48 h.The results showed that the survival rate of Lactobacillus plantarum,Lactobacillus paracasei and Lactobacillus rhamnosus was 92.5%,89.7%,and 93.3%respectively after freezing for one month.4.Clinical trial of Lactobacillus in the treatment of cow endometritis.Twenty cows with clinical endometritis were randomly divided into two groups:Lactobacillus treatment group and control group.The results showed that the cure rate was 60%and the effective rate was 90%of Lactobacillus group.