Preparation Of Probiotics Against Bovine Colibacillosis In Yak

Diarrhea of Yak Calves is the most common disease in the breeding industry in Tibet.Escherichia coli is one of the main causes of diarrhea of Yak Calves.This study focused on the concept of healthy breeding.We want to use one strain of Lactobacillus lac-2 and one strain of Bacillus subtilis kc-4,which are acid resistant,bile salt resistant and have good antibacterial effect on E.coli O₇₈,to make probiotics against yak diarrhea.The results are as follows.（1）A preliminary study of the effect of probiotics on the prevention and treatment of yaks’diarrhea disease caused by E.coliIn order to obtain the best prevention and treatment plan,the yak calves were challenged with 10⁹ CFU of E.coli O₇₈ twice a day by grouping testThe preventive test showed that lac-2 of 10×10⁹CFU/ml had the best preventive effect on E.coli O₇₈.The yak calves were challenged with10⁹ CFU E.coli O₇₈ twice a day for 3 days,and no diarrhea occurred within 15±0.5 days.The treatment test showed that:5×10⁹cfu/ml lac-2+5×10⁹cfu/ml kc-4 mixed bacterial solution had the best effect in the treatment of calf yak diarrhea caused by E.coli O₇₈.The diarrhea of Yak Calves caused by E.coli O₇₈ could be cured by gavage three times a day for up to 3±0.5days.（2）The production of probiotic preparations against yaks’E.coliIn this study,Lac-2 and Kc-4 were used to obtain probiotics dry powder by vacuum freeze-drying test.The best cryoprotectant screened was 20%skim emulsion,and the best ratio of protective agent and fermented sediment was 1:1.The physical and chemical properties of lac-2 lyophilized powder were tested.The results showed that the dosage of lac-2 lyophilized powder was 0.64×10⁹cfu/g,the viable rate was 90.29%,the moisture content was 11.82%,the protein content was 2.32 g/100g,the fat content was 1.20 g/100g,the dispersibility was good,and there was no Escherichia coli pollution.The results showed that the dosage of lac-2 and kc-4 lyophilized powder was 0.56×10¹⁰ CFU/g,the viable rate was 90.34%,the moisture content was 12.35%,the protein content was 2.68 g/100g,the fat content was 1.68 g/100g,the dispersibility was good,and there was no Escherichia coli pollution.The preservation test of freeze-drying agent showed that the viable rate of probiotics was 70.5%after 12 months of cold storage at-20℃.In order to ensure the shelf life of probiotics for one year,the first dose of probiotics was 2 g/bottle.（3）Preliminary study of the mechanism of anti-yak diarrhea in probioticsIn this experiment,ELISA method was used to detect the serum zonulin content of yak at different stages of challenge and treatment; paraffin sections were made to observe the pathological changes of each intestinal segment;fluorescent quantitative method was used to detect the m RNA expression of ZO-1 and MUC1 protein in each intestinal segment.ELISA test showed that the level of serum zonulin was significantly increased after 4 days of E.coli O₇₈ challenge（P<0.01）,and decreased after 2 days of probiotics treatment（P<0.05）;there was no significant difference between the level of serum zonulin and that on 4 days of florfenicol treatment（P>0.05）.After 2 days of treatment,the serum zonulin content in the florfenicol treatment group was significantly higher than that in the probiotics treatment group（P<0.05）.The results of paraffin section showed that probiotics was better than florfenicol in recovering the intestinal pathological changes of Yak Calves caused by E.coli O₇₈.Fluorescence quantitative test showed that E.coli O₇₈ could change the permeability of tight junction of intestinal epithelium by inhibiting the expression of ZO-1 and MUC1.The relative expression of ZO-1 andMUC1 protein m RNA in intestinal mucosa was negatively correlated with the expression of zonulin.In conclusion,in this experiment,lac-2 and kc-4 were freeze-dried in vacuum to obtain probiotics.The dosage of lac-2 lyophilized powder was0.64×10⁹cfu/g;the dosage of lac-2 and kc-4 lyophilized powder was 0.56×10¹⁰cfu/g.the probiotics prepared by both of them were 2 g/bottle.The two probiotics were still effective at-20℃for 12 months.The former can keep healthy for 15±0.5 days by gavage twice a day for 3days,while the latter can cure E.coli O₇₈ disease by gavage three times a day for 3±0.5 days at most.The results of ELISA and fluorescence quantitative test showed that the probiotics could improve the permeability of intestinal epithelial tight junction by reducing the content of serum zonulin and increasing the protein content of ZO-1 and MUC1,so as to cure E.coli O₇₈ Objective of diarrhea.This experiment proved that it is feasible to use probiotics instead of antibiotics to prevent and treat E.coli O₇₈ diarrhea in Yak Calves,and it can be used as one of the alternative drugs to prevent and treat this kind of disease.