Isolation,Identification And Immunological Protection Of Porcine Escherichia Coli O145 Vaccine

Diarrhea is an important cause of death of piglets.Piglet yellow and white dysentery are caused by pathogenic E.coli,and they are the most common bacterial diarrhea,of which morbidity and mortality are very high.They are serious threats to the development of swine industry.Antibiotics are the most common method in clinical treatment for piglet colibasillosis,but due to the use of a large number of antibiotics,bacterial drug resistance is becoming more and more serious,which often resulting in ineffective treatment.Vaccine is the most effective way to prevent and control the infection of virus,bacteria and other pathogenic microorganisms.In order to provide technical support for the prevention and control of colibasillosis of piglets in large-scale pig farms in Liaoning Province,we have isolated and identified Escherichia coli from piglets with diarrhea at the age of 7 days,and prepared inactivated vaccine for the Escherichia coli with aluminum hydroxide adjuvant and propolis adjuvant.In this study,bacterial identification culture,biochemical test,drug sensitivity test,pathogenicity test,16Sr DNA PCR amplification,Genetic evolutionary tree analysis,sequencing and sequence comparison were carried out in the liver of piglets with diarrhea from a large-scale breeding farm in liaoning Province.Results:The characteristics of bacterial identification culture,morphological observation and biochemical test results were consistent with the characteristics of Escherichia coli.Through the PCR amplification,sequencing and sequence alignment analysis,the homology with CP031919.1(serotype O145)was the highest(99.44%).The strains from Liaoning were closely related to those from Nanjing,Yangzhou,Hunan and Hebei.The results of drug sensitive test showed that the drug resistance of the isolated bacteria is serious.It is resistant to polymyxin B,cefazolin,azithromycin,amoxicillin,spectinomycin,ciprofloxacin,erythromycin,polymyxin E,rifampicin and cotrimoxazole,doxycycline and so on,except that it is sensitive to cefotaxime and is intermediate to amikacin.The virulence genes of the isolates were detected and the results showed that the isolates carried iss,iuc D and Fim H virulence genes.In the pathogenicity test,20 SPF mice were divided into control group and experimental group.Experimental group mice were intraperitoneally injected with 0.3ml live bacterial solution which has a concentration of 2.5×10~9CFU/ml,and control group mice were intraperitoneally injected with 0.3 m L sterile PBS solution.Results:Mice in the experimental group began to die after 12 h of the intraperitoneal injection of live bacteria solution,and all died at the 30 h,while mice in the control group survived healthily,indicating that the isolated strain was Escherichia coli with high pathogenicity.Inactivated vaccine with aluminum hydroxide adjuvant and inactivated vaccine with propolis adjuvant were prepared with using the isolated strains as antigen,and their safety,immunogenicity and animal challenge protection test were performed.Results:Two kinds of vaccines with different adjuvant had certain protective effect against virulent strains of mice.The protective rate of aluminum hydroxide adjuvant vaccine was 70%,and that of the propolis adjuvant vaccine was 80%,indicating that the protective rate of propolis adjuvant vaccine was better than that of aluminum hydroxide adjuvant vaccine.The results of immunoglobulin detection showed that compared with the control group,the serum Ig A antibody content in the propolis adjuvant group and the aluminum hydroxide adjuvant group was significantly increased at 14 d,21 d,28 d,35 d and 42 d after immunization,the difference was extremely significant(P<0.01).The Ig A antibody content of propolis adjuvant vaccine group was significantly higher than alumina hydroxide adjuvant vaccine at 21and 28 days after immunization(P<0.01).The 42 d after immunization,the difference between propolis adjuvant group and aluminum hydroxide adjuvant group was significant(P<0.05).On the 14,21,28,35 and 42 d after immunization,the serum Ig G antibody content in the immunized group was significantly increased compared with that in the control group(P<0.01).The 21 d after immunization,Ig G antibody content in propolis adjuvant vaccine group was significantly higher than that in aluminum hydroxide adjuvant vaccine group(P<0.01).At28 d,35 d and 42 d after immunization,there was significant difference between propolis adjuvant group and aluminum hydroxide adjuvant group(P<0.05).Conclusion:in the study,we have succeeded in the isolation of the pathogenic E.coli from the diarrhea piglets in the breeding pig farm,and the preparation of aluminium hydroxide adjuvant vaccine and propolis adjuvant vaccine in mice has good immunogenicity and high protective rate.What is more,the imminogenicity and protective rate of the propolis vaccine adjuvants is better than that of the aluminum hydroxide adjuvant vaccine.The vaccine laid a foundation for effective prevention and control of piglet colibacillosis.