Preparation And Immunogenicity Analysis Of Bovine Pathogenic Escherichia Coil Ghost

Escherichia coli（E.coil）is the most common bacterial pathogen in cattle farming,especially for calves.Some of calves should affect by sequel when recover from E.coil infection and other serious could die quickly,which cause huge economic losses for farmers.Currently,antibiotics is the main drug to prevent and treat bacterial infectious diseases.However,the abuse of antibiotics has led to the production of multi-drug resistant strains and serious environmental pollution,which have made a great challenge to global public health security.Therefore,we must find different ways differ from using antibiotic to prevent and treat bacterial infectious diseases.At present,vaccination is an effective way to prevent pathogenic bacterial infections.Nowadays,the inactivated vaccine is the most common vaccine on clinical trial.However,Immune protection of traditional inactivated vaccine is unsatisfactory owing to its inactivation method.Bacterial ghosts（BGs）could stimulate humoral and cellular immune response because of the preservation of bacterial surface antigens’natural conformation.Therefore,BGs have excellent performance and application prospect in the field of vaccines.In this study,we isolated E.coli from rectal swabs and organs of diseased cattle in a farm,and then conducted drug sensitivity tests and virulence gene determinations of these E.coli.Finally,we screened trial strains through mouse challenge experiments.Next,we constructed the trial strains ghost through genetic engineering and prepared its inactivated vaccine at the same time.At last we evaluated the efficacy of the BGs through a mouse model.There were 24 isolates finally proved as E.coli.The result showed that the drug resistance rates of cefotaxime and ampicillin were the highest,up to 83.33%and79.20%;all isolates were sensitive to cefoxitin.The resistance rate of other drugs were higher than 60%.The results of virulence determination showed that gene iut A related to iron uptake,was the most detection virulence,whose rate was up to 91.67%.And the rate of gene papc and stx2 was 79.17%and 16.67%.Then we selected E.coli with more virulence factors and challenged 2×10⁹CFU bacterial fluid through intraperitoneal,all strains can cause the death of mice.Finally,E.coli M1 was selected as the trial strain.E.coli M1 ghost and inactivated vaccine were injected by intramuscular.The result of immunization trail showed that different doses of immunization groups in twice vaccination trial can raise the anti-E.coli Ig G content.And after booster vaccinate,the content of antibodies was remarkably higher compared to the primary immunization.The result of immunity protective test showed that the protection rate of immune group inclued inactivated vaccine 1×10⁹CFU group and bacteria ghost1×10⁹CFU,5×10⁹CFU,10¹⁰CFU group was 20%.It was also found that immunization with high dose of inactivated vaccine did not raise its protection rate.We also made booster vaccinate after secondary immunization in inactivated vaccine1×10⁹CFU group and bacteria ghost 1×10⁹CFU group,the result found that triple vaccinate had no effect on the protection rate.However,the content of some antibodies was higher than secondary immunization.In summary,the isolated pathogenic Escherichia coli M1 ghost and inactivated vaccine can effectively raise igG and igM antibodies content in mice and have some immune protective effect.