Construction Of Fusion Strains Of Expressing Two Different Serotypes Of Duck E. Coli And Research On The Immunogenicity

With the rapid development of China's poultry industry, prevalence of poultry E. coli disease is growing, it caused great economic losses to poultry supplies, while it has a greater threat to the health of consumers, therefore, effective prevention of avian colibacillosis have important public health significance. The immune disease prevention in the fight against E. coli is important. However, there are many E. coli serotypes, and different regions have different advantage serotypes. Therefore, a multivalent vaccine preparation has expanded application. However, multivalent vaccine has the shortcomings of the lowest unit titer. The fusion vaccine preparation is able to solve this problem. Through building Escherichia coli fusion strains of duck which could simultaneously express two different serotypes, and studying their physiological characteristics, biochemical characteristics, serotype identification and immunology characteristics, the integration strains were selected for preparing bivalent vaccines.Two strains of duck E. coli with different serotypes and complement drug resistance were identified by serotype identification combined with susceptibility paper method. Serotype identification test results showed that there were many kinds of E. coli serotypes, including dominant serotypes of O78 and O2. Susceptibility test results showed that E. coli resistance on the part of Sichuan Province had a very serious situation,7 antimicrobial agents had 90% resistance including penicillin G, streptomycin, tetracycline, doxycycline, erythromycin, and cotrimoxazole Midecamycin, multiple drug resistance were also serious. Through the serotype identification and susceptibility test, duck E. coli strains of ZYD5 and ZYD8 were selected as the test strains. The screening markers for ZYD5 and ZYD8 fusions were tested through the MIC determination. They were 40μg/mL gentamicin and 30μg/mL ciprofloxacin hydrochloride.Bacterial protoplast fusion technology was used to build duck E. coli fusion strains to remove the cell wall by protoplast lysozyme, to promote integration of fusion by PEG, to regenerate fusion by hypertonic selective medium. Screening of fusion was conducted by the serum phenotype, physiological and biochemical characteristics, growth characteristics, staining properties and stability. This study identified that the work concentration of protoplast enzyme was 0.25 mg/mL. The work time was 15 min by the pre-experiment. The regeneration rate of the positive fusions was 1.63%. Ultimately, three fusions which showed simultaneous expression of parent serotypes were obtained. Experiments showed that fusion strain showed a typical form of E. coli morphology, the biochemical characteristics were consistent with the biochemical characteristics of E. coli. The fusion strains obtained middle drug resistance and slightly lower growth compared to parent strains, and they were genetically stable.The parent strains and fusion strains were inoculated to experimental animals to compare the virulence. The inactivated propolis vaccines were produced to immunize ducklings. The immune protection rate and antibody levels were determined to evaluate the immune effect of fusion vaccines. The LD50 of parent strains with ZYD5, ZYD8, R-ZYD109, R-ZYD113 and R-ZYD267 were 1.0×106 CFU/0.5mL,2.5×107 CFU/0.5mL,3.1×108 CFU/0.5mL, 2.7×108 CFU/0.5mL and 3.5 X 108 CFU/0.5mL. Virulence test of fusion strains indicated that the virulence of fusion strains was decreased. With the exception of inactivated vaccine, attenuated vaccine could be developed.The virulence protection rate of ZYD bivalent vaccine, R-ZYD109, R-ZYD113 and R-ZYD267 vaccine on ZYD5 was 50% to 60% at first immunization, and 60% to 80% at second immunization. The virulence protection rate of the above four vaccines on ZYD8 was 40% to 50% at first immunization, and 60% to 80% at second immunization. It indicated that the vaccines were all showed certain immune protection effect and two immunizations could obtain greater immune protection rate than one immunization. At 7 d from the time of injection into duckings with ZYD bivalent vaccine and propolis inactivated vaccine of fusions with O2 and O78 serotypes, the antibody level against ZYD8 and ZYD5 began to rise gradually. At 14 d, the antibody level of two immunization group was higher than one immunization group. The antibody level of two immunization group and one immunization group reached the highest level at 28 d, and then they began to slow down. At 56 d, the antibody level of two immunization group and one immunization group was higher than the level of same group at 14 d. Antibody level detection showed that the protection of fusion strain vaccines and the parental bivalent vaccines were no significant differences (P>0.05), it proved that fusion vaccine could substitute parental bivalent vaccine to protect the ducklings.The successfully construction of duck Escherichia coli fusion strains which could simultaneously express two different serotypes are helpful to improve the unit antigen content in vaccine. The method could simplify the process of vaccine production, while saving time and culture material, and enhance the immune effect and economic benefits. This study provides certain experimental basis for the development of new avian colibacillosis vaccine containing multivalent protective antigen.