Preliminary Study On Biological Characteristics And Inactivated Vaccine Of Escherichia Coli From Mink In Shandong Province

In recent years,with the rapid development of Shandong mink breeding industry,the number of mink raised in Shandong has exceeded the sum of other provinces.However,with the increase in the degree of intensification of the mink farming industry,the incidence of mink continues to rise and has become an important factor restricting the development of the mink industry.At present,mink colibacillosis has become a major problem in the mink breeding industry.The widespread abuse of antibacterial drugs makes antibiotics less effective in treating colibacillosis.Therefore,it is especially important to develop effective Escherichia coli vaccine to prevent the disease of Escherichia coli in minks.In this study,56 strains of Escherichia coli were identified by plate lineation,colony morphology observation,microscope observation and automatic biochemical identification apparatus with preserved strains from Shandong Animal Disease Prevention and Control Center.Then,11 kinds of serotype O were screened by 16S r RNA sequencing,and the serotype was determined by glass plate agglutination test.The drug sensitivity test and pathogenicity test were carried out on the dominant serotype strains,and two strains with high virulence were screened for vaccine research.Half lethal dose(LD₅₀)was measured for dominant serotype O91 and O103 strains,respectively,to obtain the challenge dose for the final vaccine efficacy test.Serotype results showed that 56 strains of Escherichia coli belong to 11 serotypes,namely O103,O113,O91,O121,O8,O26,O39,O80,O16,O111,O45.Among them,O91and O103 are the dominant serotypes,accounting for 44.6%of the finalized serotypes.The results of drug susceptibility experiments showed that the dominant serotype Escherichia coli is highly resistant to penicillin antibiotics,with a resistance rate of over 88%,a resistance rate of 52%to cephalosporins,and a resistance rate of 44%to aztreonam.The resistance rates of gentamicin and tobramycin were 32%and 40%,respectively,the resistance rates to ciprofloxacin and levofloxacin were 64%and 56%,and the resistance rates to compound trimethoprim were 52%.Sensitive to penem drugs.According to the results of preliminary screening of pathogenicity,H1807021a and H200113Fd were selected for vaccine research.The LD₅₀of H1807021a and H200113Fd were 5.82×10⁷CFU、5.14×10⁷CFU.Compared with the previous reports,the pathogenicity of E.coli from mink was stronger.In this study,the candidate strains were cultured in vitro,and it was found that the antigen concentration reached the highest when cultured at 37°C for 12 hours;inactivated with 0.4%formaldehyde solution for 24 hours,the inactivation was complete.Comparing different immunization doses,it is found that the effect of immunizing mice with 4×10⁸CFU is the best;through the comparison of the immunization effects of one immunization and two immunizations,the optimal number of immunizations is finally obtained as two.The immunological efficacy test was carried out twice with the inoculation dose of 4×10⁸CFU.Compared with the control group,the protection rate of the vaccine group reached 100%,which proved that the preparation of the bivalent inactivated vaccine of Escherichia coli mink was initially successful.In this study,the biochemical characteristics,serotypes,drug resistance and pathogenicity of E.coli isolated from mink in Shandong Province were preliminarily understood,and bivalent inactivated vaccine of E.coli isolated from mink was prepared,which provided important theoretical basis and technical support for the prevention and treatment of E.coli disease in mink.