Preparation And Potency Assessment Of Yolk Antibody Against Bovine Pathogenic Escherichia Coli

Bovine colibacillosis is one of the most important causes of death caused by bacterial infection in cattle.The disease can be transmitted horizontally and vertically,and occurs frequently in newborn calves.Futherore,this bacterium can be co-infected with other pathogens,which causes huge economic losses to the cattle industry.At present,there is no commercial vaccine in the market,and antibiotics are mainly used for clinical treatment,but the resulting drug resistance and drug residues run counter to the national policy of green and healthy breeding.Based on the requirements of biological treatment products for bovine colibacillosis,this study intends to prepare specific yolk antibodies by isolating the bacteria from clinical samples and analyzing their morphological and biochemical characteristics,then immunize laying hens with immunogens to implore specific yolk antibodies.At last,experimental animal models are established to evaluate the biological efficacy of the prepared yolk antibodies.The conclusions are as follows:1.Strains were isolated according to the routine bacterial isolation and identification methods in laboratory.Ordinary medium and special medium for Escherichia coli（E.coli）were used for bacterial culture,and morphological characteristics of solates were observed.The results showed that the isolated were milky colonies on LB solid medium with regular,opaque,smooth and moist borders.On Mac Conkey Agar medium,the isolates were pale pink,opaque and dull.On eosameilan medium,the isolates were metallic gray green colonies.On trypsinogen medium,the isolates appeared as opaque,moist,smooth and white colonies.The short rod-like and blunt ends were observed by Gram staining microscopy,which was consistent with the Gram staining characteristics of E.coli.After biochemical identification of the isolates,it was found that the isolates can decompose sugar and produce gas and acid,which were in accordance with the biochemical characteristics of E.coli.The predicted bands were amplified by 16S r RNA universal primer and specific primer for Fim D gene,and further sequencing showed that the isolates were bovine E.coli.2.Based on the isolate,the inactivated bovine E.oli vaccine was prepared to immunize laying hens.The whole immunization procedure was carried out for four times.Eggs during immunization were collected and yolks were seperate.Based on the previous exploration of our research group,water dilution-ammonium sulfate secondary precipitation method was used as the initial yolk antibody purification procedure,then followed by dialysis purification.The result of SDS-PAGE gel electrophoresis showed that the size of heavy chain was 70k Da and the size of light chain was 25k Da,which were consistent with the expected sizes.The average protein content was 20.21 mg·m L^-1,and the maximum was 30.683 mg·m L^-1 in the whole immunization period.The protein content curve showed that the overall protein content showed an increasing trend,with fluctuations in the middle,and reached the maximum level in the fourth immunization.3.In order to further evaluate the biological efficacy of bovine E.coli yolk antibody prepared in this study,a mouse model was established to evaluate the protective efficacy of the yolk antibody against E.coli infection using prevention/challenge and challenge/treatment tests.The results of prevention test showed that the protection rate of yolk antibody in mice reached 100%.The treatment test showed that the cure rate of yolk antibody in mice reached 80%.These result indicated that yolk antibody can play an effective role in the control of bovine E.coli.