Preparation And Potency Assessment Of Yolk Antibodies Against Bovine Viral Diarrhea Virus And Bovine Enterovirus

Bovine Viral Diarrhea Virus（BVDV）and Bovine Enterovirus（BEV）are two important pathogens that cause viral infections in cattle.The two viruses have been found to be clinically mixed,causing more serious economic losses in husbandary.Based on the current need for the prevention and treatment of these two viruses,it is necessary to explore a safe,effective and environmentally friendly therapeutic biological product.Immunoglobulin of yolk（Ig Y）,as a low cost,high yield and stable biological agent,has been used in the prevention and control of some viral diseases.In this study,we propose to use BVDV HN-1577 and BEV HN-1817 strains to prepare BVDV-BEV diphasic vaccine and yolk antibody,then a mouse infection mode was constructed to evaluate the biological effectiveness of the yolk antibody.The yolk antibodies provided technical support for the prevention and control of BVDV and BEV.METHODS:The lyophilized BVDV and BEV were reviveded and cultured with bovine kidney cells（Madin-Darby bovine kidney,MDBK）respectively;and the two viruses were inactivated with formaldehyde solution at appropriate concentrations.The inactivated viruses were mixed in equal amounts with Freund’s adjuvant,and the BVDV-BEV diphasic vaccine were prepared by manual emulsification using a three-way valve and a 5 m L syringe.A combination of intramuscular and subcutaneous neck injections was used to immunize laying hens for four times with the prepared vaccine.SDS-PAGE gel electrophoresis was used to assess the purity of crudely extracted yolk antibodies.The yolk antibodies were obtained by secondary crude extraction of the collected eggs in using saturated ammonium sulfate solution;then the protein content of the yolk antibodies was determined according to the BCA kit instructions;finally,A BALB/c mouse model was constructed to evaluate biological efficacy of Ig Y as a preventive and therapeutic agent against the BVDV and BEV viruses.The single dose of the challege inoculation was 10 ID₅₀,and the single dose of Ig Y injection was 0.2 m L.Result:The virus titers of BVDV HN-1577 and BEV HN-1817 were 1×10^4.3TCID₅₀/m L and 1×10^8.4TCID₅₀/m L repectively on MDBK cells.The BVDV inactivation conditions were in 0.2%formaldehyde and shaking at 37℃（100 r/min）for 24 h.The BEV inactivation conditions were in 0.25%formaldehyde and shaking at37℃（100 r/min）for 60 h.The prepared diphasic inactivated vaccines were tested according to the relevant requirements of the Chinese Veterinary Pharmacopoeia.The results of protein concentration of yolk antibody measured by BCA kit showed that the protein content in yolk antibody gradually increase during the four immunization procedures and reached at the highest value of 80.74 mg-m L-1 after the last immunization.The molecular sizes of heavy and light chains were 67 k Da and 26 k Da,respectively.According to the results of protective efficacy of Ig Y,in BALB/c mice infection model the anti-BVDV-BEV yolk antibody achieved 100%preventive protective in mice;the therapeutic rate reached 86.7%.These results proved that the Ig Y has excellent protetive and therapeutic effects against BVDV and BEV in BALB/c mice.Conclusion:The Ig Y prepared in this study has good preventive and therapeutic effects of BVDV and BEV in mice,which provides a strong technical guarantee for the prevention and treatment of the disease.