Preparation Of Inactivated Contagious Ecthyma Virus Vaccine And Evaluation Of Its Immune Effect

Objective:Contagious ecthyma virus mainly affects sheep and goats.Papules,blisters,pustules,ulcers and thick scabs can be formed on the skin and mucous membrane of the mouth of the diseased lamb.Due to open wounds,infected animals are very easy to be secondary infection,which has caused great harm to the sheep industry in China.Vaccination is the main means of prevention and control of the disease,but there is no commercially available vaccine for the prevention of contagious ecthyma.The purpose of this study was to prepare inactivated contagious ecthyma vaccine using lamb testicular cells and to evaluate the immune effect of the vaccine by observing and counting the morbidity of pregnant sheep and lambs after immunization.Methods:Contagious ecthyma virus was proliferated from the testicular cells of lambs.The venom was concentrated by tangential ultrafiltration membrane and the TCID₅₀was calculated by reed-Muench method.Fluorescent quantitative polymerase chain reaction external standard absolute quantitative detection method was established to detect the gene copy number of infectious impetigo virus in the virus venom to prove the concentration effect.The venom was inactivated withβ-propiolactone.The inactivated vaccine of infectious contagious ecthyma virus was prepared by mixing the venom with 206 adjuvant in a ratio of1:1.Immunological test was conducted in sheep farms with frequent infectious contagious ecthyma virus and the incidence of natural infection was observed.Results:（1）The primary cells of the testis of lambs were successfully isolated and cultured.Cell boundaries were blurred 24 h after inoculation.After inoculation 48 h,the color of nucleus was deepened.After inoculation 72 h,a large number of cells were shed and cell aggregation was like grape.（2）The tangential flow ultrafiltration membrane system was successfully used to concentrate the virus venom.The virus titer of the original solution was 10^6.3TCID₅₀/0.1 m L,and the virus titer of the concentrated solution was 10^7.5TCID₅₀/0.1 m L.（3）Fluorescent quantitative PCR external standard absolute quantitative detection method was successfully established.The virus gene copy number of the original virus solution,the concentrated virus solution of 5 times,the concentrated virus solution of 10 times and the filtered solution were detected.The results were 6.6×10⁴copies/μL,1.3×10⁵copies/μL,3.9×10⁵copies/μL and 8.0×10²copies/μL,respectively,which proved that tangential flow ultrafiltration membrane system had good concentration effect.（4）The safe inactivated vaccine of contagious ecthyma virus was successfully prepared,and the incidence of natural infection was observed after immunization.The overall incidence was 28%in the immunized group and 100%in the non-immunized group.Conclusion:Tangential flow ultrafiltration membrane has a good effect on 10 times concentration of virus stock,and has a significant effect on retaining virus particles and increasing virus concentration.Contagious ecthyma virus fluorescence quantitative PCR pustules external standard absolute quantitative detection method can be simple and rapid quantitative detection of the virus gene copy number.Contagious ecthyma virus inactivated vaccine had a significant protective effect on lambs in the experimental sheep farm.