Construction Of Fusion Strains Of Two Different Serotypes Of Duck Salmonella And Research On The Immunogenicity

With the rapid development of poultry breeding,the epidemic situation of Salmonella is also more serious,which has caused great economic losses to the actual production.Therefore,it is very important to effectively control Salmonella pneumoniae.However,there are more than 2 500 serotypes of Salmonella,and the dominant serotype is different in each place.Multivalent vaccine had low unit titre,and its manufactur process workload,the fusion strain can improve this issue.The purpose of is to construct the fusion strains,which can also express duck Salmonella from two different serotypes,and initial research on the immunological fusion strains,select suitable fusion strains for the preparation of vaccine.The study first saved 19 strains isolated from different sichuan areas of salmonella serotype identification and drug sensitive test.Gentamycin and Carbenicillin as drug resistance tag induced salmonella S10(6,7:1,v:1,5,GenR,Cars)and S16(4,5,12:i:1,2,Gens,CarR).By drawing the growth curve of S10 and S16,choose that the best time for the preparation of protoplasts was 12?14 h.Through the MIC of S10 and S16,choose that the ultimate density of gentamicin was 16 ug/mL,and the Carbenicillin was 64 ug/mL.The structure of the fusion strain was made by using lysozyme to remove the cell wall to prepare protoplast,and the fusion agent PEG was used to prepare the fusion.The primary screening of the fusion strain was the use of hypertonic selective medium.Fusion of screening from main serotype,biochemical characteristics,colony morphology and bacteria and double PCR method,two typical fusion strains were obtained.The study with two parental strains and two strains of fusion strains were compared the virulence of infected Ducklings.Prepared propolis inactivated vaccines with two parental strains and two strains of fusion strains was used to immunize ducklings.By comparison three kinds of vaccine protection rate and antibody level,the final evaluation of the immune effect of fusion vaccine,final evaluation of immune effect of fusion vaccine.The LD50 of two parent strain S10,S16 and fusion R-SS1,R-SS2 were 1×107CFU?2.2×107CFU?2.1×106CFU?1×107CFU.The protective rate of two parental strains bivalent vaccine,R-SS1 vaccine and R-SS2 vaccine against S10 was 60%to 70%,and the protective rate against S16 was 50%to 70%,which indicated that the three kinds of propolis inactivated vaccines had the protective effect.The indirect ELISA method was used to detect the antibody level.The ducklings were injected to the two parental strains bivalent vaccine,R-SSi vaccine and R-SS2 vaccine,measured the immune antibody levels were higher than matched group,the difference was remarkable(P<0.01).Starting from 3 days after the first immunization,antibody levels against S10 and Si6 began to rise.At 14 d,the antibody level of two immunization group was higher than one immunization group.At 14 to 21 d,the level of antibody and the growth rate of the 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.At 28 to 42 d,the antibody level of two immunization group and one immunization group began to slow down.The antibody level result display showed that two parental strains bivalent vaccine and fusion vaccines were no significant difference.Proved that the fusion vaccines can replaced the two parental strains bivalent vaccine.This study provide a certain experimental basis for the development of the new vaccine against avian influenza with multivalent protective antigen.