Preparation And Evaluation Of Chicken Coccidiosis Recombinant Bivalent Vaccine

Field chicken coccidiosis infections are mostly mixed infections.In order to better prevent and control the mixed infection of chicken Eimeria maxima and Eimeria tenella,the purpose of this test is to prepare a combination of the common antigen IMP1C of E.tenella and E.maxima and immune adjuvant EDA bivalent subunit vaccine,and evaluate the immuneeffect.Methods:fourrecombinantplasmids,p Et28a-EDA-Et IMP1C,p Et28a-EDA,p Et28a-EDA-Em IMP1C and p Et28a-Em IMP1C-EDA-Et IMP1C were constructed by combining the common antigen IMP1C of E.maxima and the E.tenella with the EDA molecular adjuvant,respectively,for prokaryotic expression.Three intramuscular injections of the expressed proteins were administered to the experimental chickens(12 chickens/group)at a dose of 150μg per chicken,and the serum one week after immunization was used for detection of cytokines such as IL-12,IL-2,IFN-γand Ig G,and the whole blood after immunization was used for detection of CD4~+and CD8~+T lymphocyte subsets.After three immunizations,the test chickens were fed with 2×10~4E.tenella and E.maxima spore-formed oocysts,and the lesions were dissected and observed on the fifth day after challenge and pathological sections were made to collect the challenge The feces of the next 5～10 days were counted for Coccidia oocysts by Mc Master method,the number of deaths was observed and recorded,and the body weight before and after the challenge was weighed.Results:(1)The prokaryotic expression vector was successfully constructed and the corresponding proteins were expressed.The proteins were purified by Ni2+column chromatography and urea dialysis,and the sizes of p Et28a-EDA-Et IMP1C and p Et28a-EDA-Em IMP1C proteins were38k Da,p Et28a-Em IMP1C-EDA-Et IMP1C proteins were 63.8k Da and p Et28a-EDA proteins were 10.8k Da.(2)In terms of cellular immunity,the contents of IL-2 and IFN-γin the serum of the Em IMP1C-EDA-Et IMP1C group were higher than those of the EDA-Et IMP1C group and EDA-Em IMP1C group.The highest number of CD4~+T lymphocytes in the whole blood was in the Em IMP1C-EDA-Et IMP1C group,and the difference of CD8~+T lymphocytes in the protein-immunized group was not significant(P>0.05),but it was significantly higher than that in the EDA adjuvant group and the blank PBS group(P<0.05).The highest serum IL-12 concentration was found in the Em IMP1C-EDA-Et IMP1C group,followed by the EDA-Et IMP1C group and the EDA-Em IMP1C group,which were significantly higher than the EDA adjuvant group and the blank PBS group(P<0.05).(3)In terms of humoral immunity,the Ig G antibody of Em IMP1C-EDA-Et IMP1C group was diluted at a ratio of 1:1600,and the concentration was still higher than that of EDA-Et IMP1C group and EDA-Em IMP1C group.(4)The chickens in the Em IMP1C-EDA-Et IMP1C group showed significant weight gain.The weight gain rate of the E.tenella group was 82.11%and the weight gain rate of the E.maxima group was 85.86%.The intestinal lesions of the E.tenella group were scored 1.19,and the intestinal lesions of the E.maxima group were scored 0.89.The reduction rate of coccidia oocyst excretion in the E.tenella group was 84.01%,and the reduction rate of coccidiosis oocysts in the E.maxima group was79.12%.The E.tenella resistance index was 176 and the E.maxima resistance index was 183.The results showed that the anti-E.tenella and E.maxima bivalent vaccine group can successfully activate humoral and cellular immunity,and produce cross-immunity protection.The effect of resisting E.tenella and E.maxima infection was better than the monovalent vaccine group.