Evaluation Of Immunoprotective Efficaies Of Five Antigens Of Eimeria Maxima In Chckens

Avian coccidiosis is an intestinal protozoan disease caused by various Eimeria species with simultaneous infection in chickens.E.maxima is one of the most common species in commercial broilers farm.Coccidiosis seriously reduces egge production and body weight gains,inducing huge economic loss worldwide.As conventional control strategies still rely severely on chemoprophylaxis,live vaccines or attenuated vaccine.However,the concerns of drug residues,drug resistance,and high cost of live vaccine direct our attentions to new generation vaccine,such as recombinant vaccine and DNA vaccine.Consequently,it is extremely urgent to seek new methods for the prevention and control of coccidiosis.Immunoprophylaxis can effectively overcome the shortcomings of drug prophylaxis.In particular,novel vaccines(DNA vaccines and subunit vaccines)have attracted extensive attention.Chicken coccidia are obligate intracellular parasite and cellular immunity of the body plays a major role in the defense against coccidia invasion.Th1 cytokines play a vital role in cellular immunity.Studies have shown that coccidiosis can inhibit the expression of Th1 cytokines in infected chicken.In our previous study,we also found that the expression of Th1 cytokines was inhibited after E.maxima infection in chickens.Four inhibitory antigens of Th1 cytokines were screened from the cDNA library of E.maxima sporozoites,namely EmSAG,EmHPSP-1,EmHPSP-2 and EmHPSP-3.Meanwhile,a stimulating antigen of Thl cytokines EmARM-? was also screened.This study was performed to further evaluate the immunogenicity of these five antigens and their immunoprotective efficacies against E.maxima infection.1 Expression of five recombinant antigens and detection of EmHPSP-3 expression in sporozoitesThe gene of Th1 cytokine inhibitory antigen EmHPSP-3 was amplified by PCR.The sequence analysis of EmHPSP-3 by BLAST revealed that the open reading frame contained 1536 bp and could encode 512 amino acids.The prokaryotic expression plasmid pET-32a-EmHPSP-3 was constructed by ligating EmbHPSP-3 into a prokaryotic expression vector.For pET-32a-EmHPSP-3 and 4 laboratory-preserved prokaryotic expression plasmids(pET-32a-EmHPSP-1,pET-32a-EmHPSP-2,pET-32a-EmSAG and pET-28a-EmARM-?)expression,purification and recombinant proteins of these five antigens were obtained.Immunoblotting assay indicated that these five recombinant proteins were recognized by the artificially infected chicken serum of E.maxima.The expression of EmHPSP-3 in the sporozoite stage was detected by immunofluorescence assay,and it was found to be mainly expressed on the surface of E.maxima sporozoites.2 Detection of transcription of eukaryotic expression plasmids of five antigens in chickensThe eukaryotic expression plasmid pVAXl-EmHPSP-3 was constructed.Two-week-old chickens were injected with 100?g of recombinant plasmid pVAX1-EmHPSP-3,pVAX1-EMSAG,pVAXl-EmHPSP-1,pVAX1-EmHPSP-2 and pVAX1-EmARM-?.The vector control group was injected with pVAX1.One week later,RNA was extracted from thigh muscular region at the injection site,non-injection site and the vector control group.The transcription of these five genes at the injection site was detected by reverse transcription-PCR,and the target gene was found to be successfully transcribed in immunized chickens.3 Evaluation of immune response and protective efficacies induced by the 5 antigens in forms of eukaryotic recombinant plasmid and recombinant proteinAt 2 weeks of age,chickens in recombinant protein group were immunized with 200?g of recombinant porteins EmHPSP-1,EmHPSP-2,EmHPSP-3,EmSAG and EmARM-?,chickens in the eukaryotic recombinant plasmid group were immunized with 100?g of recombinant plasmid p VAX1-EmHPSP-1,pVAXl-EmHPSP-2,pVAXl-EmHPSP-3,pVAX1-EMSAG and pVAX1-EmARM-? respectively,meanwhile,pVAX1 empty vector,pET-32a vector protein and PBS control group was given in experiment design.One week later,a booster immunization was given follwing the same way as the primary immunization.On the 7th day after each immunization,blood samples of the chickens were collected for specific IgG levels detection using indirect ELISA.Splenic lymphocytes of the chickens were collected for T cell subsets detection using flow cytometry.The results showed that the specific IgG level and the proportion of CD4+/CD3+T cells and CD8+/CD3+T cells from the immmunized group were significantly increased compared with the control groups.The IgG levels were significantly increased by the booster immunization.The results suggested that the 5 antigen could effectively induce immune reponse in forms of eukaryotic recombinant plasmid and recombinant protein.Seven days post booster immunization,all chickens were orally infected with E.maxima sporulated oocysts with a dose of 1×105 except for the nonchallenged control group.Meanwhile,the body weight of all chickens was recorded.Six days after challenge infection,all the chickens were weighted and sacrificed to observe enteric leisions induced by challenged infection.The body weight gain,oocyst production and anticoccidial index(ACI)of each group were calculated to evaluate protective efficacies of the 5 E.maxima antigens.The result showed that immunization with the 5 E.maxima antigens in forms of eukaryotic recombinant plasmid and recombinant protein significantly alleviated enteric leision and body weight loss,reduced oocyst output and induced ACIs of more than 160 compared with the control groups.This result suggested that the 5 E.maxima antigens of EmSAG,EmHPSP-1,EmHPSP-2,EmHPSP-3 and EmARM-? were able to induce partial protective efficacies against E.maxima and were the candiate antigens for developing DNA or recombinant protein vaccine against infection by E.maxima.