Study On Immunogenicity Of Eimeria Brunetti Microneme-2 Protein And Apical Membrane Antigen-1

Coccidiosis is a major parasitic disease responsible for substantial economic losses in the commercial poultry due to mortality,diarrhea,weight loss,morbidity,poor feed conversion,mal-absorption,inefficient,feed utilization,impaired growth rate and reduced egg yield.Eimeria brunetti is one of the species of Eimeria that causes hemorrhagic intestinal coccidiosis in young poultry.Lesions are limited to lower small intestine and leads to bloody diarrhea,poor feed conversion,weight loss,and mortality in severe cases.Until now,only a few genes of this coccidian and its immunogenicity have been reported.The research for new antigens and evaluation of its protective efficiency against challenge with E.brunetti will open the development of a new approach of vaccines against this parasite.In this research,Eimeria brunetti was isolated by single oocyst technique and identified using Polymerase Chain Reaction(PCR)assay.Then genomic mRNA was extracted from sporulated oocysts for the synthesis of cDNA.E.brunetti microneme-2 protein(EbMIC2)and E.brunetti apical membrane antigen-1(EbAMA1)gene specific primers were designed with Oligo Primer Premier 5.0 software base on E.brunetti MIC2(accession number AB723700)and AMA1(accession number AB723701)of GenBank,respectively.876 bp of EbMIC2 and 1656 bp of EbAMAl specific fragments were amplified by PCR and then recovered,purified and ligated into pMD19-T cloning vector.The recombinant plasmids were identified by PCR,restriction endonuclease enzymes analysis and sequencing.Sequence analysis showed that the ORF of EbMIC2 gene was 876 base pairs(bp)in length,encoded 291 amino acids with 32 kDa,and the ORF of EbAMA1 gene was 1656 bp in length,encoded 551 amino acids with 60 kDa.The homology of the nucleotide sequence and deduced amino acids sequence of the EbMIC2 gene were 99.43 and 98.63%,respectively and the homology of the nucleotide sequence and deduced amino acids sequence of the EbAMAl gene were 99.52 and 98.55%,respectively,compared with the published one on GenBank.The ORFs of EbMIC2 and EbAMA1 were inserted into the EcoRI and Hind?enzymes sites of the prokaryotic expression vector pET-28a(+)and were transformed into E.coli BL21(DE3)strain.Expression of the recombinant was induced by 0.8M 1×IPTG in E.coli,and the expressed products were analyzed by SDS-PAGE and Western blot.The results indicated that the recombinant EbMIC2 and EbAMA1 proteins were recognized strongly by serum from naturally infected chicken with E.brunetti.Rat rcEbMIC2 and rcEbAMAl antisera respectively bound to bands of about 36 kDa and 64 kDa in the somatic extract of E.brunetti sporozoites.The ORFs of EbMIC2 and EbAMA1 were also inserted into eukaryotic expression vector pVAX1 for producing recombinant plasmids pVAX1-EbMIC2 and pVAX1-EbAMA1.The expression results of aim genes in vivo were detected by RT-PCR and Western blot.Experimental chickens were injected intramuscularly with 100?g(concentration 1?g/?l)plasmids per chicken at 14 and 21 age days.The challenged control group was injected with sterile TE buffer at the same injection site and pVAX1 vector was used as a control.Unchallenged control group was not immunized and challenged.One week after the second immunization,serum from 10 chickens from each group was collected for the analysis of antibody and cytokine levels,the other chickens in each group were inoculated orally with 1×105 sporulated oocysts of E.brunetti except the unchallenged control group.Seven days after challenge,all chickens were weighed and slaughtered for ileum collection.Immunization was analyzed on the base of oocyst decrease ratio,lesion score,anti-coccidial index(ACI)and body weight gain.The results revealed that recombinant plasmids pVAX1-EbMIC2 and pVAX1-EbAMA1 decreased lesions score of intestine,oocyst output and increased oocyst decrease ratio and body weight.The ACIs of plasmid pVAX1-EbMIC2 and pVAXl-EbAMA1 were respectively 179 and 178,showing good protection,while the ACIs of EbMIC2 and EbAMA1 recombinant proteins were 159 and 160,respectively,showing partial protection.Serum from chickens immunized with plasmid pVAX1-EbMIC2 and pVAX1-EbAMA1 showed significant high level of IgG antibody compared with control groups(p<0.05),concurrently IgG antibody of chicken immunized with recombinant EbMIC2 and EbAMA1 proteins were significantly induced.On the other hand,IFN-?,IL-10,IL-17 and TGF-? of serum level from chickens immunized with plasmids pVAX1-EbMIC2,pVAX1-EbAMA1 and recommbinant proteins showed significant high level compared with control groups(p<0.05).However,no significant difference of IL-4 was observed in chickens immunized with plasmids pVAX1-EbMIC2;pVAXl-EbAMAl and recombinant EbMIC2;EbAMA1 proteins as compared with the control groups(p<0.05).These results suggested that EbMIC2 and EbAMAl possessed good immunogenicity and could become a candidate E.brunetti antigen for the development of prophylactic vaccine against chicken coccidiosis.We also performed the evaluation of passive immunization of the 2 proteins against chicken coccidiosis.Coccidia-free chickens were injected intramuscularly two times with recombinant plasmids pVAX1-EbMIC2 and pVAX1-EbAMA1,respectively.Seven days after the last booster injection,the chicks were bled to collect the serum and then IgG titer was determined using ELISA assay.The IgG titer of the serum from chicken immunized with recombinant plasmids pVAX1-EbMIC2 and pVAX1-EbAMA1 all showed more than 10,000.Experimental chicken groups were respectively administered intravenously(?)into the wing vein with the above prepared immune serum at 21 days of age with different doses rate of 30,50 and 100?l.While,chickens in the unchallenged control group and challenged control group were injected with sterile TE buffer.After injection at the same day,all chickens except the unchallenged control group were inoculated orally with 1 × 105 sporulated oocysts of E.brunetti.Seven days after challenge,all chickens were weighed and slaughtered for ileum collection to determine several parameters same to that in active immunization above.The results indicated that anti-sera collected from chickens immunized with recombinant plasmids pVAX1-EbMIC2 and pVAX1-EbAMA1 were effective to protect chickens from coccidiosis depending on the doses of antiserum used.