| E. maxima is recognized as one of the three coccidians that distributed universallyin intensive farms.The current control strategy against coccidiosis in poultry isdominated by prophylactic application of anticoccidial drugs. However, increasinggovernment regulations and bans on the use of coccidiostats, the appearance ofmulti-drug-resistant strains of Eimeria, concerns over drug residues in poultryproducts, the lack of new pipeline products and disadvantages of live vaccinesincluding the potential reversion to virulence, an early reduction in weight gain andvariable efficacy between batches suggest that new approaches are required. Althoughseveral attempts have been made to develop subunit vaccines or DNA vaccines, thereis no available vaccine against coccidiosis. A recent approach using live bacteria ascarriers to deliver and express Eimeria antigens with the long-term aim of controllingavian coccidiosis shows great potential for large-scale control of infectious diseases inthe livestock industry.Mycobacterium bovis BCG, an attenuated M. bovis strain, isparticularly attractive for the delivery of heterologous antigens based on itsremarkable safety record and intrinsic adjuvant properties. Since both M. bovis BCGand Eimeria spp. are intracellular micro-organisms, we rationalized that recombinantBCG would be appropriate for the development of a vaccine against coccidiosis.Apical membrane antigen1(AMA1), secreted by micronemes, appears to be essentialduring the invasion of host cells and is currently founded in Toxoplasma gondii,Neospora caninum,Eimeria tenella, and Eimeria maxima. AMA1in Plasmodium isone of the most promising malaria vaccine candidate and currently a hot research, hasimportant significance in the development of malaria vaccines.Based on the fact that the lack of an ideal genetically engineered vaccine againstcoccidiosis,two rBCG strains pMV261-AMA1and pMV361-AMA1expressingAMA1gene of E. maxima,were constructed and their immune protective effect wereevaluated in this study.Subsequently, safety of rBCG/pMV261-AMA1on chickenwere analyzed. For enhancement of the protective efficacy of recombinant BCG in chickens, rBCG co-expressing AMA1and IFN-γgene were constructed, and itsefficacy against homologous infection in chickens were evaluated. This report shouldprovide a new method for the future development of vaccines against coccidiosis.Cloning and bioinformatics analysis of Eimeria maxima AMA1gene:Basedon the published ORF of E. maxima AMA1gene，a pair of primers were designed andAMA1gene was cloned.Subsequently，the structure and functions of this gene wasanalyzed by bioinformatics method. The recults showed AMA1had an open readingframe(ORF) of1623bp encoded a secreted protein with a26th amino acid residuessignal peptide. while there were one transmembrane peptides，three N-glycosylationsites and34phosphorylation sites. AMA1had eight disulfide bond in tis extracellularregion. AMA1had the secondary structure mainly composed of helix，random coiland had the tertiary structure including ten helixs and14th-pleated sheet.Construction of pMV261-AMA1and pMV361-AMA1expression vectorsand expression in BCG:The AMA1gene segment of E. maxima was generated byPCR with primers designed based on sequences of E. maxima AMA1,pMV261andpMV361. The resulting plasmids pMV261-AMA1and pMV361-AMA1wereelectrotransfected into BCG and expressed successfully in rBCG.Western blottingindicated that the MAA1protein had a good immunogenicity.Protective efficacy of recombinant Mycobacterium bovis BCG expressingapical membrane antigen1against homologous infection:Day-old birds wereimmunized twice with rBCG/pMV261-AMA1, rBCG/pMV361-AMA1or BCG viaoral, intranasal, and subcutaneous routes, then orally challenged with homologous E.maxima sporulated oocysts. Gain of body weight, fecal oocyst output, lesion scores,serum antibody responses, numbers of splenocyte CD4+and CD8+T cells, and gutcytokine transcript levels were assessed as measures of protective immunity. Thesubcutaneous and intranasal routes were superior to the oral route based onaugmented weight gain, reduced fecal oocyst shedding, and decreased intestinallesions. Intranasal rBCG immunization induced a strong humoral and cellularresponse directed against homologous E. maxima infection. This study provides datafor the use of rBCG to develop a prophylactic vaccine against coccidiosis.Safety of rBCG/pMV361-AMA1on chickens:Chickens were immunizedsubcutaneously with rBCG/pMV361-AMA1at different dose. The safety of rBCG on chicken was evaluated based on weight gain, organ coefficient, blood biochemicalparameters and tissue structure of organs.The results showed that no significantdifference were observed in weight gain and organ coefficient between differentgroups(p>0.05). The D-BIL content of group1, group2and group3were highersignificantly compared with that of group4(p <0.05), and there were no difference inother blood biochemical parameters between different groups(p>0.05). High doses ofrecombinant BCG immunization had some impact on the chicken heart,and themyocardial cell presented a large number of lymphocytes infiltration. The liver, spleenand other organs had no changes in the different groups.Construction of pMV261-IFN-γ-Linker-AMA1and expression in BCG:Thevector of pMV261-IFN-γ-Linker-AMA1was constructed with chicken IFN-γgene,E.maxima AMA1gene by SOE-PCR, and was electrotransfected into BCG and selectedby kanamycin. After induction period at45℃, the recombinant proteins wereseparated by SDS-PAGE and Western boltting was done for immunoblot analysis. Aband with about65kDa was detected from rBCG/pMV261-IFN-γ-Linker-AMA1,indicatinging that the fusion protein IFN-γ-AMA1had expressed successfully in BCG.Protective immunity of rBCG/pMV261-IFN-γ-Linker-AMA1against Emaxima challenge:Chickens were immunized intranasally with rBCG/pMV261-IFN-γ-Linker-AMA1,rBCG/pMV261-AMA1and BCG, respectively. The efficacy ofimmunization was evaluated on the basis of oocyst output, cecal lesion scores andbody weight. The results showed that the recombinant BCG immunization had certainimmune protective efficacy against homologous challenge.The immune protectiveeffect with rBCG/pMV261-IFN-γ-Linker-AMA1were superior to that with rBCG/pMV261-AMA1,and the result indicated that IFN-γplayed a role in enhancing theimmune protection effect. |
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