| In vivo,the avian Eimeria exhibit a high degree of site specificity in chickens.The specific sites of E.acervulina,E.praecox,E.maxima,E.necatrix,E.brunetti,E.mitis and E.tenella are the duodenum,upper,middle,lower of small intestine and caecum of the chicken,respectively.The proteins on the surface of sporozoites have been suggested as ligands reacting with receptors on the epithelial cells of intestines,which mediates the site specificity in the invasion process of host cells by Eimeria parasite.However,the ligands of the parasite and the receptors of host remain largely undetermined.In this study,E.mitis was used as a model to determine the role of microneme proteins(MICs)in the site specificity for invasion at lower of small intestine.The result of this study is of great significance to elucidate the specific molecular mechanisms of site specificity in E.mitis,and provides candidate antigens for the developing new type vaccine against E.mitis.1.Identification of Eimeria mitis sporozoite proteins interacting with epithelial cells of lower intestine by LC-MS/MSIn this study,the soluble protein of E.mitis was incubated with epithelial cells of lower intestine.Co-immunoprecipitation(Co-IP)and LC-MS/MS were performed to identify sporozoite proteins interacting with epithelial cells of lower intestine.The protein sequences identified were analyzed by Gene Ontology(GO).The results showed that 12 identified proteins were related to invasion,including microneme protein,14-3-3 protein,rhoptry neck protein,SAG family member,calmodulin-containing protein and so on.A total of 91 proteins were successfully annotated,of which 56 proteins were associated with binding activity(61.54%of the total proteins),and 22 proteins had a catalytic activity(24.18%of the total protein).The identified proteins might play a role in the invasion of host cells by E.mitis.These findings provide basis to further understanding of parasite-host interaction in the invasion of host cells by E.mitis,and better elucidating of pathogenesis of E.mitis.2.The molecular characterization and protective efficacy of EmiMIC3 in chickensThe present study was performed to analyze the molecular characterization and protective efficacy of microneme 3 of Eimeria mitis(EmiMIC3).EmiMIC3 gene was cloned from sporozoites of Emitis and its MARs(microneme adhesive repeats domain)were predicted.Recombinant EmiMIC3(rEmiMIC3)was expressed and purified,and then was analyzed by western blot with anti-E.mitis chicken serum.Meanwhile,native EmiMIC3 from sporozoites was analycollaboration zed by anti-rEmiMIC3 rat serum.Immunofluorescence assay was performed to determine expressions of EmiMIC3 in E.mitis sporozoites and merozoites.The rEmiMIC3-induced changes of T lymphocytes subpopulation were determined using flow cytometry.Serum cytokines and IgG levels in immunized chickens were determined by ELISA,respectively.Finally,immunization and challenge experiment was conducted to evaluate the protective efficacy of rEmiMIC3.The results revealed that the deduced open reading frame(ORF)of EmiMIC3 is composed of 1145 amino acids,possessing 9 MARs.EmiMIC3 gene was submitted to GenBank(accession number:MG888670).EmiMIC3 is expressed in sporozoites and merozoites,located at the apex of E.mitis sporozoite.Western blot assay revealed that the rEmiMIC3 was recognized by serum from chicken infected with E.mitis,meanwhile native EmiMIC3 from sporozoites was also recognized by rat serum against rEmiMIC3.Following vaccination with rEmiMIC3,higher lever of IFN-y,IL-10,IL-17 and TGF-Pwere induced compared to the controls.Higher proportion of CD4+and CD8+T lymphocytes,and higher level of IgG antibody were also induced compared to controls.Vaccination with rEmiMIC3 prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection.Our result not only enriches protective candidate antigen of E.mitis,but also provides available E.mitis protective antigen for developing multivalent anticoccidial vaccines against the mixed infection of coccidia in clinical coccidiosis.3.The molecular characterization and protective efficacy of EmiMIC2 in chickensThe present study was performed to analyze the molecular characterization and protective efficacy of microneme 2 of Eimeria mitis(EmiMIC2).EmiMIC2 gene was cloned from sporozoites of E.mitis.Recombinant EmiMIC2(rEmiMIC2)was expressed and purified.And then was analyzed by western blot with anti-E.mitis chicken serum.Meanwhile,native EmiMIC2 from sporozoites was analyzed by anti-rEmiMIC2 rat serum.Immunofluorescence assay was performed to determine expressions of EmiMIC2 in E.mitis sporozoites and merozoites.The rEmiMIC2-induced changes of T lymphocytes subpopulation were determined using flow cytometry.Serum cytokines and IgG levels in immunized chickens were determined by ELISA,respectively.Finally,immunization and challenge experiment was conducted to evaluate the protective efficacy of rEmiMIC2.The results revealed that the ORF of EmiMIC2 is composed of 724 amino acids,possessing 1 vWFA superfamily domain and 4 TSP1 domains.EmiMIC2 is expressed in sporozoites and merozoites,located at the apex of E.nzitis sporozoite.Western blot assay revealed that the rEmiMIC2 was recognized by serum from chicken infected with E.mitis,meanwhile native EmiMIC2 from sporozoites was also recognized by rat serum against rEmiMIC2.Following vaccination with rEmiMIC2,higher lever of IL-2,IL-4,IL-10 and TGF-? were induced compared to the controls.Higher proportion of CD4+ and CD8+T lymphocytes,and higher level of IgG antibody were also induced compared to controls.Vaccination with rEmiMIC2 prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection.4.The molecular characterization and protective efficacy of EmiEtmic-2/7h in chickensThe present study was performed to analyze the molecular characterization and protective efficacy of etmic-2/7h of Einzeria mitis(EmiEtmic-2/7h).EmiEtmic-2/7h gene was cloned from sporozoites of E.mitis.Recombinant EmiEtmic-2/7h(rEmiEtmic-2/7h)was expressed and purified,and then was analyzed by western blot with anti-E.mitis chicken serum.Meanwhile,native EmiEtmic-2/7h from sporozoites was analyzed by anti-rEmiEtmic-2/7h rat serum.Immunofluorescence assay was performed to determine expressions of EmiEtmic-2/7h in E.mitis sporozoites and merozoites.The rEmiEtmic-2/7h-induced changes of T lymphocytes subpopulation were determined using flow cytometry.Serum cytokines and IgG levels in immunized chickens were determined by ELISA,respectively.Finally,immunization and challenge experiment was conducted to evaluate the protective efficacy of rEmiEtmic-2/7h.The results revealed that the ORF of EmiEtmic-2/7h is composed of 295 amino acids,possessing 1 Etmic-2 superfamily domains.EmiEtmic-2/7h is expressed in sporozoites and merozoites,located at the apex of E.mitis sporozoite.Western blot assay revealed that the rEmiEtmic-2/7h was recognized by serum from chicken infected with E.mitis,meanwhile native EmiEtmic-2/7h from sporozoites was also recognized by rat serum against rEmiEtmic-2/7h.Following vaccination with rEmiEtmic-2/7h,higher lever of IFN-?,IL-2,IL-4 and TGF-? were induced compared to the controls.Higher proportion of CD4+and CD8+T lymphocytes,and higher level of IgG antibody were also induced compared to controls.Vaccination with rEmiEtmic-2/7h prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection.5.The molecular characterization and protective efficacy of EmiAMAl in chickensThe present study was performed to analyze the molecular characterization and protective efficacy of apical membrane antigen 1 of Eimeria mitis(EmiAMA1).EmiAMAl gene was cloned from sporozoites of E.mitis.Recombinant EmiAMA1(rEmiAMA1)was expressed and purified,and then was analyzed by western blot with anti-E.mitis chicken serum.Immunofluorescence assay was performed to determine expressions of EmiAMAl in E.mitis sporozoites and merozoites.The rEmiAMAl-induced changes of T lymphocytes subpopulation were determined using flow cytometry.Serum cytokines and IgG levels in immunized chickens were determined by ELISA,respectively.Finally,immunization and challenge experiment was conducted to evaluate the protective efficacy of rEmiAMA1.The results revealed that the ORF of EmiAMA1 is composed of 90 amino acids,possessing 1 AMA-1 superfamily domains.EmiAMA1 is expressed in sporozoites and merozoites,located at the apex of E.mitis sporozoite.Western blot assay revealed that the rEmiAMAl was recognized by serum from chicken infected with E.mitis.Following vaccination with rEmiAMA1,higher lever of IFN-y,IL-2,IL-4,IL-10,IL-17and TGF-P were induced compared to the controls.Higher proportion of CD4+and CD8+T lymphocytes,and higher level of IgG antibody were also induced compared to controls.Vaccination with rEmiAMA1 prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection.6.The role of MICs and the domains in the process of invasion by E.mitisMICs play crucial role in the early stage of invasion of host cells by Eimeria parasites.In previous study,we found that EmiMIC3 is composed of 9 copies of Microneme adhesive repeat(MAR).In order to determine the role of EmiMIC3 and its EmiMARs during the invasion of host cells by E.mitis,we cloned and expressed EmiMIC3 and EmiMARs proteins respectively.Immunofluorescence analysis showed that EmiMIC3 bound to the epithelial cells from lower intestine of the chicken.Cell ELISA analysis showed that EmiMARs bound to the epithelial cells from lower intestine of the chicken,and the binding capacity of EmiMAR4 was the strongest.Immunohistochemical analysis showed EmiMIC3 specifically bound to lower intestine of the chicken,not the other parts of intestine(duodenum,upper,middle,and caecum).EmiMAR4 from EmiMIC3 bound to lower intestine,not the other parts of intestine.However,EmiMIC2,EmiEtmic-2/7 and EmiAMA1 did not bind to any parts of the intestine.In addition,in vitro and in vivo experiments showed that rEmiMIC3 and anti-rEmiMIC3 serum could significantly block the invasion of E.mitis sporozoites.Therefore,EmiMIC3 and its EmiMARs play important roles in the process of invasion by E.mitis sporozoite and the site specificity of E.mitis. |
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