| Coccidiosis is a major worldwide veterinary health problem that is caused by an intracellular protozoan parasite,Eimeria.Clinical coccidiosis causes a reduction in weight gain, poor feed-conversion, high levels of mortality and often can make the difference between profit and loss to a farmer raising a flock of broiler chickens for the market.Currently, seven species of Eimeria are universally accepted. Among them, Eimeria maxima, together with E.acervulina and E.tenella, is the most prevalent specie and responsible for the most economic damage. Different Eimeria species parasitize different parts of chicken intestine and this phenomenon displayed highly specific. Typically,E.acervulina majorly parasitize duodenum, E.maxima majorly parasitize jejunum, E.tenella majorly parasitize cecum and E.brunetti majorly parasitize rectum. Previous research showed that E.tenella microneme 3 (EtMIC3) was the key molecular that guide E.tenella to parasitize cecum specifically, however, the key molecular that guide E.maxima to parasitize jejunum specifically remains unknown. Thus, further research on the interaction of E.maxima sporozoites and the corresponding host cells could help understanding the E.maxima invasion process and generating strategies to control coccidiosis caused by E.maxima. In the present research, the E.maxima sporozoites soluble proteins that could bind to chicken epithelial cells were identified and four E.maxima microneme proteins were cloned and the protective immunity were evaluated. Our research could help screen new vaccine candidates to prevent coccidiosis caused by E.maxima.1. Proteomic analysis of Eimeria maxima sporozoite proteins interaction with jejunal epithelial cells by shotgun LC-MS/MSIn the present study, the proteins of chicken jejunal epithelial cells cultured with soluble proteins of E.maxima sporozoites were analyzed by Western blot. The co-Immunoprecipitation (co-IP) assay and shotgun LC-MS/MS were employed for proteomic analysis. The results showed that four protein bands (70, 90, 95 and 130 kDa) of jejunal epithelial cells cultured with the soluble proteins of E.maxima sporozoites were detected by the sera of rats immunized with the soluble proteins of E.maxima sporozoites.The co-IP dilutions were further analyzed by the shotgun LC-MS/MS. Total 35 proteins of E.maxima were identified with more than 2 unique peptide count from Eimeria maxima protein database using MASCOT engine searches. The 35 identified proteins were annotated according to Gene Ontology Annotation in terms of molecular function,biological process and cellular localization. The results revealed that 22 (62.86%) of the 35 annotated peptides occurred in binding activity and 15 (42.86%) occurred in catalytic activity. Our findings provided an insight into the interaction between E.maxima and the corresponding host cells and is of great significance in the understanding of molecular mechanisms of E.maxima invasion.2. Molecular characterization and the protective immunity evaluation of Eimeria maxima microneme 3 (EmMIC3) geneThe current study was to evaluate the protective immunity of Eimeria maxima microneme 3 (EmMIC3) gene against experimental E. maxima infection. The full sequence of EmMIC3 gene was obtained using the method rapid-amplification of cDNA ends(RACE). The complete open reading frame (ORF) of EmMIC3 was 1986 base pairs (bp)encoding a protein of 661 amino acids. The ORF of EmMIC3 was inserted into the prokaryotic recombinant plasmid pET-32a(+)-MIC3 and was successfully expressed in Escherichia coli (E.coli) BL21 (DE3). The immune protective effects and the passive immune protective effects of rat anti-rEmMIC3 sera against E.maxima challenge were evaluated respectively. The challenge experiment results showed that immune with rEmMIC3 could obviously alleviate jejunum lesions, body weight loss, increase oocyst decrease ratio and provide ACI of 186.02. The anti-EmMIC3 antibody titers of rEmMIC3 immune group was significantly higher throughout the experiment as compared to the control groups (P < 0.05). Serum from chickens immunized with rEmMIC3 displayed significantly high levels of IL-2 and IL-4 compared to those of negative controls (P < 0.05).The CCK-8 assay results showed that the splenocytes from different concentrations of rEmMIC3 groups all displayed significantly greater proliferation compared with the controls (P<0.05). The results of passive immune protection against E.maxima infection showed that passively immune with the three different dilutions of rat anti-MIC3 sera could obviously alleviate jejunum lesions, body weight loss, increase oocyst decrease ratio and provide ACIs of more than 183, respectively. All these results suggested that immunization with EmMIC3 was effective in imparting outstanding protection against E.maxima challenge and EmMIC3 could be an effective antigen candidate for the development of new vaccines against E. maxima.3. Cloning, expression and the protective immunity evaluation of Eimeria maxima microneme 2 geneIn the present study, the E. maxima microneme 2 (EmMIC2) gene was cloned. The open reading frame (ORF) of EmMIC2 was 888 base pairs (bp) encoding a protein of 295 amino acids. Then EmMIC2 was subcloned into pET-32a(+) and pVAX1,respectively.RT-PCR and western blot assays were both employed to check the in vivo transcription and expression of the eukaryotic recombinant plasmid pVAX1-MIC2 and the results revealed that the target gene was successfully transcribed and expressed in vivo. The immune protective effects against E.maxima challenge of rEmMIC2 and pVAX1-MIC2 were evaluated. The challenge experiment results showed that both rEmMIC2 and pVAX1-MIC2 could obviously alleviate jejunum lesions, body weight loss,increase oocyst decrease ratio and provide ACIs of more than 165, The anti-EmMIC2 antibody titers of both rEmMIC2 protein and pVAX1-MIC2 greups were significantly higher as compared to PBS and pVAX1 control (P < 0.05). The splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with the controls (P < 0.05). Serum from chickens immunized with pVAX1-MIC2 and rEmMIC2 protein displayed significantly high levels of IL-2, IFN-γ, IL-10, IL-17, TGF-β and IL-4 (P < 0.05) compared to those of negative controls. All the above results suggested that immunization with EmMIC2 was effective in imparting partial protection against E.maxima challenge and it could be an effective antigen candidate for the development of new vaccines against E.maxima.4. Molecular characterization and the protective immunity evaluation of Eimeria maxima microneme 7 geneIn the present study, the E. maxima microneme 7 (EmMIC7) gene was cloned. The open reading frame (ORF) of EmMIC7 was 519 base pairs (bp) encoding a protein of 172 amino acids. Then EmMIC7 gene was subcloned into pET-32a(+) and pVAX1, respectively.RT-PCR and western blot assays were both employed to check the in vivo transcription and expression of the eukaryotic recombinant plasmid pVAX1-MIC7 and the results revealed that the target gene was successfully transcribed and expressed in vivo. The immune protective effects against E.maxima challenge of rEmMIC7 and pVAX1-MIC7 were evaluated. The challenge experiment results showed that both rEmMIC7 and pVAX1-MIC7 could obviously alleviate jejunum lesions, body weight loss, increase oocyst decrease ratio and provide ACIs of more than 167. The anti-EmMIC7 antibody titers of both rEmMIC7 protein and pVAX1-MIC7 groups were significantly higher as compared to PBS and pVAX1 control (P < 0.05). The splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with the controls (P < 0.05). Serum from chickens immunized with pVAX1-MIC7 and rEmMIC7 protein displayed significantly high levels of IL-2, IFN-y, IL-10, IL-17, TGF-(3 and IL-4 (P < 0.05) compared to those of negative controls. All the above results suggested that immunization with EmMIC7 was effective in imparting partial protection against E.maxima challenge and it could be an effective antigen candidate for the development of new vaccines against E.maxima.5. Molecular characterization and the protective immunity evaluation of Eimeria maxima apical membrane antigen 1 (EmAMAl) geneThe current study was conducted to evaluate the protective immunity of Eimeria maxima apical membrane antigen 1 (EmAMA1) gene against experimental E. maxima infection. The EmAMA1 gene was cloned and the open reading frame (ORF) of EmAMA1 was 1620 bp encoding a protein of 539 amino acids. The sequence analysis results showed that an apical membrane antigen-1 superfamily domain was contained (No. 66-539 amino acids). The domain (EmAMA1FD) was then amplified and subcloned into pET-32a(+) and pVAX1, respectively. The prokaryotic recombinant plasmid pET-32a(+)-AMA1FD was successfully expressed in Escherichia coli (E.coli) BL21 (DE3). RT-PCR and western blot assays were both employed to check the in vivo transcription and expression of the eukaryotic recombinant plasmid pVAX1- AMA1FD and the results revealed that the target gene was successfully transcribed and expressed in vivo. The immune protective effects against E.maxima challenge of rAMA1FD and pVAX1-AMA1FD were evaluated. The challenge experiment results showed that both rAMA1FD and pVAX1-AMA1FD could obviously alleviate jejunum lesions, body weight loss, increase oocyst decrease ratio and provide ACIs of 176.68 and 180.35, respectively. The results showed that the anti-EmAMA1FD antibody titers of both rAMA1FD and pVAX1-AMA1FD groups were significantly higher as compared to the control groups (P < 0.05). The CCK-8 assay results showed that the splenocytes from different concentrations of rEmAMA1FD groups displayed significantly greater proliferation compared with the controls (P<0.05). Serum from chickens immunized with rAMAlFD and pVAX1-AMA1FD displayed significantly high levels of IL-2, IL-4 and TGF-β (P<0.05) compared to those of negative controls. All these results suggested that immunization with AMA1FD was effective in imparting partial protection against E.maxima challenge and EmAMA1 could be an effective antigen candidate for the development of new vaccines against E.maxima.6. The binding capacity of four recombinant MIC proteins to different parts of chickens intestineThe present research was to evaluate the binding capacity of the four previously generated recombinant MIC proteins to different parts of chicken intestine. The frozen sections of chicken duodenum,jejunum,cecum and rectum were prepared and freshly used to incubate with the purified rEmMIC3, rEmMIC2, rEmMIC7 and rEmAMAlFD respectively. Immunofluorescent assay was employed to evaluate the binding capacity of the four recombinant MIC proteins to different parts of chicken intestine. The results revealed that rEmMIC3 could only bind to chicken jejunum, but not duodenum, cecum or rectum, however rEmMIC2, rEmMIC7 and rEmAMAlFD could not bind to any parts of chicken intestine. Our results indicated that receptor specific to EmMIC3 might lie on the jejunal epithelial cells and EmMIC3 might be the key molecular that guide E.maxima to parasitize jejunum only. Thus, EmMIC3 might play a key role in the process of E.maxima invasion. |
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