Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.
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ABSTRACT: Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines.With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed.Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2?×?106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32?×?107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite.Our results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.
<h4>Background</h4>Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of ...[more]
Project description:BACKGROUND:Avian coccidiosis is often caused by co-infection with several species of Eimeria worldwide. Developing a multivalent vaccine with an antigen common to multiple Eimeria species is a promising strategy for controlling clinical common co-infection of Eimeria. In the previous study, 14-3-3 was identified as one of the immunogenic common antigen in E. tenella, E. acervulina and E. maxima. The aim of the present study was to evaluate the immunogenicity and protective efficacy of Ea14-3-3 in the form of DNA vaccine against infection with three species of Eimeria both individually and simultaneously. RESULTS:After vaccination with pVAX-Ea14-3-3, the Ea14-3-3 gene was transcribed and expressed in the injected muscles. Vaccination with pVAX-Ea14-3-3 significantly increased the proportion of CD4+ and CD8+ T lymphocytes and produced a strong IgY response in immunized chickens. Similarly, pVAX-Ea14-3-3 stimulated the chicken's splenocytes to produce high levels of Th1-type (IFN-?, IL-2) and Th2-type (IL-4) cytokines. The vaccine-induced immune response was responsible to increase weight gain, decreased the oocyst output, and alleviated enteric lesions significantly in immunized chickens as compared to control group, in addition to induce moderate anti-coccidial index (ACI). CONCLUSION:These results indicate that Ea14-3-3 is highly immunogenic and capable to induce significant immune responses. Furthermore, Ea14-3-3 antigen can provide effective protection against infection with Eimeria tenella, Eimeria acervulina, Eimeria maxima both individually and in combination with three Eimeria species. Significant outcomes of our study provide an effective candidate antigen for developing a multivalent Eimeria vaccine against mixed infection with various Eimeria species under natural conditions.
Project description:Clinical chicken coccidiosis is mostly caused by simultaneous infection of several Eimeria species, and host immunity against Eimeria is species-specific. It is urgent to identify common immunodominant antigen of Eimeria for developing multivalent anticoccidial vaccines. In this study, sporozoite proteins of Eimeria tenella, Eimeria acervulina and Eimeria maxima were analyzed by two-dimensional electrophoresis (2DE). Western bot analysis was performed on the yielded 2DE gel using antisera of E. tenella E. acervulina and E. maxima respectively. Next, the detected immunodominant spots were identified by comparing the data from MALDI-TOF-MS/MS with available databases. Finally, Eimeria common antigens were identified by comparing amino acid sequence between the three Eimeria species. The results showed that analysis by 2DE of sporozoite proteins detected 629, 626 and 632 protein spots from E. tenella, E. acervulina and E. maxima respectively. Western bot analysis revealed 50 (E. tenella), 64 (E. acervulina) and 57 (E. maxima) immunodominant spots from the sporozoite 2DE gels of the three Eimeria species. The immunodominant spots were identified as 33, 27 and 25 immunodominant antigens of E. tenella, E. acervulina and E. maxima respectively. Fifty-four immunodominant proteins were identified as 18 ortholog proteins among the three Eimeria species. Finally, 5 of the 18 ortholog proteins were identified as common immunodominant antigens including elongation factor 2 (EF-2), 14-3-3 protein, ubiquitin-conjugating enzyme domain-containing protein (UCE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In conclusion, our results not only provide Eimeria sporozoite immunodominant antigen map and additional immunodominant antigens, but also common immunodominant antigens for developing multivalent anticoccidial vaccines.
Project description:Coccidiosis is one of the most serious diseases of livestock and birds in the world. Vaccination with live-parasite anticoccidial vaccines with genetic manipulation improving the immunogenicity of vaccine strains would be the best means for controlling coccidiosis in breeder and layer stocks, even in fast-growing broilers. Profilin from apicomplexan parasites is the first molecularly defined ligand for Toll-like receptor 11 (TLR11) and TLR12 in mice and is a potential molecular adjuvant. Here, we constructed a transgenic Eimeria tenella line (Et-EmPro) expressing the profilin of Eimeria maxima, the most immunogenic species of chicken coccidia, and evaluated the adjuvant effects of EmPro on the immunogenicity of E. tenella We found that immunization with the transgenic Eimeria parasites, compared with the wild type, elicited greater parasite antigen-specific cell-mediated immunity, characterized by increased numbers of interferon gamma (IFN-γ)-secreting lymphocytes. The transgenic parasite also induced better protective immunity against E. tenella challenge than the wild type. In addition, the diversity of the fecal microbiome of the birds immunized with the transgenic parasite differed from that of the microbiome of the wild-type-immunized birds, indicating interactions of Eimeria with the gut microbiome of chickens. Our results showing enhanced immunogenicity of E. tenella by use of EmPro as a molecular adjuvant derived from the most immunogenic affinis species represent a large step forward in the development of the next generation of coccidiosis vaccines using Eimeria as a vaccine platform expressing molecular adjuvants and potentially other pathogen antigens against not only coccidiosis but also other infectious diseases.
Project description:BACKGROUND:Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that infects chickens. In the present research, the surface antigen gene of E. maxima (EmSAG) was cloned, and the ability of EmSAG to stimulate protection against E. maxima was evaluated. METHODS:Prokaryotic and eukaryotic plasmids expressing EmSAG were constructed. The EmSAG transcription and expression in vivo was performed based on the RT-PCR and immunoblot analysis. The expression of EmSAG in sporozoites and merozoites was detected through immunofluorescence analyses. The immune protection was assessed based on challenge experiments. Flow cytometry assays were used to determine the T cell subpopulations. The serum antibody and cytokine levels were evaluated by ELISA. RESULTS:The open reading frame (ORF) of EmSAG gene contained 645 bp encoding 214 amino acid residues. The immunoblot and RT-PCR analyses indicated that the EmSAG gene were transcribed and expressed in vivo. The EmSAG proteins were expressed in sporozoite and merozoite stages of E. maxima by the immunofluorescence assay. Challenge experiments showed that both pVAX1-SAG and the recombinant EmSAG (rEmSAG) proteins were successful in alleviating jejunal lesions, decreasing loss of body weight and the oocyst ratio. Additionally, these experiments possessed anticoccidial indices (ACI) of more than 170. Higher percentages of CD4+ and CD8+ T cells were detected in both EmSAG-inoculated birds than those of the negative control groups (P?<?0.05). The EmSAG-specific antibody concentrations of both the rEmSAG and pVAX1-EmSAG groups were much higher than those of the negative controls (P?<?0.05). Higher concentrations of IL-4, IFN-?, TGF-?1 and IL-17 were observed more in both the rEmSAG protein and pVAX1-SAG inoculated groups than those of negative controls (P?<?0.05). CONCLUSIONS:Our findings suggest that EmSAG is capable of eliciting a moderate immune protection and could be used as an effective vaccine candidate against E. maxima.
Project description:The recent cloning of chicken genes coding for interleukins, chemokines, and other proteins involved in immune regulation and inflammation allowed us to analyze their expression during infection with Eimeria. The expression levels of different genes in jejunal and cecal RNA extracts isolated from uninfected chickens and chickens infected with Eimeria maxima or E. tenella were measured using a precise quantitative reverse transcription-PCR technique. Seven days after E. tenella infection, expression of the proinflammatory cytokine interleukin-1beta (IL-1beta) mRNA was increased 80-fold. Among the chemokines analyzed, the CC chemokines K203 (200-fold) and macrophage inflammatory factor 1beta (MIP-1beta) (80-fold) were strongly upregulated in the infected ceca, but the CXC chemokines IL-8 and K60 were not. However, the CXC chemokines were expressed at very high levels in uninfected cecal extracts. The levels of gamma interferon (IFN-gamma) (300-fold), inducible nitric oxide synthase (iNOS) (200-fold), and myelomonocytic growth factor (MGF) (50-fold) were also highly upregulated during infection with E. tenella, whereas cyclooxygenase 2 showed a more modest (13-fold) increase. The genes upregulated during E. tenella infection were generally also upregulated during E. maxima infection but at a lower magnitude except for those encoding MIP-1beta and MGF. For these two cytokines, no significant change in expression levels was observed after E. maxima infection. CD3+ intraepithelial lymphocytes may participate in the IFN-gamma upregulation observed after infection, since both recruitment and upregulation of the IFN-gamma mRNA level were observed in the infected jejunal mucosa. Moreover, in the chicken macrophage cell line HD-11, CC chemokines, MGF, IL-1beta, and iNOS were inducible by IFN-gamma, suggesting that macrophages may be one of the cell populations involved in the upregulation of these cytokines observed in vivo during infection with Eimeria.
Project description:IntroductionAvian coccidiosis, caused by apicomplexan protozoa belonging to the Eimeria genus, is considered one of the most important diseases in the intensive poultry industry worldwide. Due to the shortcomings of live anticoccidial vaccines and drugs, the development of novel anticoccidial vaccines is increasingly urgent.MethodsEimeria maxima rhomboid-like protein 1 (EmROM1), an invasion-related molecule, was selected as a candidate antigen to evaluate its protective efficacy against E. maxima in chickens. Firstly, the prokaryotic recombinant plasmid pET-32a-EmROM1 was constructed to prepare EmROM1 recombinant protein (rEmROM1), which was used as a subunit vaccine. The eukaryotic recombinant plasmid pVAX1.0-EmROM1 (pEmROM1) was constructed as a DNA vaccine. Subsequently, 2-week-old chicks were separately vaccinated with the rEmROM1 and pEmROM1 twice every 7 days. One week post the booster vaccination, induced cellular immune responses were determined by evaluating the mRNA level of cytokines including IL-2, IFN-γ, IL-4, IL-10, TGF-β, IL-17, and TNFSF15, as well as the percentages of CD4+ and CD8+ T cells from spleens of vaccinated chickens. Specific serum antibody level in the vaccinated chickens was determined to assess induced humoral immune responses. Finally, the protective efficacy of EmROM1 was evaluated by a vaccination-challenge trial.ResultsEmROM1 vaccination significantly upregulated the cytokine transcription levels and CD4+/CD8+ T cell percentages in vaccinated chickens compared with control groups, and also significantly increased the levels of serum-specific antibodies in vaccinated chickens. The animal trial showed that EmROM1 vaccination significantly reduced oocyst shedding, enteric lesions, and weight loss of infected birds compared with the controls. The anticoccidial index (ACI) from the rEmROM-vaccination group and pEmROM1-vaccination group were 174.11 and 163.37, respectively, showing moderate protection against E. maxima infection.DiscussionEmROM1 is an effective candidate antigen for developing DNA or subunit vaccines against avian coccidiosis.
Project description:BACKGROUND: Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. RESULTS: Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s) encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. CONCLUSION: Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health.
Project description:Abiotic stress significantly impacts growth and yield of crop plants. It is imperative for crop improvement to discover and utilize stress-tolerant functional genes. In this study, genes responding to abiotic stresses, such as freezing, salt and osmotic stress, were screened from a cDNA yeast library that was constructed from the drought- and heat-tolerant wheat variety Hanxuan 10. After screening for surviving clones we isolated 7,249, 4,313 and 4,469 raw sequences, corresponding to 4,695, 2,641 and 2,771 genes following each treatment. Venn diagrams revealed 377 overlapping genes. GO analysis suggested that these genes were mainly involved in the metabolic and stress signal pathways. KEGG pathway enrichment analysis indicated that the isolated genes predominantly belonged to pathways concerning energy and metabolism. Overlapping gene TaPR-1-1 within the pathogenesis-related (PR) protein family was selected for detailed characterization. Although previous studies had shown that PR genes function during pathogen attack, our results demonstrated that TaPR-1-1 expression was also induced by freezing, salinity, and osmotic stresses. Overexpression in yeast and Arabidopsis showed that TaPR-1-1 conferred tolerance to these stresses. We concluded that screening cDNA yeast libraries following abiotic stress is an efficient way to identify stress-tolerance genes.
