BackgroundCoccidiosis 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.MethodsProkaryotic 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.ResultsThe 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-γ, TGF-β1 and IL-17 were observed more in both the rEmSAG protein and pVAX1-SAG inoculated groups than those of negative controls (P < 0.05).ConclusionsOur findings suggest that EmSAG is capable of eliciting a moderate immune protection and could be used as an effective vaccine candidate against E. maxima.
Eimeria tenella is a protozoan parasite endemic in chickens and is one of the causative agents of avian coccidiosis. The aim of this research was to determine if poly (D, L-lactide-co-glycolide) ( PLGA ) nanoparticles carrying recombinant TA4 protein of E. tenella ( rEtTA4 ) could improve the level of protective immunity against E. tenella challenge. Recombinant TA4 protein was expressed and purified. Poly (D, L-lactide-co-glycolide) loaded with rEtTA4 ( PLGA-rEtTA4 ) nanoparticles was prepared and was delivered to 2-week-old layer chickens via intramuscular inoculation. Chickens injected with PBS and PLGA nanoparticles were served as control groups. The rEtTA4 and PLGA-rEtTA4 nanoparticles induced changes of serum cytokines, IgY levels, and T lymphocytes subpopulation, and the protective efficacy against E. tenella challenge was evaluated. Results showed that both rEtTA4 and PLGA-rEtTA4 vaccination groups induced significantly higher levels of specific EtTA4 IgY antibody and IL-17 and higher proportion of CD8 + T lymphocytes. However, no significant differences were observed in the proportion of CD4 + T lymphocytes compared with the PBS control. Chickens immunized with rEtTA4 and PLGA-rEtTA4 prominently increased the BW gains and decreased oocyst output compared with chickens immunized with PBS and PLGA after oral challenge with E. tenella . Poly (D, L-lactide-co-glycolide) encapsulated rEtTA4 nanoparticles–immunized chickens significantly induced higher levels of interferon gamma, IL-6, and IL-17 and a little bit higher proportion of CD8 + T lymphocytes compared with rEtTA4 subunit vaccine–immunized chickens. Thus, PLGA encapsulated rEtTA4 nanoparticles appeared to have great potential to enhance the immune response and improved the protective efficacy against E. tenella infection. Our results provided available protective subunit vaccine rEtTA4 and PLGA loaded with rEtTA4 nanoparticles against coccidiosis and suggested that PLGA nanoparticles could be an effective adjuvant to enhance the protective efficacy of rEtTA4 subunit vaccine.
Eimeria acervulina is one of seven Eimeria spp. that can infect chicken duodenal epithelial cells. Eimeria microneme protein 3 (MIC3) plays a vital role in the invasion of host epithelial tissue by the parasite. In this study, we found that chicken (Gallus gallus) ubiquitin conjugating enzyme E2F (UBE2F) could bind to the MIC3 protein of E. acervulina (EaMIC3), as screened using the yeast two-hybrid system, and that it might be the putative receptor protein of EaMIC3. The UBE2F gene was cloned from chicken duodenal epithelial cells. The recombinant protein of UBE2F (rUBE2F) was expressed in E. coli and the reactogenicity of rUBE2F was analyzed by Western blot. Gene sequencing revealed that the opening reading frame (ORF) of UBE2F was 558 base pairs and encoded a protein of 186 amino acids with a molecular weight of 20.46 kDa. The predicted UBE2F protein did not contain signal peptides or a transmembrane region, but had multiple O-glycosylation and phosphorylation sites. A phylogenetic analysis showed that the chicken UBE2F protein is closely related to those of quail and pigeon (Coturnix japonica and Columba livia). A sporozoite invasion-blocking assay showed that antisera against rUBE2F significantly inhibited the invasion of E. acervulina sporozoites in vitro. Animal experiments indicated that the antisera could significantly enhance average body weight gains and reduce mean lesion scores following a challenge with E. acervulina. These results therefore imply that the chicken UBE2F protein might be the target receptor molecule of EaMIC3 that is involved in E. acervulina invasion.
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