| Avian coccidiosis is an enteritic disease, which caused by protozoan parasites of Eimeriidae, Eimeria, affects chicken growth and production, and brings about significant economic loss. It was reported that, the application of coccidian vaccine to stimulate host immune responses is the main approaches to control coccidiosis. Nowadays, nucleic acid vaccine (DNA vaccine) has been demonstrated had the ability to improve host cellular immune response and keep long time of immune memory. Furthermore, DNA vaccine, collaborated with cytokine injection, could exert more significant immune response and provide better immune protection. In this paper, two kinds of main immune antigen genes of E. acervulina (3-1E and α-tubulin genes) were selected to construct eukaryotic expression plasmids, which contains fused two immune antigen genes, and immunized chickens with interleukin18(IL-18) collaboratively, and its immune modulate mechanism to E. acervulina infection was researched. The results were as follows:1. The total RNA was extracted from sporozoites of E. acervulina SH strain, the a-tubulin gene was amplified by RT-PCR. The results showed that α-tubulin cDNA nucleotide of SH Strain of E. acervulina contained an open reading frame (ORF) and was1362bp in length. Sequence analysis demonstrated that it had99.4%and99.5%homology with the reference genes of E. acervulina strain (GenBank Accession Number HQ259108) in nucleotide acid and amino acid level, respectively.2. With gene splicing overlap extension technology (SOE PCR), the3-1E and a-tubulin genes was jointed together with a flexible peptide (G4S-G4S-G4S) and constructed successfully the fused gene eukaryotic expression plasmid, PVAX1-3-lE-α-tubulin. In vitro transfect experiment with indirect immunofuorenscence technique, anti-3-1E and a-tubulin polyclonal antibodis were used respectively and specific positive responses were observed. It showed that constructed eukaryotic expression plasmid with fused genes could express these two proteins and kept their biological activities. This provide solid basis for the followed experiment.3. Two hundreds of2-week-old SPF chickens were randomly divided into the following8groups with different treatments:Group1, as the control group, was injected intramuscularly in thigh muscle with PBS without coccidia challenge (B Group); Group2was injected with PBS with coccidia challenge (H Group); Group3immunized with PVAX empty vector (PVAX Group); Group4injected with PVAX-IL-18eukaryotic expression plasmid (IL-18Group); Group5immunized with PVAX-3-1E plasmid (3-1E Group); Group6immunized with PVAX-a-tubulin plasmid (WDB Group); Group7immunized with PVAX-3-1E-α-tubulin plasmid (3W Group); and Group8immunized with PVAX-3-lE-a-tubulin plasmid and injected with PVAX-IL-18collaboratively at the same time (3W+Group). Immunization was boosted with the respective constitute after one week. Seven days after booster immunization, SPF chickens were challenged with5×104E. acervulina sporulated oocysts. The immune protection efficacy for Eimeria infected chickens were observed, and immune protection mechanism was discussed.3. The proliferation response of T and B-lymphocytes of spleen, thymus and peripheral blood showed the similar trends during the experiment period. After second vaccination, T and B lymphocyte proliferative function of chickens in group of IL-18,3-1E and WDB was all very significantly higher (p<0.01) than those in group B, H and PVAX. Furthermore,3W group was remarkably higher than those in group IL-18,3-1E and WDB (p<0.01). And the T lymphocyte proliferative function in group3W+was significantly higher than other groups, including the fused genes and single gene immunization groups (p<0.01). The results of T and B lymphocyte proliferative function showed that IL-18could improve the DNA vaccine immunization and enhance the T and B lymphocyte immunofunction.In the experiment, T lymphocyte subpopulation in blood and immune organs of chickens ware assayed by flow cytometric analysis. The results showed that there were significant increase in the percentage of CD4+T lymphocyte subpopulation in spleen, thymus and peripheral blood after primary vaccination, and then kept the slow increase trends afterwards. Specifically,3W group kept the highest value among all groups. In which, The percentages of CD4+T cell in group IL-18,3-1E and WDB were remarkably higher than those in group B, H, and PVAX (p<0.01或p<0.05) from28days to the end of the experiment. The group3W was significantly higher than those of group IL-18,3-1E and WDB (p<0.01), and the group3W+was remarkably higher than all other groups (p<0.01or p<0.05)The percentage of CD8+T lymphocyte subpopulation in blood and immune organs of chickens demonstrated different changes as those of CD4+T lymphocyte subpopulation. Namely, the significant increase of percentage of CD8+T lymphocyte subpopulation was observed at35days (not28days of CD4+T lymphocyte), and had small modification (even decrease) at42days, and had a small scale of increase at49days. Statistic analysis showed that the percentage of CD8+T lymphocyte subpopulation in blood and immune organs of chickens in group IL-18,3-1E and WDB was significantly higher than that in group B, H and PVAX (p<0.01) post primary vaccination, group3W was remarkably higher than that in group IL-18,3-1E and WDB (p<0.01) and group3W+showed highest value compared with other groups (p<0.01). The results of CD4+and CD8+T lymphocyte subpopulation assay showed that IL-18could enhance the CD4+and CD8+T lymphocyte number and modulate the host immune function. The following assay of peripheral blood specific antibody against3-1E and WDB with ELISA support the above results.The immune protection efficacy of different kinds of eukaryotic plasmids for Eimeria infected chickens was evaluated. The average body weight gain of chickens before challenge in all groups, there was not significantly difference (p>0.05). However, after challenge, The average body weight gain of chickens in group3-1E, WDB,3W and3W+was significantly higher than those in group H and PVAX (p<0.05). Ocysts per gram in feces of infected chickens in group3-1E, WDB,3W and3W+was significant or very significantly lower than that in group H (p<0.05or p<0.01). And the ACI of group3-1E, WDB, IL-18,3W and3W+was respectively166.51,162.97,165.59,180.77,194.32, and was all higher than ACI in group H. The ACI in group3W was more than180, which indicate it could provide significant protection efficacy. |
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