| Avian coccidiosis, caused by intestinal infection with Eimeria spp. invading intraepithelial cells, is spread worldwide and economically the most important parasitic disease of the poultry industry. At present, the controls of Eimeria infections are still mainly based on the anticoccidial drugs and live vaccines. However, these measures have many drawbacks. It is necessary to develop alternative control strategies against coccidiosis. DNA vaccine has been confirmed that it can provoke both humoral and cell-mediated immune responses and prevent various diseases including coccidiosis. It shows a new way to develop vaccine. A few of coccidial protective antigens such as EtMICs, SO7,5401, cSZ-1, 3-1E have been identified and their protective effects have been confirmed. A 37 kDa protein of E. acervulina lactate dehydrogenase of was identified that was partially protective and considered an vaccine antigen candidate. This protein was present at similar levels throughout four different parasitic stages (oocysts, sporozoites, schizonts and merozoites).In this study, E. acervulina LDH gene was amplified by RT-PCR from the first generation schizonts of E. acervulina. The ORF of E. acervulina LDH had 993 nucleotides and was predicted to encode a protein of 37 kDa. After confirm by PCR amplification, endonuclease cleavage and sequence analysis, the results showed that fragment had the open reading frame (ORF) of 993 bp in length and encoded 330 amino acids, which shared nucleotide sequence similarity of 98% with that of E. acervulina LDH in GenBank. By the means of DNA recombination technique, LDH gene was cloned into prokaryotic expression vector pET28a (+) and transformed into BL21 (DE3). The recombinant protein was expressed by inducing with IPTG As the results of SDS-PAGE indicated, the recombinant protein was successfully expressed in E. coli with the relative molecular weight of about 41 kDa. The results of SDS-PAGE of the pellet and supernatant after ultrasonication showed that most of the recombinant protein existed in the inclusion body.By the means of DNA recombination technique, three DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γand pVAX-LDH-IL-2 were constructed. Two-week-old chickens were injected intramuscularly (IM) in leg muscle with recombinant plasmids after they were identified. One week post-inoculation, vaccine injected tissues, non-injected tissues and pVAX1 plasmid injected tissues were collected. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Results indicated that all DNA vaccines could be well transcripted and expressed in injected tissues.Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, recombinant LDH protein and inclusion bodies by leg intramuscular injection, respectively. A booster immunization was given 1 week later. Challenged control group and unchallenged control chickens were injected with sterile TE buffer at the same injection site. pVAX1 plasmid alone was given to chickens as plasmid control. All chickens except the unchallenged control group were challenged orally with 1.2×105 sporulated oocysts of E. acervulina 7 days following second injection. All chickens were euthanized to determine the effects of immunization on the 6th day post-challenge. The efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and anti-coccidial index (ACI). The results indicated the DNA vaccines could protect chickens from E. acervulina infection and pVAX-LDH-IFN-γand pVAX-LDH-IL-2 could induce better protection.Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAXl plasmid alone and sterile water, respectively. On day 7 post-second immunization, spleens were removed from five chickens and lymphocytes were prepared. Lymphocyte proliferation responses were determined by MTT. The results showed that splenocytes from DNA vaccinated chickens displayed significantly greater proliferation compared with plasmid control group chickens and water control group. This indicated that DNA vaccines constructed in this study could stimulate the immune system especially cell-mediated response of chickens.Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. On day 7 and 14 post primary immunization, spleens and cecal tonsils were removed from five chickens of each group. Splenocytes were dually stained with mouse anti-chicken CD3+, CD4+ and CD8+ monoclonal antibody. Percentage of CD3+,CD4+/CD3+ and CD8+/CD3+ were determined by flow cytometric analysis. The results showed that percentage of CD3+T lymphocytes in lymphocytes obtained from spleen and cecal tonsil 7 days post primary injection showed no differences (p>0.05). However, on day 7 following booster, percentages of CD3+T cells of vaccinated groups in both spleen and cecal tonsil were significantly higher than that of control groups (p<0.05). This indicated that DNA vaccines constructed in this study could enhance T lymphocytes response of chickens.Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. On day 7 post-second immunization, spleens and cecal tonsils were removed from chickens and lymphocytes were prepared. Total RNA was extracted from lymphocytes and cDNA was synthesized. The changes of IFN-γ, IL-2, IL-4, TNFSF15, IL-17D, TGF-β4 mRNA were determined by real-time PCR. The relative quantification of cytokine gene mRNA compared with the internal control gene GAPDH was calculated using 2-ΔΔCT method. The results showed that the levels of IFN-γ, IL-2, TNFSF15, IL-17D and TGF-β4 mRNA in spleens and cecal tonsils of chickens were significantly higher following DNA vaccination. This indicated that DNA vaccines constructed in this study could promote the secretion of cytokines.Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. Whole blood from chicks was collected by cardiac puncture at weeks 1,2,3,4,5 and 6 post-second immunization and the serum was collected. The serum IgG antibody levels were determined by ELISA and E. acervulina LDH recombinant protein was used as coating antigen. The results showed that anti-LDH antibody titers were higher in chickens vaccinated with DNA vaccines compared to those of water control and plasmid control chickens during weeks 1-6 post-second immunization (P <0.05). Antibody titers were significantly greater in groups vaccinated with pVAX-LDH-IFN-γand pVAX-LDH-IL-2 than group immunized with pVAX-LDH (P<0.05). Chickens vaccinated with pVAX-LDH-IL-2 developed the highest antibody titer compared with the other two vaccinated groups. Non-specific antibody was detected in water control and plasmid control chickens throughout the experiment. These results indicated that DNA vaccines could induce specific antibody in chickens.The results of immune protective effects and mechanism of DNA vaccines on chickens showed that DNA vaccines could simulate the systemic and local immune system of chickens and induce both cell-mediated and humoral responses to resist coccidiosis. |
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