| The obligate intracellular Gram-negative bacterium Chlamydia psittaci often causes avian chlamydiosis and influenza-like symptoms in humans. However, the commercial subunit C. psittaci vaccine could only provide a partial protection against avian chlamydiosis due to poor cellular immune response. The nonpathogenic herpesvirus of turkeys (HVT), isolated from domestic turkeys, is considered as one of the most potent vectors for polyvalent live vaccines. In this study, an indirect PmpD-N ELISA for detecting C. psittaci antibody and a SYBR Green I real-time PCR for detecting HVT were developed in pioneer experiment, and a recombinant HVT vaccine expressing the N-terminal fragment of PmpD of C. psittaci by modifying the HVT genome within a BAC were generated. Then, the immunogenicity and vaccine efficacy were evaluated in SPF chickens.Development and application of the enzyme-linked immunosorbent assay (ELISA) for detecting antibodies of C. psittaci:First of all, N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) were coated onto plates. Then, optimizaition of antigen, primary antibody and cut-off value were determined. The sensitivity and specificity of the PmpD-N coated ELISA were confirmed to be 97.9% and 100%. Also, a good repeatability was determined, while the concordance was 98.1% compared to that of MOMP ELISA. This assay could be applied to identify the C. psittaci infection, surveillance and vaccine efficacy test.SYBR Green I real-time PCR assays for detecting HVT:The HVT quantify assay targeting the unique sorf 1 gene was established using a real-time PCR approach. Standard curve was optimized with 0.99 correlation index. The melting curve displayed a good specificity. The minimum template concentration of 7×101 copies/ul can be detected and the maximum Ct value was only 1.44% in the repeatability test. HVT and MDV CV1988 were able to be recognized. It was confirmed be an effective, low cost and reliable method for rapid quantification and proliferation of wild HVT and recombinant HVT in vivo and.in vitro.Construction of recombinant HVT expressing PmpD-N:An infectious recombinant derivative of HVT was constructed to express the PmpD-N of C. psittaci with CMV or EF-1α promoter. The recombinant virus was recovered from primary chicken embryo fibroblast (CEF) cells. The expression of obove two recovered viruses were analyzed by immunoblot and immunofluorescence. Also, other charecterization were determined in vitro. Two recombinant viruses with two different promoters (rHVT-CMV-pmpD and rHVT-EF-1α-pmpD) were recovered. The expression of PmpD-N was determined by Western blot and IFA, and the PmpD-N expression mount of rHVT-CMV-pmpD was four times as compared to that of rHVT-EF-1α-pmpD. Under immunofluorescence microscopy, PmpD-N protein was located in the nucleus, cytoplasm and on the cell surface. The rHVT-pmpD-N construct was maintained after 20 passages in vitro culture. Growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT. Macrophages could be activated by rHVT-pmpD-N.Efficacy test of recombinant HVT expressing PmpD:Twenty one-day-old SPF chickens were given subcutaneously with 8000 PFU rHVT-pmpD-N one time, while each group of ten birds with HVT vector or CEF was used as the control group. Later on, ten of the birds with rHVT-pmpD-N were inoculated intraperitoneally with very virulent MDV RB-1B on day 8, and another ten of those were infected intra-tracheally with C. psittaci CB7 to evaluate the protection efficacy on day 36. As for HVT replication, no significant difference was found between wild HVT group and rHVT-pmpD-N group using SYBR Green I real-time PCR assay. SPF chicks inoculated subcutaneously with rHVT-pmapD-N displayed an increasing PmpD-N-specific antibody and a vigorous PmpD-N-specific lymphocyte proliferation response in comparison with HVT vector or CEF cells. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized animals when compared to parental HVT. All immunized chickens were protected upon challenge with very virulent strain of Marek’s Disease virus (MDV) RB-1B, while none of the control group chickens was protected. Post challenge with C. psittaci, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. Our study showed that the recombinant HVT live vaccine may provide a good immunity protection against both very virulent MDV and C. psittaci.In conclusion, the PmpD-N ELISA coated by PmpD-N protein was developed successfully to detect C. psittaci antibody in early infection stage; the SYBR Green I real-time PCR assay was established for detecting and quantifying HVT replication; while the rHVT-pmpD-N vaccine is a promising approach to provide the good protection against very virulent MDV and C. psittaci infection. |
PDF Full Text Request