Studies On Humoral Immune Responses And Dynamic Distribution Of Duck Plague Virus UL24DNA Vaccine In The Vaccinated Ducklings

This study focuses on the proliferation and purification of duck plague virus (DPV), the preparation and purification of rabbit anti-DPV polyclonal serum, the expression of DPV UL24DNA vaccine and its molecular adjuvant LTB gene in COS-7cells, the humoral immune responses induced by these DNA vaccines and dynamic distribution of DPV UL24DNA vaccines and attenuated DPV vaccine in tissues and organs of ducklings. The main immunization procedure was as follows:①each duckling intramuscularly received at a total dose of200μg of naked DNA vaccine pVAX-UL24, pVAX-LTB-UL24or pVAX-UL24plus pVAX-LTB dissolved in sterilized normal saline;②in oral groups, each duckling in group A1, A2or A3was inoculated with1ml SL7207(pVAX-LTB-UL24) at doses of1011,1010and109CFU respectively and duckling in group A4or A5was inoculated intragastrically with SL7207(pVAX-UL24) or SL7207pVAX-UL24plus SL7207pVAX-LTB at1010CFU;③in control groups, each ducklings received1010CFU SL7207(pVAX1),0.5mL PBS or0.2mL attenuated DPV vaccine respectively. Sera samples, spleen, thymus, bursa of Fabricus (BF), Harderian gland, lung, pancreas, liver, duodenum, jejunum, ileum, appendix and rectum were collected. Indirect ELISA method was established to detect specific anti-DPV IgG in sera. And dynamic distribution of DPV UL24DNA vaccines and attenuated DPV vaccine were detected in the tissues and organs of vaccinated ducklings. The results were showed below:1. The proliferation and purification of DPV in duck embryo fibroblast monolayer cells (DEF) and the preparation and purification of rabbit anti-DPV polyclonal serum: the DEF cells were infected by DPV with notable cytopathic effect, such as being rounded, shrinking, falling off and the formation of plaque. Normally, the cytopathic effect appears at36h post-infection and at48h post-infection,80%of DEF cells showed cytopathic effect. The purification of DPV was conducted by polyethylene glycol6000(PEG6000) precipitation method and sucrose ultracentrifugation. After the rabbits were immunized with purified DPV antigens, the rabbit anti-DPV polyclonal serum was obtained and the titer of it was1:32, which was carried out by double diffusion test. The DPV IgG was extracted by the caprylic acid-ammonium sulfate method.2. The expression of DPV UL24DNA vaccines and its molecular adjuvant LTB gene in COS-7cells:COS-7cells were transiently transfected with the recombinant plasmids pVAX-UL24, pVAX-LTB-UL24and pVAX1using cationic liposome.48h later, indirect immunofluorescence experiments demonstrated that specific fluorescent granules dispersed in nucleus and cytoplasm in the cells transfected with pVAX-UL24or pVAX-LTB-UL24, whereas no fluorescence was detected in the cells infected with plasmid pVAX1, indicating that UL24was expressed in cell. Similarly, at48h after COS-7cells were transiently transfected with the recombinant plasmids pVAX-LTB, pVAX-LTB-UL24and pVAX1using cationic liposome, total cellular RNA was extracted and the transcription of LTB gene was detected using RT-PCR, which was about430bp in length.3. The establishment of indirect ELISA to detect specific anti-DPV IgG in sera and the study of humoral immune responses induced by DPV UL24DNA vaccines:after optimization, the concentration of covered antigen was0.25μg/ml, the dilution of the examined serum was1:160, temperature and time of incubated the examined samples were37℃1h. The enzyme linked antibody dilution is1:2000and temperature and time of incubated it were also37℃1h. DPV UL24DNA vaccines can induce significantly higher humoral immune responses either immunized orally or intramuscularly than PBS or SL7207(pVAX1)(P<0.05), but ducklings in oral groups showed better humoral immune responses than those in intramuscularly groups (P<0.05), Taken together, DPV DNA vaccines carried by attenuated Salmonella Typhimurium SL7207can enhance humoral immune responses and LTB gene was a superior adjuvant. Compared with attenuated DPV vaccine, humoral immune responses were lower induced by DPV UL24DNA vaccines.4. The establishment of indirect immunohistochemistry method and indirect immunofluorescence assay:after optimization, the dilution of both primary and secondary antibody were1:100, temperature and time of primary antibody incubated the examined samples were4℃overnight, temperature and time of secondary antibody incubated it were37℃1h.The established IHC and IFA were sensitive, which can correctly detect the distribution of DPV UL24antigen in tissues and organs of immunized ducklings.5. The dynamic distribution of oral DPV UL24DNA vaccines in tissues and organs of immunized ducklings:in oral groups at1w post-immunization, there were a large number of brown particles in the lung, but the number of positive cells gradually declined. The positive signals were observed in the spleen, Harderian gland, BF and the intestinal tract at1w post-immunization and the number of positive cells was reached the peak at3w post-immunization, followed by constant decrease at6w post-immunization. The liver and pancreas had a quite few positive cells during the whole experiment.6. The dynamic distribution of DPV UL24DNA vaccines after intramuscular injection in tissues and organs of immunized ducklings:there were positive signals in spleen, Harderian gland, BF and liver at1w post-immunization and the strongest positive signals appeared at3w post-immunization. A large number of brown particles were found in the intestinal tract at3w post-immunization, while there were a few positive cells in the thymus and pancreas during the whole experiment. The number of positive cell in all organs was decreased at6w post-immunization, but remaining detectable at10w.1. The dynamic distribution of attenuated DPV vaccine in tissues and organs of immunized ducklings:DPV viral antigen can be detected in all organs of ducklings subcutaneously immunized with attenuated DPV vaccine at1w post-immunization. The positive signals became stronger, were strongest at2w and3w post-immunization and remained detectable at10w post-immunization. Compared with DPV UL24DNA vaccines, the positive cells which distributed more widely and had longer duration, in organs of ducklings immunized with attenuated DPV vaccine were more.