Studies On Antigenic Expression Phase And Distribution Regularity Of DPV GC Gene Vaccine In The Vaccinated Ducklings

In this paper, the study concentrates on the purification of duck plague virus (DPV), the detecting methods of DPV gC antigens, antigenic expression phase and distribution regularity of DPV gC gene vaccine, and proliferation and distribution regularity of attenuated DPV vaccine.①purification and microstructure morphology observation of DPV;②development and application of an indirect immunohistochemical method (IHC) and an indirect immunofluorescence assay (IFA) for detection of DPV gC antigens in paraffine sections;③the 20-day-old ducklings were immunized with different doses of pcDNA-DPV-gC gene vaccine via gene gun bombardment (6μg,3μg and 1μg) and intramuscular injection (200μg,100μg and 50μg), respectively;④the 20-day-old duckings were immunized with 100μg liposome/DNA gene vaccine and chitosan/DNA gene vaccine via intramuscular injection, oral administration and nasal administration, respectively;⑤In the control groups, the 20-day-old ducklings were subcutaneouly immunized 0,5 mL attenuated DPV vaccine, and other groups were intramuscularly inoculated with 0.5 mL sterile physiologic saline and 100μg blank plasmid. At intervals of 4h,12h,1d,3d,5d,7d,2w,4w,6w and 10w post-vaccination (p.v.),two ducklings were randomly euthanatized from the immunization group.After euthanasia, heart, liver, spleen, lung, kidney, pancreas, brain, esophagus, thymus, bursa of Fabricus (BF), Harderian gland, duodenum, caecum, rectum and injection site were collected. The control groups were processed in the same way.Development and application of the IHC and IFA were detected antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the vaccinated ducklings, and the same methods were prepared to detect proliferation and distribution regularity of attenuated DPV vaccine. The results were showed as follow:1.The purification of DPV could be operated by conventional differential centrifugation and discontinuous sucrose-density gradient centrifugation. Many purification virus were examined by negative staining microscopy under transmission electron microscope(TEM). Some definite structures of mature DPV particles could be clearly observed with typical morphous structure of Herpervirus. Rabbit anti-DPV polyclonal serum was obtained from the rabbits immunized with purified DPV antigens, and the titers of specific antibody were more than 1:32 in the final injection. The DPV IgG was extracted by saturated ammonium sulfate method and purified through High-Q chromatograghy.2.Development and application of the IHC and IFA are subjective, sensitive and specificity, which is a reliable method for detecting dynamic distribution of DPV gC antigens, and the DPV gC protein could be distributed in the tissues of the vaccinated ducklings at 1 d p.v.,and exist in those at least 10 w p.v..3.The antigenic expression phase and distribution regularity of pcDNA-DPV-gC with gene gun bombardment and intramuscular injection:The positive immunoreactivity in different dose groups was found in the liver, duodenum, caecum, rectum and injected spot at 1 d p.v., and the immunoreactivity of injected spot was stronger than other organs.In addition, a drastic reduction of DPV gC antigen levels were observed after the highest immunoreactivity at 2 w p.v.,but still had a detectable vaccine antigen level in the liver, brain, duodenum, caecum and rectum at 10 w p.v.. Interestingly, the pancreas was scarcely detected positive signals at different time points; The antigen levels have a conspicuous discrepancy among the different tissues.The positive immunogenicity was mainly found in the liver, spleen, bursa of Fabricus, brain, duodenum, caecum and rectum, which served as the principal sites for antigen localization. The target cells had a ubiquitous distribution, especially in the hepatocyte of liver, the lymphocyte of splenic white pulp and red pulp, the lymphocyte of cortical substance and punctate substance of bursa of Fabricius, the neuroglia cells of brain pallium, cellula epithelialis and lamina propria mucosae of intestinal tract.4.The antigenic expression phase and distribution regularity of liposome/DNA gene vaccine and chitosan/DNA gene vaccine:The positive signals were observed in the liver, bursa of Fabricius (BF),duodenum, caecum and rectum in the intramuscular injection group at 1 d p.v.,and in the nasal administration group, the positive staining reaction were found in the lung at 12 h p.v.,and the DPV gC proteins were observed in the Harderian gland and BF at 1 d p.v. Moreover,the positive staining were firstly found in the esophagus in the oral administration group at 12 h p.v.,and the DPV gC proteins were observed in BF, duodenum, caecum and rectum at 1 d p.v. The positive immunogenicity was mainly found in the lung, esophagus, Harderian gland, BF, duodenum, caecum and rectum. The target cells had a ubiquitous distribution, especially in the cellula epithelialis of lung and esophagus, the lympholeukocyte of BF and Harderian gland, lamina propria and cellula epithelialis of intestinal tract.5.The antigenic expression phase and distribution regularity in the different doses of pcDNA-DPV-gC gene vaccine:The signal intensity and duration time of different doses were in below order:6μg groups>3μg groups>1μg groups,200μg groups>100μg groups>50μg groups. The results explained that there were the positive dependablity between the immunization doses and the dynamic expression regularity of DPV gC antigens.6.The antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the different immunization-routes:The DPV gC antigens could distribute in the tissues of ducklings via the different immunization routes(gene gun bombardment and intramuscular injection).The expression level of DPV gC antigens in the gene gun groups were higher than the intramuscular injection groups in the early immunization period, especially DPV gC proteins of the skin were more than the muscle of injection site. The distinction could not be obvious because the number of positive immunoreactivity in all tissues of the gene gun groups and intramuscular injection groups had dramatically increased in the intermediate immunization stage. However, a drastic reduction of DPV gC antigen levels were observed, and most of tissues could not found the positive signals in the gene gun groups and intramuscular injection groups at 10 w p.v.. The antigen levels have a conspicuous discrepancy among the different tissues when the ducklings were immunized liposome/DNA gene vaccine and chitosan/DNA gene vaccine via the different immunization routes (intramuscular injection, oral administration and nasal administration). For example, the positive immunoreactivity of Harderian gland in the nasal administration groups were higher than the intramuscular injection groups and oral administrations groups, and the antigen levels of esophagus in the oral administration groups were more than the intramuscular injection groups and nasal administration groups. The signal intensity and duration time of different doses were in below order:intramuscular injection groups> nasal administration groups>oral administration groups.7. The antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the different immunological adjuvant:The different immunization adjuvant could promote antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine. The result showed that the persistence time and the positive signal intensity in the chitosan groups were more than the liposome groups. Chitosan was better immunological adjuvant as pcDNA-DPV-gC gene vaccine.8.The proliferation and distribution regularity of attenuated DPV vaccine:The first signals of DPV-specific antigens were observed in the liver and spleen at 12 h p.v., but no positive staining cells could be observed in the controls birds.The number of positive immunoreactivity in all tissues had dramatically increased to reach peak levels between 1 d and 7 d p.v.. The highest levels of positive staining reaction were found between 2 w and 4 w p.v.. The attenuated DPV vaccine appeared intense pantropic to the tissues of the vaccinated ducklings. The liver, BF, thymus, duodenum, caecum, rectum were served as the principal sites for antigens localization, but the heart, pancreas and esophagus had lower detection ratio.