Mucosal And Systemic Immune Response To The Distribution Of Virulent Or Attenuated Duck Plague Virus In Inoculated Ducklings

Duck plague (DP) is an acute, contagious herpesvirus infection of ducks, geese and swans with high morbidity and mortality. In duck-producing areas of the world where the disease has been reported, DP has produced significant economic losses in domestic and wild waterfowl. Vaccination has been used as a preventive measure and also for controlling disease outbreaks. Attenuated DPV vaccine has been developed and used extensively with good success in the world, as it provides protection immediately after vaccination. A number of studies have shown the importance of the systemic immune responses in the resistance and clearance of DPV infection. However, the mucosal immune responses against DPV infection remain to be unknown, which will hind the elucidation of the immune mechanism of attenuated DPV vaccine. The mucosal immune system is known to provide an effective barrier between the host and various pathogens that is regulated in a different fashion to that of systemic immunity. The study on the mucosal immune system of fowl will provides valuable insight into the controlling of disease and development of mucosal vaccine. However, There are only a little studies focus on the mucosal immune system of chickens. To understand better the mucosal immune responses of ducks, The 20-day-old ducklings were vaccinated by subcutaneous, oral, and nasal administration with attenuated DPV vaccine, quantitative analysis of vaccine virus and humoral and cell-mediate mucosal immune response in birds were estimated. Above detection on infected ducklings with virulent DPV was also estimated in this study, in order to evaluate the mucosal immune response against DPV infection. The effect of vaccination on the intestinal microbial community structure feature was also estimated using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR, and the contents are summarized as follws:1.Replication kinetics of DPV loads in ducklings were examined by using real-time quantitative PCR. The results indicated that vaccine virus can spread to all tested samples and replicated quickly at 12 hours postinoculation (p.i.), and the route of vaccine administration had significant effect on the sequential tissue distribution of duck plague virus in ducklings, and the lymphoid organs and intestine tissues are the primary tissue sites of DPV replication independent of the different virulence of virus. The vaccine virus levels reached peak at 24 hours p.i. followed by a stead decline, which manifest that the stimulation of host immune response may suppress the virus replication. A low-level of DPV vaccine persistence in lymphoid organs at the terminal of experiment will contributed to long-term immune protection. Live DPV vaccine were detected from direct-contact ducklings, which may bring about contact immunization and strengthen host mucosal immune response to ducklings vaccinated by systemic administration. The virus loads in infected ducklings with virulent DPV increased untill the birds died or agonal as a result of infection.2.The mucosal and systemic humoral immune responses in blood, bile, and the respiratory and digestive tract fluids of ducklings were evaluated using indirect enzyme linked immunosorbent assay (ELISA). The results showed that sIgA immunoglobulin is the predominant antibody in mucosal immune system stimulated by attenuated DPV vaccine, while IgM immunoglobulin was first detected in all samples examined, and IgG immunoglobulin is the most important antibody in systemic immunity. The exposure of the mucosa to live DPV vaccine can lead to specific antibody production in the local as well as distant mucosal surface, which suggest that DPV vaccine by mucosal administration can induce both MIS and CMIS. The speed and loads of virus vaccine exposure to mucosa have a close correlation with the induction of local mucosal humoral immunity. Specific antibody can be detected in tested samples of vaccinated ducklings at 60 days p.i., particularly the high antibody titer of IgG in blood and sIgA in intestinal fluids. Little or a little antibody were detected in infected ducklings with virulent DPV, which indicated that the pathological lesions of intestinal mucous membrane can suppress the effective stimulation of host mucosal immune response.3.Immunohistochemical localization of IgA producing-cells in respiratory and digestive tract, and lymphoid organs of ducklings were detected using indirect immunoperoxidase staining methods. The results showed that the numbers of IgA-producing cells increased by various rates in all samples examined of vaccinated ducklings, and the numbers of IgA-producing cells originated from a local mucosal membrane that exposure to DPV vaccine early were the most. These results may indicated that attenuated DPV vaccine can induce the increase of the numbers of IgA producing-cells in ducklings, and the choice of immune routes is critical for the stimulation and proliferation of IgA-producing cells. The numbers of IgA-producing cells in lamina propria of intestine, particularly in duodenum, were higher than that in orther tissues and organs. This results suggesting that the lamina propria of intestine is the predominant efective sites of mucosal immune response of duck. The replication kinetics of IgA-producing cells in vaccinated or direct-contact vaccinated ducklings were corresponds to the kinetics of specific sIgA antibody, suggesting the stimulation and proliferation of IgA-producing cells have a close corelation with the induction of specific sIgA antibody. The numbers of IgA-producing cells in infected ducklings with virulent DPV decreased or slightly increased followed by a drastic decrease with the progression of the infection. This results may indicated that the pathological lesions of organs, particularly intestinal mucous membrane, have significant effect on the stimulation and proliferation of IgA-producing cells.4. Immunohistochemical localization and proliferation of DPV antigen and CD3~+ cells in respiratory and digestive tract, and lymphoid organs of ducklings were detected using indirect immunoperoxidase staining methods. The results showed that the DPV antigen was first detected in infected or vaccinated ducklings at 1 to 3 days p.i., and the target cells for DPV are epithelial and lymphoid cells independent of the different virulence of virus. The numbers of CD3~+ cells in all samples examined of artifically or direct-contact vaccinated ducklings increased by various rates at 3 to 6 days p.i. and reached maximum at 15 to 21 days p.i. The numbers of CD3~+ cells in orally vaccinated birds increased early and peak loads of cell numbers were higher compared with that in orther vaccinated ducklings, suggesting that the speed and loads of attenuated DPV vaccine exposure to local mucous membrane have significant effect on the CD3~+cells level. The CD3~+ cells were found predominantly in lamina propria of intestine, and many CD3~+ cells were also found in the lamina propria of trachea and lymphoid nodes of lymphoid organs. The results suggested that the lamina propria of mucosa is the most important effective site of CD3~+ cells, and intestine may play critical role in induction of mucosal immune system of duck. The numbers of CD3~+ cells in infected ducklings with virulent DPV decreased or slightly increased followed by a drastic decrease with the progression of the infection.5. The structural dynamics of intestinal microbial communities of vaccinated ducklings have been monitored using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR. The results showed that the steady pattern of microbial structural within duodenum, jejunum, and ileum of controlled healthy ducklings formed relatively early than that within cecum and rectum. The vaccine virus inoculated by systemic and mucosal administration has significant effect on the intestinal microbial community structure by various degree. The results also showed that the microflora in duodenum can formed new steady structure quickly with DPV antigen, and sIgA antibody after vaccination. The results of sequence analysis of DNA fragments in PCR amplified bands showed all bacterial in intestine of controlled and vaccinated birds were normal intestinal microflor, and aerobe or facultative aerobe, including Escherichia, Erwinia, and Bacillus became predonderant bacilli in all parts of intestine. However, the anaerobe or facultative anaerobe, including Lactobacillus and Enterococcus became predonderant bacilli in cecum or ileum.