The Study Of Mucosal And Systemic Immune Response Of Chickens Induced By Different Routes Of Vaccination With Attenuated NDV Vaccine

Newcastle disease (ND) caused by Newcastle disease virus (NDV) is an acute, septicaemic and highly contagious disease which is one of the most important diseases in poultry throughout the world. At present, although there many good vaccine to prevent ND, the phenomenon that the flock are infected with NDV still occur due to irrelevance of vaccination route and approach, so it still severely influences the development of the poultry industry. It is commonly thought that humoral immunity of chickens plays an important role in resistance to the infection with NDV. However, there is a phenomenon in practice that the flock is not protected after challenge with virulent NDV when there are high level antibodies in serum. Why? It remains to be explored. The mucosal immunity is an important part of immune response of chickens'immune system and play very important roles to local and general immunity of chickens. As studies about the immunologic mechanism of attenuated NDV vaccine have long been focusing on the systemic immune responses in the resistance to NDV, and there have been only few studies about the developing regularity of specific mucosal immunity, it is hard to reveal completely the elucidation of the immune mechanism of attenuated NDV vaccine. Thus this study elaborated on the developing regularity of specific antibodies and antibody positive cells in local mucosal immune tissues of the respiratory and digestive tract of experimental chickens vaccinated with attenuated NDVvaccine. The main results are summarized as follows:1. The purification of Chicken SIgA and the initial study of its antiviral activityChicken secretory IgA (SIgA) was coarsely isolated from bile by ammonium sulfate precipitation and polyethylene glyco(lPEG)extraction and finally purified by the gel filtration with Sephadex G-200 and ion exchange chromatography with DEAE. The purity and activity of purified SIgA were analyzed by SDS-PAGE electrophoresis and agar gel diffusion tests. Then the anti-NDV activity of chicken SIgA was detected by egg inoculation test, the viral neutralization test of embryonated eggs and therapeutic test of chickens. The results indicated that the final concentration of purified chicken SIgA was 3.8mg/mL, the molecular weight of heavy chain and light chain was approximately 67kD and 28kD respectively. And after NDV was treated with chicken SIgA, the HA titer of NDV in the allantoic fluids of eggs could significantly be reduced, and the result of this test was not significantly different from the test result with the serum group (P>0.05). The neutralization index of chicken SIgA was 6.2 and lower than that of the serum group. In addition, the protective rate of SPF chickens challenged with virulent NDV was 46.7% with purified SIgA treatment and also not significantly different from the serum group (P>0.05). In the end, the result indicated that chicken SIgA has the ability to neutralize NDV.2. The mucosal and systemic humoral immune response in chickens induced by different vaccination routes with attenuated NDV vaccineAn indirect ELISA assay was established to measure NDV specific antibodies in serum, bile, secretions of respiratory and digestive tracts in 7-day-old SPF chickens after vaccination through different routes with attenuated NDV vaccine. And the systemic humoral immune response of chickens was detected by HI test. The results indicated that NDV specific SIgA,IgG and IgM could be detected in all samples of vaccinated chickens. IgM could be detected at 3d post vaccination and decreased rapidly after reached the maximum at 3d post vaccination. It was the earliest anibody at early infected time. The levels of IgG were the highest and could be detected at 7d post vaccination and reached the maximum at 21d post vaccination. It was the earliest anibody at early infected time. SIgA could be detected at 7d post vaccination and reached the maximum at 28d post vaccination. The levels of SIgA were the highest in bile, secretions of respiratory and digestive tract. It was the predominant anibody in local secretions. The exposure of the mucosal to NDV vaccine could lead to specific antibody prodution in the local as well as distant mucosa, which suggested that NDV vaccine can induce both MIS and CMIS. Serum HI titers of chickens induced by ocular-nasal vaccination with attenuated NDV vaccine were significantly higher than those by drinking water vaccination. And the levels of specific SIgA in trachea and lung washes by ocular-nasal vaccination were significantly higher than those by drinking water vaccination, however, the levels of specific SIgA in duodenum, jejunum and ileum washes by drinking water vaccination were significantly higher than those by ocular-nasal vaccination. In conclusion, these results indicated that the strength of mucosal immune response in different mucosal parts induced by different vaccination routes was different from each other. In other words, the strength of mucosal immune response in the respiratory tract induced by ocular-nasal vaccination was stronger than that by drinking water vaccination, on the contrary, the strength of mucosal immune response in the digestive tract induced by drinking water vaccination was stronger than that by ocular-nasal vaccination.3. The kinetics of IgA, IgM and IgG positive cells in local mucosa and immune organ of chickens induced by different vaccination routes with attenuated NDV vaccineIn this study, an indirect immunoperoxidase staining method to localizate IgA , IgM and IgG positive cells positive cells was successfully established by utilizing routine paraffin sections based on optimization of various reaction conditions. And with this method, the distribution and number of antibody positive cells was observed in paraffin sections of spleen, trachea, duodenum, jejunum, ileum and cecal tonsil of 7-day-old SPF chickens vaccinated by different routes with attenuated NDV vaccine. The results indicated that the shape of IgA , IgM and IgG positive cells stained with yellow was round or oval. These positive cells distributed in the marginal zone and medullary cord of spleen, the lamina propria of tracheal and intestinal villi mucosa. Antibody positive cells were induced to yield in the lamina propria of intestine of chickens by both ocular-nasal and drinking water vaccinations. The number of IgG positive cells were highest in spleen and the number of IgA positive cells were highest in mucosa. And the number of IgM positive cells began to increase significantly 3d post vaccination and reached the peak at 14d, the number of IgG positive cells began to increase significantly 7d post vaccination and reached the peak at 21d and he number of IgA positive cells began to increase significantly 7d post vaccination and reached the peak at 28d. There are more IgA positive cells in duodenum than in jejunum and ileum, which indicated that the lamina propria of duodenum is the predominant effective part of the alimentary tract.4. The effect of the immunosuppression on mucosal immune response induced by attenuated NDV vaccine in SPF chickensIn this study, one-day-old SPF chickens were treated to induce the immunosuppression by bursectomy and injecting cyclophosphamide (CY). At 7 days of age experimental chickens were vaccinated with attenuated NDV vaccine by drinking water. At different periods after vaccination, the relative weights of immune organs, the numbers of peripheral blood lymphocytes, the titers of serum HI antibody, levels of NDV specific SIgA (in bile, tracheal and duodenal washing) and the number of IgA positive cells in lamina propria of intestine were detected. The aim of this experiment is to compare the effect of two immunosuppressive methods on the humoral and mucosal immunity. The results indicated that two immunosuppressive methods could significantly reduce serum HI titers and result in severe immunosuppression of systemic humoral immunity. And the suppressive effect of CY was stronger than bursectomy. In addition, the two methods significantly decreased levels of NDV specific SIgA in bile, tracheal and duodenal washing and the numbers of IgA positive cells in lamina propria of intestine, especially within 3 weeks after vaccination. So bursectomy and CY could also induce the immunosuppression of mucosal immunity. And the result of challenge experiment with virulent NDV indicated that local mucosal immunity might play an important role in resistance to NDV in chickens.5. The effect of the immunopotentiators on mucosal immune response induced by attenuated NDV vaccine in chickensIn this study, attenuated NDV vaccines were respectively mixed with CpG ODN, astragalus polysaceharide (APS) and levamisole (LMS) and vaccinated 14 days of age chickens by ocular-nasal route. To evaluate the immunologic enhancement of three immunopotentiators on the humoral and mucosal immune response induced by attenuated NDV vaccine, the titers of serum HI antibody, the levels of specific SIgA in local secretions and the numbers of IgA positive cells in small intestine mucosa were detected. The results indicated that three immunopotentiators all could enhance serum HI titers and the immunologic enhancement of LMS was stronger than CpG ODN and APS. And three immunopotentiators also could all enhance levels of NDV specific SIgA in bile, trachea and duodenum washes and the immunologic enhancement of CpG ODN was stronger than APS and LMS. The above results sufficiently proved that three immunopotentiators could enhance not only the humoral immune response but also the mocosal immune response induced by attenuated NDV vaccine. In conclusion, this study illustrated the characteristics and variability of mucosal immunity in various mucosal parts in chickens induced by different vaccination routes with attenuated NDV vaccine though the kinetics of IgM, IgG and SIgA in local secretions and the generation regularity of IgM, IgG and IgA positive cells in local mucosa. It will provide important theoretical guidance and technical support for studying the characteristics of mucosal immunity of chickens and epidemic prevention from ND by vaccines and has theoretical and practical significance in scientific prevention from ND.