Effect Of Oral Immunization With Inactivated Avian H9N2 Influenza Virus On The Local Digestive Tract And Systemic Immunity In Ducks

In order to evaluate the effects that the inactivated avian H9N2 influenza virus(IAIV) vaccine with adjuvant on the immune response of the local digestive tract (mainly pharynx and small intestine) and the systemic immunity, ducklings were oral immunized with IAIV mixed with adjuvant CpG DNA or glucose.125 seven-day-old ducklings were randomly divided into five groups. On being 10 days old, each duck of the first group was oral immunized with NaCl liquol 0.3ml. Each bird of the second group was oral immunized with IAIV 0.3ml only. Each bird of the third group was oral immunized with IAIV 0.3ml and glucose. Each bird of the fourth group was oral immunized with IAIV 0.3ml in combination with CpG. Each of the fifth group was oral immunized with IAIV 0.3ml containing CpG and glucose. The ducks on becoming 14 days old from all groups were vaccinated again. The ducks were decapitated and sample were collectioned at 3rd,5th and the 7th week after the first immunization. Firstly, to investigate the local cell-mediated immunity in the digestive tract, the number of small intestine intraepithelial lymphocyte (IEL) and mast cells and IL-2 and IL-6 in the digestive tract were detected; To investigate the humoral immunity in local digestive tract, the area of IgA and IgG secreting cells and the level of AIV-specific IgA and IgG antibody in the digestive tract were detected; And then, to investigate the systemic immunity, the level of avian influenza virus (AIV)-specific IgA and IgG antibody in serum were detected; Finally, to validate the ability for resisting the challenge of H9N2 virus by challenge test; To validate the effect of shedding and lateral spread between groups of vaccinated and unvaccinated ducks by contact transmission.1. Purification of duck immunoglobulin A(IgA) and IgG and preparation of rabbit anti-duck IgA and IgGThe immunoglobulin A(IgA) and IgG were isolated from duck bile and serum separately. The solated IgA was purified by Sephadex G200 gel filtration and then DEAE-SephadexA-52 cellulose chromatography. The purified IgA and IgG were electrophoresed on the SDS-PAGE gel. The results showed IgA and IgG is respectively composed of heavy and light chains. The molecular weight of heavy chains respectively is about 70,000～72,000 and 67,000, the IgA has a same molecular weight of light chains with IgG about 26,000. Rabbits were subcutaneouly inoculated in the back by purified IgA and IgG respectively, rabbit antiserum is proved by double agar diffusion test that the titer of the antiserum reaches tol:32.2. Effects of oral immunization with inactivated avian H9N2 influenza virus vaccine on the local cell-mediated immunity in duck digestive tractHE coloration method was applied to show IELs in the small intestine. The result discovered that the number of IELs in the small intestine increased significantly (P<0.05) with IAIV containing CpG or CpG and glucose after oral immunization; whereas oral immunization with IAIV only or IAIV and glucose could not bring effect on the number of IEL.Toluene-amine-cyanine coloration method was applied to show mast cells in the digestive tract. The result discovered that the number of mast cells in the pharynx and small intestine increased significantly(P<0.05 or P<0.01) with IAIV containing CpG or CpG and glucose after oral delivery; No significant difference on the number of mast cells in the groups with IAIV only or IAIV and glucose.RIA was applied to detect cytokines IL-2 and IL-6 in small intestinal mucosa. The result found that the level IL-2 and IL-6 increased significantly(P<0.01) with IAIV containing CpG or CpG and glucose after oral immunization; No significant changes in the animals immunized only with IAIV or IAIV and glucose.The results of three essays above prompted that oral immunization with IAIV and adjuvants may have increased significantly the local cellular immunity in duck digestive tract.3. Effects of oral immunization with inactivated avian H9N2 influenza virus vaccine on the local humoral immunity in duck digestive tractSPA-HRP immunohistochemical technique was applied to show IgA and IgG secreting cells. The result discovered that the area of IgA and IgG secreting cells increased significantly (P<0.05 or P<0.01) with IAIV containing CpG or CpG and glucose after oral immunization; whereas the effect on the area change of IgA and IgG secreting cells was exiguity after oral immunization with IAIV only or IAIV in combination with glucose. Indirect enzyme-linked immuno-technique was applied to detect the levels of AIV-specific IgA and IgG antibody in the mucosa of pharynx and small intestine. The result showed the levels of the IgA and IgG antibody in the mucosa of pharynx and small intestine increased significantly(P<0.01) with IAIV in combination with CpG or CpG and glucose after oral immunization; But no significantly changes were found in the animals immunized with IAIV only or IAIV and glucose.The results of two essays above prompted that oral immunization with IAIV and adjuvants may have increased significantly the local humoral immunity in duck digestive tract.4. Effects of oral immunization with inactivated avian H9N2 influenza virus vaccine on the systemic immunity in duck.HI test was applied to detect the level of AIV-specific HI antibody. The result showed that the HI degree increased significantly (p<0.05) with IAIV in combination with CpG or CpG and glucose after oral immunization; whereas the level of HI was no significant changes after oral immunization with IAIV only or IAIV and glucose.Indirect enzyme-linked immuno-technique was applied to detect the levels of AIV-specific IgA and IgG antibody in the serum. The result found that the levels of the IgA and IgG antibody increased significantly (p<0.05) with IAIV in combination with CpG or CpG and glucose after oral immunization; there were no changes after oral immunization with IAIV only or IAIV and glucose.The results of two essays above prompted that oral immunization with IAIV and adjuvants could improve the systemic immunity level at a certain extent.5. Effects of oral immunization with inactivated avian H9N2 influenza virus vaccine on the ability for resisting the challenge of H9N2 virus30 seven-day-old ducklings were randomly divided into three groups in the challenge test. On being 10 days old, each duck of the first group was oral immunized with NaCl liquol 0.3ml. Each duck of the second group was oral immunized with IAIV 0.3ml only. Each duck of the third group was oral immunized with IAIV 0.3ml containing CpG and glucose. At the 21th day after the first immunization, to choose H9N2 virus(ELD5o was 108.32/0.1ml), the ducks were orally inoculated with the dose of 2ml per duck. There were no clinical symptoms and visible damage in all ducks after challenge. To the 3rd day after challenge, AIV was isolated using 10-day-old embryonated SPF hens'eggs. The results found that AIV were recovered from some of the samples of the ducks immunized with NaCl liquor or only with IAIV; Whereas, no virus isolation attempts were successful from the ducks by IAIV in combination with CpG and glucose. The results suggested that resistance against H9N2 virus increased significantly with IAIV containing CpG and glucose.In order to establish the occurrence of lateral spread between groups of vaccinated and unvaccinated birds,24 seven-day-old ducklings using 12 vaccinated ducks and 12 unvaccinated ducks. At the 21 th day after the first immunization,6 ducks were orally inoculated with H9N2 virus respectively in vaccinated ducks and unvaccinated ducks. Contact transmission after challenge. The result found that the number of positive ducks in unvaccinated ducks more than which in vaccinated ducks, which in contacted with unvaccinated infected ducks and vaccinated infected ducks respectively. The result indicated that oral immunization with IAIV in combination with CpG and glucose appears to be efficacious in suppressing viral shedding, and lateral spread of infection to unvaccinated and vaccinated ducks.