A novel combined adjuvant strongly enhances mucosal and systemic immunity to low pathogenic avian influenza after oral immunization in ducks.

As natural reservoirs of avian influenza viruses, waterfowl play an important role in the generation, spread, and enzootic transmission of avian influenza. To prevent avian influenza in waterfowl through a simple, noninvasive, and needle-free route, ducks were immunized orally with an inactivated avian influenza virus (H9N2, IAIV) combined with CpG DNA and high-dose glucose, and then the local and systemic immune responses of these ducks were investigated. In addition, the immune protection was assayed after viral challenge. After the oral administration of IAIV combined with CpG DNA and glucose, the expression levels of interleukin-2 and interleukin-6 in the small intestine tissues increased significantly in the early period after booster immunization relative to the levels after immunization with IAIV and a single adjuvant. Significant increases were also observed in the IgA and IgG antibody levels in the local intestinal tract tissues and serum at wk 3, 5, and 7 after the first immunization. Furthermore, enhanced hemagglutination inhibition titers were also detected in serum samples taken between the third and seventh weeks after immunization with IAIV and both adjuvants. In the viral challenge and transmission study, the prior administration of IAIV combined with both CpG DNA and glucose reduced the viral titers observed for the cloaca swabs and colon tissues of challenged ducks and prevented virus transmission between ducks. Our study suggests that the combination of CpG DNA and high-dose glucose can improve immunization with inactivated H9N2 virus by enhancing the local and systemic immune responses and reducing viral shedding.