Immunological Studies Of The Recombinant Lactic Acid Bacteria Anchoring NDV Hn Protein

Neweastle disease virus(NDV) is an acute epidemic disease infected via alimentary canal andrespiratory tract, typically causing dyspnoea, disordered nervous function, mucosal and serosal haemorrhage.Neweastle disease (ND) is one of severe poultry epidemic disease in the world, that have threaten to thepoultry farming and been harmful to the economy.The recombinant L.casei anchoring the NDV HN antigen protein was reconfirmed. After pLA-HN/L.casei culturing it in the MRS medium for expression of the target protein, SDS-PAGE showed the99KDa protein band which indicated the HN-pgsA fusion protein was expressed. The immunofluorescencemicroscopy and Flow Cytometry analysis with chicken-anti-NDV polyclonal serum indicated that therecombinant protein reacted with the specific antibodies. The results showed that the HN fusion protein wasdisplayed on the cell surface of Lactobacillus casei (L.casei).The chickens were immuned with the control pLA/L.casei, recombinated pLA-HN/L.casei, recombinatedpLA-HN/L.casei plus immune enhancing agent and live attenuated LaSota vaccine via oral, intranasal oreyedrop, respectively. Specific anti-NDV HN IgG in the serum and specific anti-NDV HN secretimmunoglobulin A (sIgA) antibody in the lung, intestines fluid and collected tear, bile of chickens weredetected by indirect ELISA. T-cell proliferation responses were investigated by MTT test2weeks later afterthe third immunization. Three weeks after the last immunization with the recombinant L.casei (plus immuneenhancing agent or not) or LaSota vaccine, the chickens orally or intranasally vaccinated and controlchickens were challenged with standard-type NDV(F48E8). The results showed that special serum IgG andsIgA antibodies were detected in the vaccinated groups. One handred pesent chichkens survived in oral andintranasal LaSota immunization groups whereas91%and100%chichkens survived in oral and intranasalrecombinant pLA-HN/L.casei plus immune enhancing agent immunization groups after challenged withF48E8. Eighty two percent and91%chichkens survived in oral and intranasal recombinant pLA-HN/L.caseirespectively, while all of the chickens dead in the control group. Significant difference was observed amongthe spleen lymphocyte proliferation index (LPI) among mice of the immunization groups and control groups.In conclusion, both oral and intranasal or eyedrop immunizations with recombinant L.casei plus immuneenhancing agent or not can induce significant specific mucosal sIgA response as well as serum IgGresponses, and can evoke both mucosal immune and system immune responses. pLA-HN/L.casei may be thecandidate vaccine against NDV infection.