Establishment Of ELISA For Detection Of Chicken SIgA And Research On Local Mucosal Immune Responses To The Vaccination With H5 Avian Influenza Recombinant Newcastle Disease Virus In Chicken

Mucosal immunity of poultry is very important as the first defense barrier against infectious pathogens. The effective factor of mucosal immunity in poultry is Secretory Immunoglubulin A (SIgA), which has many functional attributes, serving to prevent adhering and multiplication of pathogens, priming biological activity in immunology to clean infective agents and preventing poultry from infection.In this study, the recombinant chicken SlgA heavy chain fragment protein was expressed and purified from E.coli, and then used to immunize BALB/c mice after. A hybridoma cell clone steadily secreting monoclonal antibody against chicken SlgA was selected based on SIgA antigen purified from chicken biles, and designed as C1. The isotype of C1 hybridoma was defined as IgG2aκtype. ELISA and Western-blotting showed that monoclne antibody from C1 is high affinity and specificity to chicken SIgA.Based on purified and biotin labeled ascitic fluid IgG of C1, an ELISA for the detection of Newcastle disease virus (NDV) and H5 Avian influenza virus (H5 AIV) specific mucosal SIgA was established. This methods then was used to investigate the specific SIgA immune responses of local mucosa to immunization of H5 influenza recombinant NDV vaccine (rL-H5) in Specific pathogen free (SPF) chickens.One group of one-week-old SPF chicken ocular-nasally received one dose of rL-H5 (1×10⁶EID₅₀), the NDV-specific SIgA and H5 AIV-specific SIgA responses could be detected in oral and upper respiratory tract secretions at 2 weeks post vaccination and 3 weeks post vaccination respectively. NDV and H5 AIV specific SIgA levels rapidly declined and close to non-immunized levels at 3 to 4 weeks post vaccination. In intestinal secretions, both NDV and H5 AIV specific SIgAs could be detected at 4 week after immunization, the responses obviously delayed in comparison with oral and upper respiratory tract responses. However, the levels of NDV and H5AIV specific SIgA in intestinal secretions were significantly higher than that of oral and upper respiratory tract secretions.Another group of 6-week-old SPF chickens ocular-nasally received the first dose of rL-H5 (1×10⁶EID₅₀) and the second dose at 16 weeks later, the results showed that NDV and H5 AIV specific SIgA responses could be detected in intestinal and oral and upper respiratory tract secretions. The level of NDV and H5 AIV specific SIgA responses were significant higher than that of one dose immunization above. Both of NDV and H5 AIV specific SIgA responses reached the peak at 2 weeks post boost vaccination and quickly declined and close to non-immunized levels at 3 weeks post boost vaccination in oral and upper respiratory tract secretions or intestinal secretions.These results above demonstrated that rL-H5 induced effectively NDV and H5 AIV HA specific mucosal SIgA immune responses in chickens, and the specific SIgA responses were elicited in intestinal tract much stronger than oral upper respiratory tract. The second dose elicited significant stranger and faster specific SIgA responses, which means rL-H5 immunized chickens response to the infection of NDV and H5AIV efficiently and quickly in local mucosal. The Specific SIgA responses induced by booster vaccination also kept very short duration, reached the peak at 2 weeks post boost vaccination and quickly declined and close to non-immunized levels at 3-4 weeks post boost vaccination. The results above partly illustrated the immune protective mechanism of rL-H5, and provided very important information for the vaccination procedure of NDV recombinant vectored live vaccine.