Detection Of The Effects Of PSIP409-NP-M1-DCpep Lactobacillus Preparation Against H9N2 Avian Influenza Virus Infection

Avian Influenza virus（AIV） spreads worldwide, which is a serious threat to poultry and human health. In many countries, Low Pathogenic Avian Influenza viruses such as H9N2 viruses become an enormous economic burden on the commercial poultry industry when they cause mild respiratory disease and reduction in egg production. Lactobacillus characterized by safe culture is a kind of multifunctional micro ecological preparation and was recognized as being naturally plasmid free. The core nucleoprotein（NP） and matrix protein（M1） has been shown to be effective vaccine candidate antigens, and highly conserved internal proteins among the different subtypes AIVs. DC peptide（DCpep）derived from a phage of display library can significantly enhance the immune response.The purpose of this study was to evaluate whether Lactobacillus preparation could enhance immune response against H9N2 AIV when utilizing Lactobacillus plantarum NC8（NC8） to present NP-M1-DCpep in chicken models. 200 μL of NC8-p SIP409-NP-M1-DCpep suspension containing 2×109 colony-forming units（CFU） was administered to the 20 day-old for 3 days chickens, and two times every 10 days. We evaluated the protective efficacy of NC8-p SIP409-NP-M1-DCpep against H9N2 AIV in chicken. The results are as follows:（1） The study of Lactobacillus preparation against H9N2 AIV challenge infectionChicken was administered by orally NC8-p SIP409-NP-M1-DCpep, Flow cytometry was performed for analyzing the frequencies of CD³⁺CD⁴⁺ T cells and CD³⁺CD⁸⁺ T cells in chicken, and the specific proliferation and transformation of spleen lymphocytes were detected by MTS colorimetric assay. Specific SIg A and Ig G Ab were analyzed by ELISA after the booster immunization. Then the chickens were euthanized, and the throat and lung tissue samples were stained with HE, Histopathological Analysis of throat and lung in chicken, and the loss of body weight were observed for 10 days after infection.The results showed that NC8-p SIP409-NP-M1-DCpep could induced the activation of CD⁴⁺ and CD⁸⁺ T cells significantly and improve proliferation and transformation of spleen lymphocytes in chickens. In addition, Lactobacillus preparation also significantly increased mucosal Ig A level in the small intestinal washings and Ig G level in serum in chicken. The injury of lung and throat tissue and body weight of chickens receiving NC8-p SIP409-NP-M1-DCpe administration were reduced significantly after infection.（2） Screening the administration route of NC8-p SIP409-NP-M1-DCpepAlthough oral administration of NC8-p SIP409-NP-M1-DCpep have proven to be effective in preventing influenza virus infection in mice, due to the acidic conditions of the stomach and the absorption degree of intestinal epithelial cells and antigen reasons, the progress of oral administration and immunization was slow. It has been suggested that intranasal administration of Lactobacillus could be effective in protection against respiratory infection due to direct augment of the respiratory immune system. Therefore, our study administered Lactobacillus by intranasal and oral route, and challenged chickens with H9N2 virues to compare two routes that cause the protective efficacy of NC8-p SIP409-NP-M1-DCpep against challenge with influenza virus.Compared with oral route, intranasal administration of NC8-p SIP409-NP-M1-DCpep provided higher protection against influenza virus infection by enhancing Secretory Ig A production, Mucosal Secretory Ig A antibodies in the respiratory tract provided cross-protection against variant respiratory virus infections, which may confer higher antiviral effects than systemic Ig G antibodies. And the protective efficacy of NC8-p SIP409-NP-M1-DCpep against challenge with influenza virus in mice according to administration route was compared, The injury of lung and throat tissue and body weight of chickens receiving nasal administration were lower than orally administered groups. The present results indicates that administration of NC8-p SIP409-NP-M1-DCpep could induce immune response protecting chickens against influenza H9N2 viruses infection significantly.