The Effect Of Avian Influenza Recombinant B.subtilis On The Immnue Efficacy And Growth Of Chickens By Oral Immunity

The spread of avian influenza(AI)is mainly through gastrointestinal and respiratory mucosal routes.Oral immunization is an effective mucosal immune pathway which can cut off the transmission of avian influenza in the mucosal site and control the spread of AIV.The AIV recombinant live vector vaccine is safe and effective,which has become one of the main directions of future research and development of bird flu vaccines.Therefore,we successfully constructed recombinant B.subtilis:WB800-HA and WB800-HA-SS.Confirmed that HA could express correctly by SDS-PAGE and Western blot.After then,we evaluated the effect on immune efficacy and growth performance of chickens by oral immunity,which provides new ideas for the development and application of HPAI vaccine.5-day-old hyline chickens were randomly divided into six groups and were immunized third(at 10,13 and 17-day-old).The first group were oral immunized with PBS as control group;the second group were oral immunized inactivated H5N1 vaccine and the third group were subcutaneously immunized the inactivated oil emulsion adjuvant H5N1 vaccine,which were positive control groups;the forth group was oral immunized B.subtilis WB800(0.3 mL1010 cfu)as negative control group;the fifth and sixth groups were oral immunized B.sultilis WB800-HA and WB800-HA-SS(0.3 mL1010 cfu)respectively.First,the number of B.sultilis in the chicken droppings were measured to analysis the colonzation in chicken gut;Second,the level of local mucosal immunity and the systemic immunity were measured by HI and ELISA;Finally,for assessing the role of recombinant B.sultilis on the growth performance of chickens,the weights and morphological change of intestinal mucosal tissues(HE staining)were investigated.1.The colonzation of B.sultilis in chicken gutAseptically collected the chicken droppings at different times after the first immunization.By identifying of morphology and 16S rDNA to statistic the number of B.sultilis,found that:after 2 h,most of B.sultilis were excreted,and then the remaining were excreted slowly,To the 48 h,B.sultilis were still to be detected.2.The detection of oral recombinant B.sultilis on local mucosal humoral immunityThe levels of specific S IgA antibody were detected from oral lavage fluid of the 3rd 5th 7th weeks and intestinal and trachea lavage fluid of the 8th week.The level of S IgA of recombinant B.sultilis in oral and intestinal lavage fluid were significantly higher than the control group(P<0.01),The level of S IgA of WB800-HA-SS in trachea lavage fluid were higher than the control group(0.01<P<0.05).Suggested that:the recombinant B.sultilis vaccines could effectively enhance the intestinal and respiratory local mucosal humoral immune response in chickens,especially for intestinal local mucosal humoral immune response.3.The detection of oral recombinant B.sultilis on systemic immunity in chickenThe results of HI assay showed that:the level of maternal antibody at the 1st week was relatively high and then began a slow decline.Subsequently,the HI titers of experiment groups had a slight rise at the 5th week,but the HI titer was low overall.The level of specific IgG in the serum of 3rd 4th 5th weeks showed that:the level of special IgG of WB800-HA and WB800-HA-SS were significant higher than WB800(0.01<P<0.05),while lower than the Inactivated vaccine group.Suggested that the recombinant B.sultilis were able to efficiently induce systemic immune response in some degree.4.The influence of oral recombinant B.sultilis on chicken growth performanceThe chicken weights were measured each week,each group was equivalent in the first four weeks;After 5 weeks later,the weight from normal B.sultilis group and recombinant B.sultilis groups were significantly higher than the control group(P<0.01);Besides,recombinant B.sultilis vaccines were slightly higher than that of the normal B.sultilis group.The morphological change of jejunum villus by HE staining showed that:the recombinant B.sultilis could significantly increase the length of intestinal villus,which was related to the chickens weight(P<0.01).The length of intestinal villus in WB800-HA-SS group was higher than the other groups(P<0.01).Besides,they also improve PP junction differentiation,development,increase the area of PP junction significantly(P<0.01).Our data suggested that the two kinds of recombinant B.sultilis vaccines,especially for WB800-HA-SS,were able to efficiently improve the growth performance of chickens and increase significantly the PP junction area,enhance the immune response.