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The influenza neuraminidase (NA) enzyme is a promising candidate for a conserved influenza vaccine, offering long-term and effective cross-protection against the disease. The aim of this study is to develop a\n                      <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                      -vectored vaccine with strong protective efficacy against H9N2 avian influenza virus. We utilized the antibiotic-free balanced-lethal system of regulated delayed-lysis\n                      <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                      (\u03c711218) as the starting strain to construct a\n                      <jats:italic toggle=\"yes\">focA<\/jats:italic>\n                      gene-deleted\n                      <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                      (\u03c7YL57) strain. To optimize the vaccine design, we fused a tetravalent NA protein (tetramer-NA) with a chicken dendritic cell-targeting peptide (chDCpep) and delivered it via \u03c711218 or \u03c7YL57, yielding the candidate vaccine strains S323 or \u0394S323, respectively. The results revealed that the successful synthesis of tetramer-NA proteins could be detected using nonreducing western blotting and that the presence of chDCpep could dramatically increase the maturation of chicken bone marrow-derived dendritic cells. The\n                      <jats:italic toggle=\"yes\">focA<\/jats:italic>\n                      deletion enhanced bacterial escape from macrophages\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      and bacterial colonization\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      . After oral immunization, the combination of chDCpep and\n                      <jats:italic toggle=\"yes\">focA<\/jats:italic>\n                      gene deletion efficiently improved the humoral and mucosal immune responses. Additionally, CD4 and CD8 T cell proliferation in the chicken spleen significantly increased, with elevated intracellular interleukin-4 and interferon-\u03b3 mRNA levels. After the virus challenge, the virus titers in the lungs and trachea of the chickens appeared to significantly decrease, as did the production of the inflammatory cytokines IL-6 and IL-1\u03b2. Moreover, improved body weight gain and histological lesions were observed. In summary, the combination of chDCpep and\n                      <jats:italic toggle=\"yes\">focA<\/jats:italic>\n                      deletion provides a novel option for the design of a\n                      <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                      -based oral vaccine against avian influenza virus infection.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      A\n                      <jats:italic toggle=\"yes\">focA<\/jats:italic>\n                      gene deletion was introduced into\n                      <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                      with delayed lysis, resulting in increased cellular escape and colonization. Afterward, a chicken DC-targeting peptide was ligated to the tetramer neuraminidase protein of the H9N2 influenza virus. The combination of the\n                      <jats:italic toggle=\"yes\">focA<\/jats:italic>\n                      gene mutation and chicken DC targeting effectively enhanced the humoral, cellular, and mucosal immune responses, yielding improved protection against H9N2 challenge in chickens.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01421-25","type":"journal-article","created":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T12:00:33Z","timestamp":1761566433000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["Protection against H9N2 challenge in chickens via\n                    <i>Salmonella<\/i>\n                    with a\n                    <i>focA<\/i>\n                    mutation delivered by a dendritic cell-targeted avian influenza virus NA protein tetramer"],"prefix":"10.1128","volume":"13","author":[{"given":"Chongbo","family":"Ge","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dmhhd41","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Animal Medicine, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Provincial Key Laboratory of Animal Microecology and Healthy Breeding, Engineering Research Center of Microecological Vaccines (Drugs) for Major Animal Diseases, Ministry of Education, Jilin Agricultural University","place":["Changchun, China"]},{"id":[{"id":"https:\/\/ror.org\/024kjbt21","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Modern Agriculture, Changchun Polytechnic University","place":["Changchun, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Sun","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dmhhd41","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Animal Medicine, 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