| Summary: | Avian pathogenic <i>Escherichia coli</i> (APEC) infection in poultry causes enormous economic losses and public health risks. Bacterial outer membrane vesicles (OMVs) and nano-sized proteolipids enriched with various immunogenic molecules have gained extensive interest as novel nanovaccines against bacterial infections. In this study, after the preparation of APEC O2-derived OMVs (APEC_OMVs) using the ultracentrifugation method and characterization of them using electron microscopy and nanoparticle tracking analyses, we examined the safety and vaccination effect of APEC_OMVs in broiler chicks and investigated the underlying immunological mechanism of protection. The results showed that APEC_OMVs had membrane-enclosed structures with an average diameter of 89 nm. Vaccination with 50 μg of APEC_OMVs had no side effects and efficiently protected chicks against homologous infection. APEC_OMVs could be effectively taken up by chicken macrophages and activated innate immune responses in macrophages in vitro. APEC_OMV vaccination significantly improved activities of serum non-specific immune factors, enhanced the specific antibody response and promoted the proliferation of splenic and peripheral blood lymphocytes in response to mitogen. Furthermore, APEC_OMVs also elicited a predominantly IFN-γ-mediated Th1 response in splenic lymphocytes. Our data revealed the involvement of both non-specific immune responses and specific antibody and cytokine responses in the APEC_OMV-mediated protection, providing broader knowledge for the development of multivalent APEC_OMV-based nanovaccine with high safety and efficacy in the future.
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