| Summary: | <i>Gallibacterium anatis</i> is a Gram-negative opportunistic avian pathogen representing an emerging threat to poultry meat and egg production worldwide. To date, no vaccine able to effectively prevent the morbidity associated with <i>G. anatis</i> infections has been developed yet. Our group previously reported that inoculation of different combinations of <i>G. anatis </i>outer membrane vesicles (OMVs), FlfA and GtxA-N proteins is effective in preventing lesions caused by <i>G. anatis </i>infections in layer chickens. Here we report the testing of the efficacy as vaccine prototypes of <i>G. anatis </i>OMVs isolated by hydrostatic filtration, a simple technique that allows the cost-effective isolation of high yields of OMVs. Layer chickens were immunized with OMVs alone or in combination with FlfA and/or GtxA-N proteins. Subsequent challenge with a heterologous <i>G. anatis </i>strain showed that immunization with OMVs alone could significantly reduce the lesions following a <i>G. anatis </i>infection. A second study was carried out to characterize the dose-response (0.25, 2.5 and 25 µg) relationship of <i>G. anatis </i>OMVs as immunogens, showing that 2.5 μg of OMVs represent the optimal dose to elicit protection in the immunized animals after a similar challenge. Additionally, administration of ≥2.5 μg of <i>G. anatis </i>OMVs induced specific IgY titers and possibly vertical transfer of immunity.
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