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The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMVβ) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme β-galactosidase (β-gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme β-gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against β-gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (β-gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on-farm mass immunization purposes, especially with regard to DNA-based vaccines and small size fish, in which i. m. administration remains unfeasible.Oral administration is the ideal route for mass vaccination in fish.Plasmid-based vaccines are readily inactivated within the digestive tract of fish.If protected in adequate nanoparticles, plasmids can resist feed processing.Feed-delivered plasmids exhibited remarkable in vivo biological activity.Feed pellets enable non-disturbing, efficient oral delivery of plasmid DNA.