Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland

Abstract As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln 125, 232 -lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad- lys ) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding β -galactosidase (Ad- lacZ ) was also evaluated. Transduction of cultured bovine cells by Ad- lacZ was confirmed by the presence of β -galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad- lys secreted immunoreactive Gln 125, 232 -lysostaphin (0.8  μ g/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10 10 plaque-forming units (pfu) of Ad- lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of β -galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10 10 pfu of Ad- lys while contralateral udder halves received Ad- lacZ . The animals were euthanized 48 h postinfusion. In both animals, extensive expression of β -galactosidase was detected in Ad- lacZ exposed teats. Immunoreative Gln 125, 232 -lysostaphin was detectable in secretions from Ad- lys exposed glands 24 h postinfusion, increasing to approximately 1  μ g/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln 125, 232 -lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.