Adenoviral-Mediated Transfer of a Lysostaphin Gene into the Goat Mammary Gland

• Published in: Journal of dairy science Vol. 85; no. 7; pp. 1709 - 1716
• Main Authors: Fan, W., Plaut, K., Bramley, A.J., Barlow, J.W., Kerr, D.E.
• Format: Journal Article
• Language: English
• Published: Savoy, IL Elsevier Inc 01.07.2002; Am Dairy Sci Assoc; American Dairy Science Association
• Subjects: Adenoviridae - genetics; adenovirus; Animal bacterial diseases; Animal productions; Animals; Anti-Bacterial Agents - biosynthesis; Bacterial diseases; Biological and medical sciences; Biotechnology; Female; Fundamental and applied biological sciences. Psychology; gene therapy; Gene transfer; Gene Transfer Techniques; Genetic engineering; Genetic technics; Genetic Vectors; Goat Diseases - microbiology; Goat Diseases - prevention & control; Goats; Infectious diseases; lysostaphin; Lysostaphin - biosynthesis; Mammary Glands, Animal - cytology; Mammary Glands, Animal - physiology; mastitis; Mastitis - prevention & control; Mastitis - veterinary; Medical sciences; Methods. Procedures. Technologies; Peptides; Staphylococcal Infections - prevention & control; Staphylococcal Infections - veterinary; Staphylococcus aureus - genetics; Synthetic digonucleotides and genes. Sequencing; Terrestrial animal productions; Vertebrates; Ad-lacZ; X-gal; mastitis; adenovirus; lysostaphin; BME-UV; hGH; gene therapy; Ad-lys; lacZ; Adenoviridae; Ruminant animal; Metalloendopeptidases; Veterinary medicine; Goat; Mammary gland; Recombinant protein; Ungulata; Dairy animal; Mastitis; Farming animal; Enzyme; Treatment efficiency; Experimental study; Milk production; Genetic transfer; Infection; Virus; Mammary gland diseases; Peptidases; Vertebrata; Lysostaphin; Mammalia; Treatment; Bacteriosis; Hydrolases; Artiodactyla; Gene therapy
• ISSN: 0022-0302; 1525-3198
• DOI: 10.3168/jds.S0022-0302(02)74244-6

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, Gln125, 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 48h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125, 232-lysostaphin (0.8μg/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 1010 plaque-forming units (pfu) of Ad-lacZ by two intramammary infusions given 48h apart. The animals were euthanized 24h 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 1010 pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48h postinfusion. In both animals, extensive expression of β-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125, 232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24h postinfusion, increasing to approximately 1μg/ml at 48h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125, 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.