Project Number: 1245-31000-103-04
Project Type: General Cooperative Agreement

Start Date: Aug 27, 2010
End Date: Mar 14, 2013

Objective:
Create triple-lytic-domain PG hydrolase-PTD fusions and test in cultured cells. Create triple-lytic-domain-PTD fusions via molecular techniques. Verify that the triple-lytic-domain-PTD fusions maintain three enzymatic activities. Test efficacy of the triple-lytic-domain-PTD fusions against extracellular staphylococci (S. aureus and CoNS) in broth cultures and against internalized S. aureus in cultured bovine mammary epithelial cells. Test efficacy of the Lyso-PTD fusion protein to cure chronic bovine mastitis. Induce S. aureus mastitis in cows and evaluate the triple-lytic-domain-PTD fusion for the ability to cure chronic mastitis via intramammary injections at dry off. Determine if the triple-lytic-domain-PTD kills staphylococci that reside in milk somatic cells. Characterize S. aureus isolates from this study that are resistant to the triple-lytic-domain-PTD. Determine if triple-lytic-domain-PTD fusion can kill staphylococci within mammary abscesses. Examine proteome differences between S. aureus grown in ex vivo milk vs. within chronically infected quarters.

Approach:
Identify pregnant lactating cattle for purchase, house and monitor lactating cattle, infect them with S. aureus, monitor for chronic status of infection, dry-off the cattle, inject the cattle with the antimicrobial fusion protein during dry-off period, birth the calves, and monitor for eradication of pathogen in colostrum and milk following birth.