Abstract #515

# 515
The efficacy of PlyC endolylsin as an alternative therapy for Streptococcus uberis mastitis in vitro.
Sara Linden1, Parimala Sharma1,2, Kasey M. Moyes*2, Daniel C. Nelson1,3, 1University of Maryland, College Park, MD, 2Institute for Bioscience and Biotechnology Research, Rockville, MD, 3Department of Veterinary Medicine, College Park, MD.

The bacteriophage endolysin, PlyC, displays antimicrobial activity against select streptococcal species and may be a promising new therapy for S. uberis-associated mastitis. This study investigated the antimicrobial activity of PlyC against a broad spectrum of S. uberis isolates, established an in vitro dose response, measured ex vivo binding and antimicrobial efficacy in S. uberis infected milk, and assessed safety and response to antibodies. For antimicrobial activity, 2 μg/mL of PlyC was incubated with 7 different strains of S. uberis isolated from cows with mastitis as well as ATCC 27958 and BAA-854 (strain 0140J). For dose-response studies, PlyC (0–16 μg/mL) were incubated with 27958. The linear proportion of the initial lytic velocity was measured using a spectrophotometric turbidity reduction assay for both antimicrobial activity and dose-response assays. To demonstrate streptococcal-specific binding in raw cow’s milk, 20 μg of AlexaFluor-555-labeled PlyC was incubated with milk containing BAA-854 and binding was visualized via fluorescent microscopy. Finally, polyclonal antibodies against PlyC (pre-immune sera and hyper immune sera [titer ranging from 1:10–1:10,000]) were mixed with 32 μg/mL of PlyC and activity was measured using the spectrophotometric turbidity reduction assay. All data were statistically analyzed using the Student’s t-test. Our results showed that PlyC possessed potent lytic activity (P < 0.01) against all strains of S. uberis tested. The dose-response profile demonstrated lytic activity (P < 0.01) at concentrations ranging from 1 to 16 µg/mL. Microscopy results indicated that PlyC specifically labeled cell walls of BAA-854 in raw milk (P < 0.01). In addition, PlyC retains full lytic activity (P < 0.01) against 27958 in the presence of high titer anti-PlyC antibodies, suggesting that even if antibodies are present, they are non-neutralizing. Taken together, the results indicated PlyC has the potential to be used as a novel therapeutic against S. uberis-associated bovine mastitis via its specificity against several strains of S. uberis, the components in milk do not interfere with activity, and it is not inhibited in the presence of antibodies.

Key Words: mastitis, PlyC, treatment