Phagocytosis Assay
Phagocytosis assay was performed as described previously [13, 14]. Briefly, S. equi SEM1 and GAS bacteria from exponential growth phase in THY were washed with phosphate-buffered saline (PBS) and labeled with 0.75 μg/ml FITC in PBS at 37°C for 20 min. The labeled bacteria were washed and resuspended at 1 x 109 cfu/ml in PBS. Ten μl of the labeled bacteria were mixed with 100 μl of non-immune heparinized horse or human blood with or without 100 μg/ml SeMac and incubated with gentle shaking at 37°C for 5, 10, or 20 min. The samples were immediately processed using an Immunolyse Kit (Beckman Coulter) according to the manufacturer’s protocol and analyzed by flow cytometry. The percentage of PMNs with fluorescent bacteria was used as a measure of phagocytosis efficiency.

Other Assays
To assess IgG endopeptidase activity of SeMac, human, mouse or horse IgG (20 μg) was incubated with E. coli lysate containing SeMac proteins or 1 μg purified SeMac or GAS M1 Mac in PBS at 37° for 90 min, and the reaction mixture was analyzed by SDS-PAGE. Western immunoblot analysis, which was performed as described previously [15], was used to assess in vitro SeMac production and the presence of SeMac-specific antibody in the horse and mouse convalescent sera. Culture supernatant proteins of S. equi, which were used to assess the in vitro SeMac production, were prepared by the method of Lei et al. [16].