Recombinant SeMac and In Vitro and In Vivo Expression of SeMac 
To characterize SeMac, the fragment of S. equi mac gene encoding mature SeMac was cloned, and recombinant SeMac was purified to >95% purity as assessed by SDS-PAGE (Fig. 3A).
To assess the in vitro production of SeMac, culture supernatant of the 10 S. equi strains was prepared by the method of Lei et al. [16], resolved by SDS-PAGE, and probed by Western immunoblot with anti-SeMac mouse antisera.
No SeMac was detected in all the samples (data not shown), suggesting that SeMac is not produced in vitro.
To test whether SeMac is produced in vivo during S. equi infection, the presence of SeMac-specific antibody was assessed by Western immunoblot analysis with convalescent sera from 3 horses suffered from strangles and mice with experimental S. equi infection.
All the convalescent sera tested had SeMac-specific antibody (Fig. 3B), indicating that SeMac is produced in vivo during infection.
