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Streptococcus equi subsp. zooepidemicus (Sz) is an important opportunist pathogen of the equine respiratory and reproductive tracts. It is highly variable with respect to sequence type and virulence factors including SzP and SzM. Recent studies in the UK have shown that many Sz strains host genes for mitogenic superantigens including szeF, szeN, szeP, seeI, seeL, and seeM. The aims of the present study were to estimate the prevalence of mitogenicity in equine Sz in North America and establish whether mitogenicity is more likely to be associated with isolates from a specific site of infection. Twenty-six percent (23/90) of strains randomly selected from over 600 Sz isolated in the United States from 1969 to 1994 were mitogenic for equine peripheral blood mononuclear cells. All but five of these mitogenic Sz encoded one or more of the superantigen genes szeF, szeN, szeP, seeI, seeL, and seeM. Homologues of seeH in S. equi were not detected in any Sz strain. Polymerase chain reaction analysis revealed the presence of seeI, szeF, szeN, or szeP in 91% (10/11) of isolates from lymph node abscesses confirming earlier reports of a significant association of superantigens with lymph node abscessation in the UK. In contrast, only 24% (4/17) of Sz isolates from the reproductive tract were mitogenic and/or hosted seeL, seeM, szeF, szeP, or szeN.Twenty-six percent of randomly selected Streptococcus zooepidemicus strains from equines in North America were mitogenic.Superantigen genes of these mitogenic strains are similar to those in Europe.Isolates from lymph node abscesses were very likely to host superantigen genes.Isolates from the reproductive tract were unlikely to host these genes.