AimsTo perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. Methods and ResultsThe potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly- and hly-), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 713% of the Staph.aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (616%) possessed all lukD-E/lukM/lukPV83 genes, 123 (477%) harboured both hly-/hly- genes, whereas almost all (973%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (694%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. ConclusionsIn this search, several putative virulence determinants have been identified in ovine Staph.aureus isolates collected in Sardinia. Significance and Impact of the StudySome of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis.