Bovine mastitis is a costly disease to the dairy industry and intramammary infections (IMI)
with Staphylococcus aureus are a major cause of mastitis. Staphylococcus aureus strains responsible for
mastitis in cattle predominantly belong to ruminant-associated clonal complexes (CCs). Recognition
of pathogens by bovine mammary epithelial cells (bMEC) plays a key role in activation of immune
responsiveness during IMI. However, it is still largely unknown to what extent the bMEC response
di ers according to S. aureus CC. The aim of this study was to determine whether ruminant-associated
S. aureus CCs di erentially activate bMEC. For this purpose, the immortalized bMEC line PS was
stimulated with S. aureus mastitis isolates belonging to four di erent clonal complexes (CCs; CC133,
CC479, CC151 and CC425) and interleukin 8 (IL-8) release was measured as indicator of activation.
To validate our bMEC model, we first stimulated PS cells with genetically modified S. aureus strains
lacking (protein A, wall teichoic acid (WTA) synthesis) or expressing (capsular polysaccharide (CP)
type 5 or type 8) factors expected to a ect S. aureus recognition by bMEC. The absence of functional
WTA synthesis increased IL-8 release by bMEC in response to bacterial stimulation compared to
wildtype. In addition, bMEC released more IL-8 after stimulation with S. aureus expressing CP
type 5 compared to CP type 8 or a strain lacking CP expression. Among the S. aureus lineages,
isolates belonging to CC133 induced a significantly stronger IL-8 release from bMEC than isolates
from the other CCs, and the IL-8 response to CC479 was higher compared to CC151 and CC425.
Transcription levels of IL-8, tumor necrosis factor alpha (TNF ), serum amyloid A3 (SAA3), Toll-like
receptor (TLR)-2 and nuclear factor B (NF- B) in bMEC after bacterial stimulation tended to follow
a similar pattern as IL-8 release, but there were no significant di erences between the CCs. This study
demonstrates a di erential activation of bMEC by ruminant-associated CCs of S. aureus, which may
have implications for the severity of mastitis during IMI by S. aureus belonging to these lineages.