Characterization of leukocytotoxic and superantigen-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leu kotoxins and superantigens) are supposed to play an important role in the i nitiation and/or the exacerbation of this disease. In order to detect all k nown and putative members of leukotoxins and SAgs (superantigens), we teste d secreted factors of different S. aureus isolates in flow cytometry-based assays. Isolates were sampled from 68 cows of different farms and cultured for 24 h in vitro. Supernatants were then coincubated with purified polymorphonucle ar granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate super natants for bovine MNC. Based on these criteria, the supernatants of S. aur eus isolates fell in three groups. The first group (n = 32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrop hils and eosinophils) in vitro. The second group of supernatants (n = 20), termed SAg-SN (superantigen-containing supernatants), induced activation an d proliferation of mononuclear cells (MNC) and, only in the presence of MNC , resulted in a selective depletion of neutrophils after 24 h in vitro. The third group of supernatants (n = 16) contained neither LTs or SAgs. Functi onally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was con firmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic acti vity, but did not reveal any MNC-activating potential. This study, therefor e, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be di fferentiated by the functional assays described. The prevalence of leukotoxin- or superantigen-producing isolates was compar able among cattle with subclinical (LT = 41%; SAg = 30.8%) mastitis. The hi gher frequency of LT-producing isolates in cases of clinical mastitis (LT = 55.2%; SAg = 27.6%) was not significant. At least, these findings argue ag ainst the dominant role of superantigens or leukotoxins in S. aureus-induce d bovine mastitis. (C) 2001 Elsevier Science B.V. All rights reserved.