Molecular epidemiology and immune evasion strategies of ruminant Staphylococcus aureus

Abstract

Infections with the Staphylococcus aureus bacteria are a common cause of mastitis in ruminants, causing major financial losses for the dairy industry. The outcome of these infections ranges from mild subclinical cases of mastitis, to severe clinical cases. It is not fully understood if and how the infecting bacteria influences the clinical severity of mastitis. Therefore, this thesis explored the molecular epidemiology and differences in immune evasion strategies of ruminant S. aureus. By comparing the genetic structure of S. aureus responsible for subclinical and clinical cases of bovine mastitis (collected in The Netherlands and 10 other European countries), a specific subtype of S. aureus (belonging to Clonal Complex (CC) 479) was identified that was associated with clinical mastitis. These bacteria produced increased levels of leukocidin LukMF’. This toxin is a potent killer of ruminant immune cells, offering a likely explanation for the increased virulence of CC479 S. aureus. In addition, genetic analysis of all collected isolates demonstrated that only a relatively small number of S. aureus CCs are responsible for bovine mastitis in 11 European countries, with 5 CCs (CC151, CC97, CC479, CC133 and CC398) representing almost 75% of collected bacteria. These ruminant-associated CCs differed greatly in their carriage of virulence genes (e.g. toxin and antibiotic resistance genes) and their distribution between countries. Although multiple virulence genes were associated with clinical mastitis, including several superantigen genes, it was not possible to differentiate between the effect of individual genes and S. aureus CC on clinical severity since carriage of these genes was highly correlated with specific CCs. Similar studies performed using a collection of S. aureus isolates obtained from mastitis cases in goats and sheep, showed no differences in genotype and LukMF’ production between clinical and subclinical isolates. There were, however, small genetic differences between S. aureus collected from goats and goats. Furthermore, we also demonstrated that mammary epithial cells (MEC), key activators of the immune response of the udder during S. aureus infections, respond differently to various ruminant-associated S. aureus CCs. Stimulation with CC133 S. aureus triggered a stronger release of cytokine IL-8 by the bovine MEC line PS than stimulation with CC151, CC479 and CC425 S. aureus. In addition, a preliminary study using primary caprine and bovine primary MEC showed that caprine and bovine MEC had a different cytokine response to stimulation with a cow-associated (CC479) and small ruminant-associated (CC133) strain. The results of this thesis demonstrated that ruminant-associated S. aureus CCs differ in their carriage of immune evasion and antibiotic resistance genes, expression of LukMF’ and their activation of ruminant MEC. Furthermore, CC479 S. aureus were associated with clinical mastitis and this strongly suggests that the type of infecting bacteria influences the clinical severity of mastitis. Knowledge of the genotype of S. aureus responsible for ruminant mastitis could therefore be useful information for veterinarians and farmers to assist them in increasing the effectiveness of mastitis treatment and reduction strategies on dairy farms.