Relative distribution of virulence-associated factors among Australian bovine Staphylococcus aureus isolates: Potential relevance to development of an effective bovine mastitis vaccine

Abstract

Staphylococcus aureus is one of the major contagious pathogens causing bovine mastitis worldwide.1 It causes contagious mastitis resulting either clinical or subclinical mastitis with increase in the number of somatic cell count (SCC) in milk. More than $130 million is lost by the Australian dairy farmers ($A200/cow/year) every year due to poor udder health caused by mastitis resulting in reduction of milk production, increase in treatment costs, veterinary consultation fees, and number of cow culls. There are multiple pathogens that have been found to be associated with bovine mastitis in Australia.2 While the relative distribution of the different pathogens causing mastitis may differ in different regions and countries, S. aureus is one of the most significant contagious bacterial pathogens causing bovine mastitis and is of concern to public health because of its potential for transmission to humans. Once the organism enters into the mammary gland, it adheres to epithelial lining and defies the host innate immune defenses by variety of virulence factors such as capsule and protein A which interfere with the process of phagocytosis.3 Once intra-mammary infection is established, damage to the mammary gland epithelial lining is initiated by ulceration and occlusion of lactiferous ducts and alveoli, infiltration of inflammatory cells in the parenchyma.4 S. aureus produces a variety of virulence factors which evade the tissue and host immune system and thereby maintain infection. These virulence factors are capsular polysaccharides, cytotoxins, superantigenic enterotoxins and MSCRAMM (microbial surface components recognizing adhesive matrix molecules). A large number of cytotoxins are produced by S. aureus which form pores in the cell membrane causing osmotic swelling leading to cell death. These cytotoxins include leukocidins, phenol soluble modulins (PSMs) and cytolysins. The cytolysins of S. aureus are α-, β-, γ-, and δ-toxins, of which α-toxin is well characterized for its contribution to biofilm formation and protective potential.5,6 β-toxin is a sphingomyelinaseC and 95% of S. aureus isolates from bovine mastitis cases produce β-toxin7 which causes damage to epithelial lining of mammary gland. Gamma (γ) and delta (δ) toxins, bicomponent toxins are synergohymenotropic toxins that act through the synergistic activity of 2 non-associated secretory proteins creating lytic pores in host cells including neutrophils and are assembled from the 2 components secreted separately by the organism as water-soluble molecules.8 Panton-Valentine Leukocidin (PVL) is encoded by 2 contiguous and cotranscribed genes, LukS-PV and LukF-PV9 and creates lytic pores in neutrophils, monocytes and macrophages adversely affecting their function. Phenol soluble modulins (PSMs) are the peptides produced by S. aureus, which are cytotoxic and proinflammatory agent. Recent finding has demonstrated that it plays a part in the formation of S. aureus biofilm.10 S. aureus produces a number of superantigens including enterotoxins (SEs), Toxic Shock Syndrome toxin and exfoliative toxins. Enterotoxins of S. aureus include the classical enterotoxins A to E and the recently identified and characterized SEG-SEU toxins.11,12 These antigens are considered as superantigens due to their ability to release inflammatory cytokines from both T cells and macrophages by binding to the surface of MHC class II proteins and T cell receptors. 13-16 The first step in establishing infection is the initial attachment of S. aureus to eukaryotic membrane and extracellular matrix proteins which is followed by colonization and subsequent infection.17 Colonization is commonly associated with a variety of adherence factors or adhesins which are known as microbial surface component recognizing adhesive matrix molecules (MSCRAMM). There are over 20 different MSCRAMMs identified, which can be expressed in S. aureus18 that mediate attachment to surface proteins of host cells including collagen, elastin, fibrinogen, thrombospondin, fibronectin, bone sialoprotein and laminin.19 Major adhesins in this group that mediate the initial attachment of S. aureus to the bovine mammary gland, providing the first critical step for establishing infection19 are clumping factors A and B (ClfA and ClfB),20 collagen adhesion (CNA),21 bone sialo binding protein (BBP)22 and the fibronectin binding proteins A and B (FnBPA and FnBPB).23 Besides these major adhesins, biofilm-associated protein (bap) has also been reported to be associated with primary attachment of S. aureus to mammary tissue.24,25 An accessory gene, agr, regulates the production of biofilms including detachment of biofilm that helps in virulence and dissemination of S. aureus in the mammary gland resulting in persistent bovine mastitis,26 whereas penicillin resistance of S. aureus is mediated by blaZ gene.27 Variability in the prevalence of virulence factors in S. aureus may result in various levels of severity and forms of mastitis in cows.28 No studies have been carried out in Australia on the virulence factors of S. aureus isolated from clinical cases of bovine mastitis. Aim of this study was to determine the relative distribution of different virulent factors of bovine S. aureus isolates in Australia including MSCRAMMS and exotoxins using conventional polymerase chain reaction (PCR) and the available serological methods.