Relationship between virulence factor genes in bovine Staphylococcus aureus subclinical mastitis isolates and binding to anti-adhesin antibodies Melania Scarpa 1 , Renata Piccinini 2 , Paola Brun 1 , Alessia Grillo 1 , Giorgio Palu ` 1 , Carlo Mengoli 1 , Valentina Dapra ` 2 , Ignazio Castagliuolo 1 and Alfonso Zecconi 2 * 1 Department of Histology, Microbiology and Medical Biotechnologies, Padua, Italy. Via Gabelli 63, 35121 Padova 2 Department Animal Pathology, Hygiene & Health, Milan, Italy. Via Celoria 10, 20133 Milano Received 15 July 2009 ; accepted for publication 27 October 2009 ; first published online 24 December 2009 Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. It is characterized by a wide array of virulence factors. The differences among strains jeopardize the development of effective vaccines against Staph. aureus mastitis. We tested the immunogenicity of a peptide subunit vaccine coding for three different adhesion factors, fibrinogen-binding protein (Efb), fibronectin-binding protein A (FnbpA) and clumping factor A (ClfA). Then we evaluated the influence of some virulence factors on the ability of specific anti-adhesin antibodies to react with sixteen Staph. aureus strains isolated from bovine subclinical mastitis. Immunization with the recombinant adhesins stimulated a strong humoural (IgG and IgA) and mucosal IgA immune response in all animals tested. Hyperimmune serum recognized with diverse efficiency the sixteen Staph. aureus strains and this circumstance correlated well with the level of expression of adhesins. Among the different virulence factors considered to classify strains, spa gene polymorphisms showed the strongest influence on isolate reactions to hyper- immune serum. Our results indicate the importance of a disease- and environment-specific analysis of isolates. Thus, as opposed to other pathogens to obtain an effective vaccine we should characterize multiple strains and identify the prevalent virulence factors expressed. Keywords : Staphylococcus aureus, adhesin, gene polymorphism, vaccine. Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis, a disease that causes relevant worldwide losses in the dairy industry (Zecconi et al. 2006a). Antimicrobial therapy is by enlarge the most frequently applied method to control contagious mastitis ; however, the efficacy of this approach to control Staph. aureus mastitis is a matter of debate (Sol et al. 1994; Sol et al. 1997). Besides resistance factors, Staph. aureus expresses a wide array of surface-associated virulence factors such as a polysaccharide capsule and proteins that promote adhesion to mammary tissues and contribute to tissue colonization and resistance to phagocytosis (Foster, 2005). In addition, Staph. aureus secretes various exotox- ins that damage the membranes of leucocytes and favour pathogen survival in host tissues (Schuberth et al. 2001 ; Rainard et al. 2003; von Eiff et al. 2004). Taking into ac- count the wide range and polymorphism of virulence genes present in the genome it is not surprising that Staph. aureus is the most frequently isolated contagious pathogen worldwide (Peacock et al. 2002 ; McLaughlin & Hoogewerf, 2006; Zecconi et al. 2006b; Josefsson et al. 2008). Several reports suggest that virulence factors poten- tially represent excellent targets for vaccine development (Aarestrup et al. 1995; Foster & Hook, 1998; Schuberth et al. 2001) but the heterogeneous expression of these molecules in different Staph. aureus isolates influences not only virulence but could be involved in the variable success of immunization protocols. These issues have contributed to the poor outcome of the field vaccine trials against Staph. aureus mastitis in dairy cows. Therefore the importance of evaluating the combination of Staph. aureus virulence factors is gaining growing attention as an indis- pensable step towards the development of effective vac- cines (Jarraud et al. 2002; Peacock et al. 2002; Shkreta et al. 2004; Haslinger-Loffler et al. 2005). *For correspondence ; e-mail : alfonso.zecconi@unimi.it Journal of Dairy Research (2010) 77 159–167. f Proprietors of Journal of Dairy Research 2009 159 doi:10.1017/S0022029909990598