Contribution of Bordetella bronchiseptica Filamentous Hemagglutinin and Pertactin to Respiratory Disease in Swine

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract.Respiratory disease in pigs is the most important health concern for swine producers today. According to the 2006 NAHMS survey, respiratory disease was the greatest cause of mortality in swine, accounting for 53.7% of nursery deaths and 60.1% of deaths in grower/finisher pigs (68). Bordetella bronchiseptica is widely prevalent in swine populations and contributes to multiple pathologies in respiratory disease. In very young pigs it causes severe bronchopneumonia with high morbidity and, if untreated, mortality. It is a primary etiologic agent of atrophic rhinitis, causing a moderate to mild reversible form, and promotes colonization by toxigenic strains of P. multocida, usually leading to severe, progressive atrophic rhinitis (11, 12). B. bronchiseptica is frequently found in nasal turbinates and lung lesions of fattening pigs who may not exhibit clinical signs of respiratory disease. Nonetheless, field surveys document that subclinical pneumonia can result in substantial economic losses due to slower weight gain, increased days to market, and reduced feed efficiency (4, 22). In addition, B. bronchiseptica infections increase the severity of respiratory disease associated with other bacterial and viral pathogens and is thus a main contributing agent in porcine respiratory disease complex, a multifactorial disease state that is consistently listed as a top research priority by the National Pork Board (3, 6, 7, 10, 11, 72).Infection begins with colonization of the ciliated epithelial cells of the upper respiratory tract. Two well-studied Bordetella virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). Both FHA and PRN are regulated by the BvgAS signal transduction system, which controls the expression of virulence determinants involved in the Bordetella infectious cycle (14). Numerous in vitro studies have demonstrated that FHA functions as an adhesin and contains several different binding domains (2, 16, 25, 27, 28, 40-42, 58, 63, 66-67, 69-71). These domains include a heparin-binding domain that facilitates binding to sulfated polysaccharides (23), a carbohydrate-recognition domain that promotes binding to ciliated epithelial cells of the respiratory tract and macrophages (52), and an Arg-Gly-Asp (RGD) domain. The RGD domain has been shown to play a key role in the upregulation of intercellular adhesion molecule 1 by epithelial cells, through an NF-κβ signaling pathway, by interacting with very late antigen-5 (28, 29). This RGD domain also plays an important role in the upregulation of CR3 binding activity by interacting with the leukocyte response integrin/integrin-associated protein located on monocytes and macrophages (27). In addition, FHA of B. bronchiseptica has been shown to be required for colonization of the rat trachea (16).PRN belongs to the type V autotransporter protein family and, similar to FHA, contains an RGD domain as well (18). Several in vitro studies have demonstrated PRN to function as an adhesin (19, 32, 36, 71); however, an exact host receptor has not been identified. The role of PRN as a protective immunogen is more clearly defined. Active immunization with purified PRN has been shown to provide protection against mortality and reduce pathology and lung colonization in mice and pigs challenged with Bordetella, and passive transfer of a PRN-specific monoclonal antibody has also been shown to provide protection in mice (33, 43, 46, 60).The overwhelming majority of studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Swine isolates of B. bronchiseptica possess unique genetic and phenotypic traits relative to isolates from other host species (20, 44, 55). Recent experiments in rats demonstrate that attachment is a multifactorial process (16, 40), and this is also likely to be true in swine. However, no definitive data exist with respect to swine, either in vivo or with swine tissue or cells in vitro. In this report, we investigate the role of FHA and PRN in Bordetella pathogenesis in swine, by constructing two mutants containing an in-frame deletion of the FHA or the PRN structural gene in KM22, a virulent B. bronchiseptica swine isolate. We compare both of these mutants to KM22 for their ability to mediate adherence in vitro and to colonize and cause disease during respiratory infection in swine.