Pathogenic Role of SEF14, SEF17, and SEF21 Fimbriae in Salmonella enterica Serovar Enteritidis Infection of Chickens

Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), and fimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14 2 strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed. Salmonellosis is the third most commonly reported infectious disease in the United States. Since the middle 1980s, most human cases reported to the National Salmonella Surveillance System administered by the Centers for Disease Control and Prevention have been attributed to Salmonella enterica serovar Enteritidis. More than 80% of food-borne serovar Enteritidis outbreaks reported since 1985 are associated with the consumption of raw or undercooked eggs (16). Salmonella species enter the host by invading the gastrointestinal mucosa, a step essential for pathogenesis (13). Fimbriae have been shown to be involved in colonization and in adherence to specific host target tissues in the early stages of infection (5, 14, 15). Studies involving fimbriae have drawn considerable interest because fimbriae can serve as potential immunogens against many pathogenic bacteria that colonize epithelial surfaces (3‐5, 20). Currently, we are exploring the use of fimbrial antigens as vaccine candidates. However, the role of fimbriae in pathogenesis of serovar Enteritidis is poorly understood. More knowledge is necessary to clarify the role of serovar Enteritidis fimbriae in vaccines designed to reduce the colonization and prevalence of this bacterium in poultry. Serovar Enteritidis produces at least five distinct fimbriae, namely SEF14 and SEF17 (type 1 fimbriae) and SEF21, LPF, and PEF (28). Of all serovar Enteritidis fimbriae, SEF14 has been studied the most. SEF14 has been shown to contribute to serovar Enteritidis adherence to mouse epithelial cells, and passive administration of SEF14 antibodies is protective in mice (26). However, in vitro and in vivo studies using isogenic mutants of serovar Enteritidis have demonstrated no role for SEF14 in pathogenesis (25, 28). Information regarding the role of SEF17 and SEF21 fimbriae in serovar Enteritidis pathogenesis is lacking. Because the factors which influence fimbrial phase variation in vivo and which influence variability in the fimbriation of cells carrying an intact fimbrial operon are not known, it is difficult to evaluate the role of fimbriae in pathogenesis. Therefore, a genetic approach was used in this study to investigate the role of the three known serovar Enteritidis fimbrial operons, sefA (7), agfA (10), and fimA (22), which encode SEF14, SEF17, and SEF21 fimbriae, respectively. The ability of these sefA, agfA, and fimA mutant strains to mediate colonization and invasion was assessed in vitro and in a chicken model. Bacterial strains, plasmids, and growth conditions. Serovar Enteritidis phage type 4 strain was originally isolated from the liver of a laying chicken (18). A spontaneous nalidixic-acidresistant (Nal r ) mutant was obtained by plating on a nalidixicacid-containing Luria-Bertani (LB) agar plate and was used as the recipient strain for conjugation. Escherichia coli S17-1/lpir was used as the donor strain. Inactivation of fimbrial genes was carried out by using suicide plasmid vector PKNOCK-Km (2). pGEM-T plasmid vector (Promega, Madison, Wis.) was used for cloning PCR fragments. Bacterial strains were grown either