SummaryThe severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S . Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signaturetagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S . Typhimurium colonization of chicks. Transposon insertions in SPI-4 compromised S . Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI-4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many 'housekeeping' genes and genes of unknown function. We conclude that S . Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.

Salmonella enterica is a facultative intracellular pathogen of worldwide importance and causes a spectrum of diseases depending on serovar-and host-specific factors. Oral infection of pigs with S. enterica serovar Typhimurium strain 4/74 produces acute enteritis but is rarely fatal, whereas serovar Choleraesuis strain A50 causes systemic disease with a high mortality rate. With a porcine ligated ileal loop model, we observed that systemic virulence of serovar Choleraesuis A50 is not associated with enhanced intestinal invasion, secretory responses, or neutrophil recruitment compared to serovar Typhimurium 4/74. The net growth in vivo of serovar Choleraesuis A50 and serovar Typhimurium 4/74 was monitored following oral inoculation of pigs with strains harboring pHSG422, which exhibits temperature-sensitive replication. Analysis of plasmid partitioning revealed that the enteric virulence of serovar Typhimurium 4/74 relative to that of serovar Choleraesuis A50 is associated with rapid replication in the intestinal wall, whereas systemic virulence of serovar Choleraesuis A50 is associated with enhanced persistence in intestinal mesenteric lymph nodes. Faster replication of serovar Typhimurium, compared to that of serovar Choleraesuis, in the intestinal mucosa was associated with greater induction of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-8 (IL-8), and IL-18 as detected by reverse transcriptase PCR analysis of transcripts from infected mucosa. During replication in batch culture and porcine alveolar macrophages, transcription of genes encoding components of type III secretion systems 1 (sipC) and 2 (sseC) was observed to be significantly higher in serovar Typhimurium 4/74 than in serovar Choleraesuis A50, and this may contribute to the differences in epithelial invasion and intracellular proliferation. The rapid induction of proinflammatory responses by strain 4/74 may explain why pigs confine serovar Typhimurium infection to the intestines, whereas slow replication of serovar Choleraesuis may enable it to evade host innate immunity and thus disseminate by stealth.

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype-and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle.Shiga toxin-producing Escherichia coli (STEC) comprises an emerging group of zoonotic enteric pathogens (41). In humans, STEC infections result in bloody or nonbloody diarrhea, which may be complicated by hemorrhagic colitis and severe renal and neurological sequelae, including hemolytic uremic syndrome (HUS) (45, 55). The pathogenesis of HUS is believed to involve the translocation of Shiga toxin (Stx1 and/or Stx2; also called verocytotoxins), produced by STEC in the intestinal lumen, to systemic sites, where they cause microvascular endothelial cell damage, fibrin-platelet thrombus formation, and thrombocytopenia (45).Cattle are an important reservoir of STEC (25), and human infections are frequently associated with the consumption of contaminated beef and dairy products (26). Severe cases of infection of susceptible calves with bovine virulent STEC strains may result in atrophy of the villi, epithelial cell damage, diffuse infiltration of neutrophils into the lamina propria and intestinal lumen, and the formation of a pseudomembrane containing blood, fibrin, cellular debris, and neutrophils (29,46). A similar histopathology occurs in some cases of STECassociated hemorrhagic colitis in humans (27,55). An understanding of the induction of intestinal inflammatory responses to STEC is important in the development of strategies to reduce the prevalence of STEC in cattle. By implication, such strategies may lower the incidence of human STEC infections.Infection of the gastrointestinal tracts of conventional cattle and 5-day-old gnotobiotic calves by STEC serotype O157:H7 is asymptomatic (5, 63). However, E. coli O157:H7 is frequently associated with human disease, and the molecular basis of STEC serotype host specificity is unknown. Infection of neonatal colostrum-deprived calves with E. coli O157:H7 may result in enterocolitis similar to that s...