Intimin, Tir, and Shiga Toxin 1 Do Not Influence Enteropathogenic Responses to Shiga Toxin-Producing Escherichia coli in Bovine Ligated Intestinal Loops

Abstract

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...

Efa-1/LifA mediates intestinal colonization of calves by enterohaemorrhagic Escherichia coli O26 : H– in a manner independent of glycosyltransferase and cysteine protease motifs or effects on type III secretion

“…Such phenotypes may reflect reduced bacterial load owing to the role of Efa-1/LifA in intestinal colonization rather than direct effects. We have developed a bovine ligated ileal loop model to evaluate the role of bacterial and host factors in the induction of intestinal inflammatory and secretory responses by EHEC (Stevens et al, 2002a;Vlisidou et al, 2004). Consistent with these studies, fluid secretion (Fig.…”

“…Plasmid pDM4 is a positiveselection suicide replicon (oriR6K mobRP4 cat sacBR; Milton et al, 1996) and was used to create chromosomal deletions by double homologous recombination. Plasmid pCVDDstx1 was similarly used to create a deletion spanning stx1AB (Stevens et al, 2002a). Plasmid pDK46 encoding the c, b and exo genes of bacteriophage l under the control of an arabinose-inducible promoter (Datsenko & Wanner, 2000) was obtained from the E. coli Genetic Stock Center, Yale University.…”

“…Fluid accumulation was analysed after incubation of the loops for 12 h in situ and is recorded as the LSM (±standard error of the LSM) volume (V, ml)/loop length (L, cm) ratio. Neutrophil recruitment was measured by isolation of polymorphonuclear (PMN) cells from jugular blood, labelling with 111 In oxinate followed by reinjection into the donor calf as described previously (Stevens et al, 2002a). PMN influx denotes the ratio of total cradioactivity associated with the contents and mucosa of test loops relative to negative control loops inoculated with sterile medium.…”

“…EHEC O26 : H-strain 193 nal R was selected for this study as it efficiently colonizes the intestines of calves (van Diemen et al, 2005(van Diemen et al, , 2007. A deletion spanning the stx1AB genes was first created using the positive-selection suicide replicon pCVDDstx1, as described previously (Stevens et al, 2002a). The resulting strain (193 nal R Dstx1) was verified by Southern hybridization and PCR with primers Stx1-3 and Stx1-6 (Table 2), and was not cytotoxic to VERO cells (data not shown).…”

“…Procedures for construction of bovine ligated ileal loops under terminal general anaesthesia are described in detail elsewhere (Stevens et al, 2002a). Each strain was tested in triplicate loops in a semi-randomized order in each of four separate calves.…”