The Early Dynamic Response of the Calf Ileal Epithelium toSalmonella typhimurium

Abstract

Ileal loops including Peyer's patch were prepared in five 28-day-old calves and infused Salmonella typhimurium strain ST4/74. Loops were fixed 5 minutes to 2 hours after inoculation, and the mucosa was examined by light and electron microscopy. Within 5 minutes, the bacteria were interacting with the follicle-associated epithelium (FAE); the surface of M cells changed to lamellipodia, engulfing many bacteria. This process proceeded rapidly to 30 minutes, involving most M cells above crypt level. Most cells were exfoliated, and many were packed with bacteria, and the domed villi became stunted. There was a rapid migration of neutrophils through the FAE into the lumen by 15 minutes. By 60 minutes, there was no further interaction between the bacteria and the FAE; at this time bacteria were present in macrophages in the lamina propria. Restitution of the FAE was complete by 2 hours in spite of the many bacteria in the cell debris overlying the epithelium. Interaction of bacteria with the absorptive villi was delayed compared with interaction with the FAE. After 15 minutes, bacteria were seen adhering to some enterocytes of the upper third of the villi; many bacteria were adhering to the surface of the enterocytes at 20 and 30 minutes, but few were seen thereafter. Adherence was patchy and largely confined to cells whose surfaces were depressed relative to others. The microvillous surface of these enterocytes was extensively remodelled. Tissue response, with uptake of bacteria into vacuoles, exfoliation of enterocytes containing bacteria, and subsequent stunting of the villi, began at 30 minutes and was severe and progressive to 2 hours. Following the initial attachment and uptake of the bacteria loss of enterocytes progressed from these initial sites; bacteria were associated with the lateral cell membrane of cells adjacent to cells being extruded and not with the microvilli of cells at new sites. In a calf 4 hours after dosing orally with the same strain, M cells were engulfing bacteria and their cell surface was changed as seen in the inoculated loops; absorptive enterocytes were also taking up bacteria as seen in the ileal loops, indicating the process seen in the loops and after oral dosage was similar. For this strain of S typhimurium, there was an initial concentration of bacilli around the domed villus epithelium. This distribution was not random but may have resulted from a specific attraction to the FAE.

An improved method for preparing thick sections for immuno/histochemistry and confocal microscopy and its use to identify rare events

“…These results confirm previous observations of Salmonellainfected bovine intestines using conventional immunohistochemistry of NBF-fixed, wax-embedded sections and transmission electron microscopy (Watson et al, 1995;Frost et al, 1997). However, unlike the previous studies, this technique can now be extended to describe the phenotype of the intestinal cells associated with the invading Salmonella.…”

Comparative study of the invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin for Caco-2 cells, HEp-2 cells and rabbit ileal epithelia

“…Invasiveness of Salmonella serotypes for gut has been studied in vivo in several animal species including guinea-pigs [1], monkeys [2], rabbits [3], cattle and pigs [4,5], chickens [6] and mice [7]. Also, successful attempts have been made to study invasiveness in gut explants of rabbit ileal mucosa [8,9], although less success was achieved with corresponding porcine and bovine explants because of technical dif®culties in handling these tissues (unpublished data).…”

Salmonella: Invasion, Evasion & Persistence

“…However, several types of bacteria and viruses are known to exploit M cells by selectively adhering to the apical surface, internalising themselves and evading mucosal immune responses (Neutra et al, 1996;Sansonetti & Phalipon, 1999). Numerous studies have shown both S. Typhi (Kohbata et al, 1986;Pascopella et al, 1995) and S. Typhimurium (Clark et al, 1994;Jones et al, 1994) preferentially target and invade mouse M cells and that S. Typhimurium also invades M cells in calf ileal loops (Frost et al, 1997). Despite these reports, there is a lack of direct evidence that human M cells are a major site of Salmonella invasion, although it is widely speculated that sites of Salmonella evasion in humans parallel mice and calf studies.…”

“…Membrane ruffles are a crucial determinate of salmonella invasion, as demonstrated in cultured epithelial cell lines (Galan & Collmer, 1999). The membrane 'ruffler' formation results from extensive rearrangement of M cell apical membranes due to the redistribution of polymerised actin to form membrane protrusions (Clark et al, 1994;Frost et al, 1997;Jones et al, 1994). This process is triggered by a active process in which the ultimate result is engulfment of Salmonella.…”