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The importance of the Escherichia coli O157:H7 translocated intimin receptor (Tir) protein in intestinal colonization and the effect of infection with Tir+ strains on protection against subsequent challenge was studied in adult beef cattle. Cattle were orally inoculated (C1) with a Shiga toxin-2++ E. coli O157:H7 strain that was Tir+ or Tir-, and 42 days later were re-challenged (C2) with the nalidixic acid (Nal) R parent strain to test whether prior infection had any effect on fecal shedding. During the first 14 days post-C1, the NalS wildtype (WT) strain was shed at significantly higher levels and for a longer period than the other strains; however, the mean levels of shedding of the NalR and Δtir complemented strains were not significantly different from that of the Tir- strains. The Δtir, Tir complemented mutant, and Δtir vector control strains inadvertently did not express flagellin, and did not effectively colonize the intestine. We were unable to determine whether Tir exposure at C1 had any effect on protection. Further, those given an initial inoculation with a non-flagellated variant of E. coli O157:H7 were more susceptible to a second challenge with motile E. coli O157:H7 than those originally inoculated with motile strains. The cause of the loss of expression of flagellin was not addressed. We suggest that either the flagellum or a factor that regulates both its production and that of some other effector has a significant function in colonization.