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Previous Article | Next Article 
Infection and Immunity, August 2006, p. 4685-4693, Vol. 74, No. 8
0019-9567/06/$08.00+0 doi:10.1128/IAI.00406-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
University of Idaho, Department of Microbiology, Molecular Biology, and Biochemistry, Moscow, Idaho 83844-3052
Received 13 March 2006/ Returned for modification 17 April 2006/ Accepted 17 May 2006
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ABSTRACT |
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92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, 
tir, and eae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa. |
INTRODUCTION |
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Cattle are considered the primary reservoir for E. coli O157:H7 and the most common source for food-borne and direct animal contact infections (5, 25, 69). Healthy cattle carry E. coli O157:H7 transiently without suffering pathological symptoms (2, 4, 26). Individual animals can passively shed E. coli O157:H7 in their feces for a short time (a few days) without establishing a colonized state or can pass fecal E. coli O157:H7 for a longer time (a month or more) if the bacteria colonize and persist (22). The conditions that lead to these different host-bacterium interactions are not understood.
It is well accepted that reducing the carriage or prevalence of E. coli O157:H7 in cattle would reduce the risk of human exposure to this pathogen (61). Recently, the recto-anal junction (RAJ) mucosa was identified as the primary site of E. coli O157:H7 colonization in cattle (47). Based on this finding, we demonstrated that culture of recto-anal mucosal swabs (RAMS) is easier and more sensitive than fecal culture to detect the bacteria (51) and that administration of E. coli O157:H7 to the terminal rectal mucosa is a reliable method to establish colonized cattle experimentally (58). Development of effective interventions to reduce bovine carriage of E. coli O157:H7 will rely on understanding the bacterial factors involved in E. coli O157:H7 colonization and persistence at the RAJ mucosa. Virulence factors that contribute to the clinical manifestations of E. coli O157:H7 infection in humans or other mammalian species, such as rabbits or gnotobiotc pigs (40, 52), may be the same factors important for E. coli O157:H7 colonization and/or persistence in cattle.
In a previous study, we identified a naturally occurring, RAJ-colonizing STEC strain prevalent in cattle and similar to E. coli O157:H7 (57). This E. coli ONT:H25 isolate shares at least five putative virulence genes with E. coli O157:H7, including stx2 (encodes Shiga toxin type 2), eae (encodes intimin), tir (encodes the translocated intimin receptor [Tir]), exhA (encodes enterohemolysin), and a pO157-like plasmid (57; unpublished data). We hypothesized that these common virulence factors contribute to the colonization and persistence of the bacteria at the bovine terminal rectal mucosa. Studies in mature, fully ruminant cattle have not investigated the role of these five most prominent virulence factors in EHEC colonization at the RAJ mucosa. Also, it is important to assess whether or not nonpathogenic E. coli without any of the aforementioned virulence factors can colonize this site. Does the O157:H7 serotype have an interaction with the terminal rectal tissue that is unique compared with other E. coli? Since many nonpathogenic E. coli strains can be cultured from RAMS samples, analysis of their persistence would be instructive in understanding the factors required for bacterial colonization.
The purpose of this study was to analyze specific virulence factors and their contribution to the persistence of E. coli at the bovine RAJ mucosa. To that end, we (i) compared the RAJ mucosa-colonizing ability of nonpathogenic E. coli K-12 with that of wild-type E. coli O157:H7, (ii) engineered isogenic gene deletion mutants that were each missing one of four virulence factors (eae, tir, ehxA, and the pO157), and (iii) compared each deletion mutant and a previously constructed Stx-negative strain with the corresponding wild-type E. coli O157:H7 strain for the ability to colonize the bovine RAJ mucosa. In all trials, animals received a single rectal application of one E. coli strain and colonization was monitored by the highly sensitive RAMS culture technique.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Red recombinase system described by Datsenko and Wanner (7). The primers used for deletion of eae, tir, and ehxA were described by Ritchie et al. (52). Briefly, the primers contained 40 to 45 nucleotides corresponding to the regions adjacent to the genes targeted for deletion and 20 additional nucleotides that annealed to the template DNA from pKD4. This plasmid carries a kanamycin resistance gene flanked by FRT sites. Electrocompetent cells were prepared by growing E. coli O157:H7 carrying pKD46 in Luria-Bertani (LB) broth (Difco, Detroit, Mich.) containing 0.1 mM L-arabinose, and the PCR products were introduced by electroporation (15). The kanamycin resistance gene was eliminated by introducing the plasmid pCP20 encoding FLP recombinase. Plasmids pKD46 and pCP20 were then cured at 42°C. This procedure resulted in a deletion of the target gene and no remaining antibiotic resistance gene. The gene deletions were analyzed by PCR and confirmed by DNA sequencing on a Perkin-Elmer thermocycler using ABI Big Dye sequencing mix according to the manufacturer's instructions and an ABI no. 3730 genetic analyzer.
To compare the hemolysin activities of wild-type 43895 and 43895 ehxA, the strains were grown on tryptose agar plates (Difco Laboratories, Detroit, Mich.) supplemented with 10 mM CaCl2 and 5% defibrinated sheep blood cells, as described previously (57). Inoculated plates were observed for hemolysis after overnight incubation in ambient air at 37°C. To test Stx production in wild-type 905 and 905 stx2, cells were grown in LB broth and an enzyme immunoassay for Stx (Premier EHEC; Meridian Diagnostics, Inc., Cincinnati, Ohio) was performed according to the manufacturer's instructions. Results were analyzed spectrophotometrically at two wavelengths, 450 and 630 nm, in a PowerWave XS reader (Bio-Tek, Winooski, Vermont). In both the hemolysin and toxin assays, E. coli K-12 (MG1655) and ATCC 43894 were used as negative and positive controls, respectively.
Extraction of pO157 and construction of a pO157-complemented strain.
The pO157-cured E. coli O157:H7 strain 277 used in this study was a gift from J. Shaw (Brooks Air Force Base, Texas). This strain was cured using standard mini-F incompatibility, a procedure that did not mutate chromosomal DNA. The complemented pO157 strain was constructed with the pO157 isolated from E. coli O157:H7 ATCC 43894 ehxA retaining the inserted kanamycin resistance gene (constructed as described above). The kanamycin resistance gene that replaced ehxA on this pO157 served as the selection marker. The pO157 was extracted from this strain by the method described by Kado and Liu (34) with slight modifications. Briefly, the E. coli O157:H7 43894 ehxA :: Kanr was streaked on a 1-cm2 area of an LB-Kan agar plate and incubated at 37°C overnight. About one-fourth of the colonies on the 1-cm2 area were scraped off and suspended in 100 µl lysing solution (3% sodium dodecyl sulfate, 50 mM Tris, with pH 12.6 adjusted by 2 N NaOH). The suspension was incubated at 55°C for 60 min and emulsified in an equal volume of phenol-chloroform. After centrifugation at 6,000 x g for 5 min at 4°C, the upper aqueous phase was transferred to a new tube. A portion of the sample was electrophoresed through a 0.7% agarose gel for plasmid detection. The remaining sample was extracted with diethyl ether. Traces of ether were removed by heating the solution to 68°C for 5 min and transferring the solution to a new tube. The plasmid DNA was precipitated by the tRNA/ethanol method described by Zhu and Dean (70). Briefly, 30 µl of DNA solution was mixed with 5 µg/µl yeast tRNA (Invitrogen) and 2 volumes of 100% cold ethanol was added to precipitate the plasmid DNA. The sample was cooled on crushed dry ice for 10 min and centrifuged at 14,000 x g for 10 min at room temperature. The pellet was washed with 80% ethanol twice, air dried, and resuspended in 2 µl of ultrapure water. The purified plasmid was transformed into the pO157-cured strain 277 by electroporation to make a complemented mutation. The complemented 277::pO157 ehxA was confirmed by plasmid extraction and PCR for the existence of the plasmid-encoded eae-conserved fragment gene (ecf) and lack of exhA on the pO157.
Animals. Five- to eight-month-old, fully ruminant Holstein steers were used in all experiments. Steers were fed alfalfa hay twice and pellet grain feeds once daily and had free access to drinking water. Animals were housed in quarantined facilities in which feed, water, bedding, manure, and waste were not in contact with other animals. All personnel followed strict biosafety procedures, and all procedures were approved by the Institutional Animal Care and Use and Biosafety Committees.
Rectal application of bacteria in cattle. Prior to application of bacteria, the steers were confirmed to be culture negative for E. coli O157:H7 by the culture method described below. Bacterial strains were grown separately in LB at 37°C for 18 h with aeration. Bacteria were applied to the bovine terminal rectal mucosa as previously described (58). Briefly, feces were manually cleared from the terminal rectum. A 10-cm by 3.5-cm diameter cylindrical sponge (Rubbermaid, St. Francis, WI) with a wooden handle was saturated with 10 ml overnight culture containing 109 CFU bacteria/ml, inserted into the anus, and gently rubbed against the terminal rectal mucosa. After swabbing, defecation was prevented for 10 min by holding the tail firmly against the anus. This presumably ensured that the applied bacteria were retained in the rectal lumen for sufficient time to attach to the mucosa.
Four separate trials were done. In the first trial, nalidixic acid-resistant (Nalr) E. coli K-12 and E. coli O157:H7 43895 Nalr were applied rectally to two different groups of four steers. In the second trial, wild-type E. coli O157:H7 43895 and three mutants, 43895 eae, 43895 tir, and 43895 ehxA, were applied rectally to each of four different groups of six steers. In the third trial, E. coli O157:H7 905 and its isogenic Stx– mutant 905 stx2 were applied rectally to each of two different groups of four steers. In the fourth trial, E. coli O157:H7 43894, 277 (pO157-cured mutant), and 277::pO157 ehxA (pO157-complemented strain) were each applied rectally to three different groups of four steers.
Bacterial culture and enumeration. Following bacterial applications, RAMS samples were taken from each steer as previously described (51, 57) on days 1, 4, 7, 11, 18, 25, and 32. Briefly, the RAMS samples were homogenized with 3 ml ice-cold sterile Trypticase soy broth (Difco Laboratories, Detroit, Mich.) and kept ice-cold until processing. Direct and enrichment procedures were used for bacterial culture. For direct culture, serial dilutions of the RAMS homogenates were plated on sorbitol MacConkey agar supplemented with cefixime, potassium tellurite, vancomycin, and MUG (SMAC-CTVM) agar; for culture of the nalidixic acid-resistant strains E. coli O157 43895 Nalr and K-12 Nalr, LB containing 25 µg/ml nalidixic acid (LB-Nal25) was used. Plates were incubated at 37°C overnight and observed for colonies not fermenting sorbitol (colorless colonies) and not hydrolyzing MUG (no florescence at 363 nm) or colonies resistant to nalidixic acid on LB-Nal25 plates. E. coli O157:H7 colonies were confirmed by a latex agglutination test (Pro-Lab Diagnostics, Toronto, Canada), and K-12 colonies recovered from cattle were confirmed by a pulsed-field gel electrophoresis (PFGE) fingerprinting method as described previously (57). E. coli O157:H7 isolates from animals that received a deletion mutant were confirmed to be carrying the appropriate gene deletion by PCR. Wild-type and deletion mutant isolates from cattle were periodically confirmed by PFGE to show that no gross chromosomal rearrangements had occurred. The CFU/swab were calculated from plate colony counts, converted to log10, and averaged for each group. Samples negative by direct culture were enriched by incubation at 37°C overnight with aeration, and serial dilutions were plated on SMAC-CTVM (or Nal25-LB where appropriate). For statistical analysis, RAMS samples positive only by enrichment culture were assigned a value of 10 CFU/swab and negative samples were assigned a value of 1 CFU/swab.
Statistical analysis.
In the first and third trials, differences in the numbers of the bacteria in RAMS samples between E. coli O157:H7 and K-12 or between E. coli O157:H7 905 and its isogenic Shiga toxin-negative strain 905 stx2 were evaluated with the Student t test. In the second and fourth trials, differences in the number of bacteria in RAMS samples between E. coli O157:H7 strains inoculated individually into steers were compared by repeated measures of analysis of variance of the logs of the group geometric mean of CFU/swab from all steers in each group. Graphpad Prism software 4.0 (San Diego, Calif.) was used for this analysis, and a Mantel-Haenszel chi square was used for comparison of the proportion of the steers that cultured positive for E. coli O157:H7 or the mutants.
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RESULTS |
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eae, tir, and ehxA genes, the corresponding open reading frames (ORFs) of strain 43895, and the "scar" regions of 80 bases within each target gene, as previously described (7) (data not shown). The complemented mutant 277::pO157 ehxA was confirmed by plasmid extraction and PCRs that were positive for the ecf gene and negative for the exhA gene (data not shown). Hemolysin assays showed colonies of 43895 ehxA with no hemolytic zones, while wild-type colonies had small, clear zones of hemolysis (data not shown). Stx production was not detected from strain 905 stx2 but was detected in the wild-type strain 905 (data not shown). All E. coli O157:H7 mutants had growth rates comparable with that of the corresponding parental wild-type strain in the laboratory in either LB or SMAC-CTVM selective medium (data not shown). Also, the two nalidixic acid-resistant E. coli strains K-12 and O157:H7 had comparable growth in LB-Nal25 (data not shown). Animal carriage of E. coli. All animals in this study were healthy and did not have ill effects from challenge with the wild type, mutant E. coli O157:H7, or E. coli K-12. All animals given a single rectal dose of wild-type E. coli O157:H7 carried the bacteria in a manner typical of experimental and natural infections with this microorganism (58).
Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. To test the ability of nonpathogenic E. coli to colonize and persist at the terminal rectal mucosa, E. coli K-12 Nalr or E. coli O157:H7 ATCC 43895 Nalr was applied rectally to two groups of four steers. Colonization at the RAJ mucosa was monitored by culture of nalidixic acid-resistant bacteria in RAMS samples over the course of the experiment. Isolates were confirmed to be E. coli K-12 by a PFGE fingerprinting method as described previously (57) (data not shown).
The number (P < 0.01) of E. coli K-12 isolates was significantly lower than that of cultured from RAMS samples of E. coli O157:H7 isolates cultured over the first 11 days after bacterial application (Fig. 1). Also, distinct patterns in the numbers of E. coli K-12 and E. coli O157:H7 isolates cultured were seen. Among steers that received E. coli O157:H7, the mean number of these bacteria cultured increased between days 1 and 4 from 3.7 log units to 4.5 log units and then gradually decreased on all subsequent sampling days (Fig. 1). This initial increase in the number of E. coli O157:H7 between days 1 and 4 indicated that O157 not only successfully attached to the terminal rectal mucosa but also multiplied at that site. In contrast, the pattern of the mean number of E. coli K-12 isolates cultured on days 1 and 4 sharply decreased from 2.8 log units to 1.4 log units and continued to decrease on all subsequent sampling days. Three of four steers given E. coli O157:H7 continued to be culture positive on day 32, whereas only one of four steers given E. coli K-12 continued to be culture positive on day 25 and all four animals in this group eliminated the E. coli K-12 by day 32 (Fig. 1). Among the other three animals that received E. coli K-12, two cleared the bacteria by day 7 and one cleared the bacteria by day 18. E. coli O157:H7 established a persistent colonization at RAJ mucosa, consistent with previous studies (57, 58, 68), and showed a distinct colonization pattern compared with the nonpathogenic E. coli K-12.
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eae, 43895 tir, or 43895 ehxA. On each sampling day, isolates from steers that received a mutant strain of E. coli O157:H7 were verified by PCR analysis to be carrying the expected deletion (data not shown). The logs of the geometric means of the CFU of E. coli O157:H7/swab in each group and the percentage of culture-positive steers in each group are presented in Fig. 2.
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ehxA strain increased between days 1 and 4 and then gradually decreased on the subsequent sampling days. In contrast, the mean numbers of both the 43895 eae and 43895 tir strains decreased between days 1 and 4 and continued to decline on the subsequent sampling days (Fig. 2A). This pattern was similar to that of the nonpathogenic E. coli K-12 (Fig. 1) and indicated that the ability of E. coli O157:H7 to colonize the bovine terminal rectal mucosa was notably impaired with deletion of eae or tir. A repeated-measures analysis of variance of these data, accounting for the bacterial strain, day postapplication, and CFU/swab, showed that the differences between 43895 eae and the wild type were significant on days 1, 4, 7 (P < 0.01), and 11 (P < 0.05), while the differences between 43895 tir and the wild type were significant on days 1, 4, and 7 (P < 0.01). There were no differences (P > 0.05) between the CFU of E. coli O157 in RAMS samples from animals receiving 43895 ehxA or the wild type on any sampling day. The Mantel-Haenszel chi-square comparison of these data indicated that the proportion of steers that were cultured positive for E. coli O157:H7 was significantly lower (P < 0.05) among animals that received the eae or tir mutants than among the steers that received the wild type. Similar analysis showed that there was no significant difference (P > 0.05) between the proportions of steers cultured positive for the ehxA mutant and those culture positive for the wild-type strain.
Stx2 did not promote initial adherence and colonization at the bovine terminal rectal mucosa.
Controversy surrounding the role of Stx in E. coli O157:H7 persistence in ruminants led us to test E. coli O157:H7 strain 905 and its isogenic toxin deletion mutant for colonization of cattle. Two different groups of four steers were inoculated rectally with E. coli O157:H7 905 (stx1/stx2+) or its isogenic Stx– strain 905 stx2. On each sampling day, isolates were confirmed by PCR to be carrying the expected stx deletion (data not shown). The logs of the geometric means of the CFU of E. coli O157:H7/swab in each group are presented in Fig. 3. There was no significant difference (P > 0.05) between the CFU of E. coli O157:H7/RAMS sample from animals given the stx2 or wild-type strain on any sampling day. The pattern of increasing numbers of E. coli O157:H7 between days 1 and 4 was observed for both strains and was similar to the recognized pattern for strains that attached and multiplied at the RAJ mucosa (Fig. 1 and 2). The results indicated that Stx did not influence E. coli O157:H7 colonization of cattle.
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DISCUSSION |
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In this study, we used a previously published, novel rectal administration of Escherichia coli O157:H7 (57, 58) for assessing colonization factors at the bovine terminal rectal mucosa. The procedure was highly reliable at reproducing the state of the RAJ colonization but also had the advantage of directly measuring the ability of genetically defined E. coli O157:H7 mutants to colonize at the RAJ mucosa. The traditional oral administration of E. coli O157:H7 to assess colonization factors not only measures survival and attachment to the terminal rectal mucosa but also measures the ability of the bacteria to survive in and pass through the gastrointestinal tract (GIT).
Genomic sequencing of E. coli O157:H7 EDL933 (ATCC 43895) reveals that this pathogenic serotype contains 1,387 unique genes not present in the chromosome of nonpathogenic E. coli K-12 MG1655 (50). The unique genes are proposed to be related to virulence or benefit the bacteria in some way. An important benefit may be to enhance survival and persistence of E. coli O157:H7 in ruminant hosts. Our findings suggested that nonpathogenic E. coli K-12, which carries no virulence factors, was more easily passed with the constant movement of digesta/feces and was more easily cleared from the terminal rectal mucosa than was E. coli O157:H7. However, it was surprising that E. coli K-12 persisted in one animal for >25 days, which suggested that multiple bacterial factors, metabolic capabilities, other flora, and/or host factors likely contribute to the persistence of E. coli at this mucosal site. Both E. coli K-12 and O157:H7 are able to utilize the same glycolytic substances (45). Nonetheless, this first comparative analysis with a nonpathogenic E. coli strain suggested that E. coli O157:H7 had a unique attaching ability and/or faster replicating ability at the bovine terminal rectal mucosa than did E. coli K-12. Analysis of the RAJ-colonizing ability of bovine commensal E. coli strains will be important for a more complete understanding of the host-bacterium dynamics at this site.
Many studies have implicated intimin and Tir as having roles in E. coli O157:H7 intestinal colonization, but no previous study has used isogenic deletion mutants placed at the terminal rectal mucosa in fully ruminant cattle. E. coli O157:H7 and many STEC strains contain the locus of enterocyte effacement (LEE) pathogenicity island that encodes a type III secretion system and effector proteins that are homologous to genes in the enteropathogenic E. coli (28, 39). Nucleotide sequence analysis identifies five major operons, named LEE1 to LEE5 (16). Intimin and Tir, encoded on the LEE5 operon, are critical for intimate adherence and the formation of characteristic attaching and effacing lesions on cells in tissue culture by EHEC and enteropathogenic E. coli (12, 13, 32). Studies to determine the roles of intimin and Tir in animals have used newborn piglets, newborn colostrum-deprived calves, and sheep (6, 8, 10, 11, 14, 33, 40, 60, 63, 65, 67). Recent studies show that E. coli O157:H7 forms attaching and effacing lesions at the bovine RAJ and that the LEE4 operon is essential for colonization of E. coli O157:H7 in calves (48). Others have shown that intimin can bind to both the bacterium-encoded Tir (11, 27, 36) and to host-encoded receptors referred to as host intimin receptors (10, 19, 27, 41). Our work with 43895 eae and 43895 tir suggested that intimin and Tir promoted the level of E. coli O157:H7 adherence at the terminal rectal mucosa and affected the percentage of colonized steers both short term (2 weeks) and long term (1 month). The Tir deletion mutant was as defective as an intimin deletion mutant in colonizing cattle in this study. Although we did not measure binding to host intimin receptors, this suggested that cattle do not provide an effective intimin receptor at the terminal rectal mucosa and that intimin binding at this site is dependent on the bacterial Tir. A similar result was recently reported in 14-day-old calves using an oral dose model of carriage (65). However, the eae gene is not present in all bovine STEC isolates, although they persist in cattle (31, 55, 66), indicating that other factors influence persistence of the bacteria in the bovine GIT.
The role of the pO157 in E. coli O157:H7 colonization and persistence in cattle has not been studied previously and is not well understood. The complete nucleotide sequence of the pO157 reveals that it contains 100 ORFs (5), of which fewer than 20 ORFs are characterized. These genes encode enterohemolysin (ehxA) (56), lymphocyte inhibitory factor (lifA and efa) (30), a putative adhesin (toxB) (62), and a zinc metalloprotease (stcE) (24). Also, work in our laboratory shows that a myristoyl transferase gene in the plasmid ecf operon is associated with lipid A modification. Its product is similar to that of the chromosomal gene lpxM. Deletion of both genes leads to reduced survival in the bovine GIT and reduced persistence in water troughs (68). Deletion of toxB results in reduced adherence to cultured epithelial cells and influenced the expression and secretion of proteins encoded by LEE but did not affect the colonization of E. coli O157:H7 in calves (60, 62). StcE mucinase activity may contribute to intimate adherence of E. coli O157:H7 to epithelial cells (24). In vivo and in vitro studies report conflicting results for the role of pO157 in adherence to epithelial cells (20, 35, 64). Fratamico et al. reported that both the plasmid-cured strain and the parent strain produce pili and adhere equally well to HEp-2 and intestinal 407 cells (20). Karch et al. reported that pO157 is associated with the expression of fimbriae that enhance bacterial adherence to epithelial cells in tissue culture (35). Tzipori et al. demonstrated that both plasmid-cured derivatives and the parent strains produce the typical mucosal lesions of bacterial attachment in piglets (64). Differences in cell lines and animal models likely contribute to these conflicting results. In this study, we demonstrated that pO157 was required for efficient colonization at the bovine terminal rectal mucosa.
Shiga toxins are a major EHEC virulence factor with potent cytotoxic activity that is responsible for many patient symptoms and can lead to death. Stx can cause damage to various cell types, including vascular endothelial, renal tubular, glomerular epithelial, and intestinal epithelial cells (29, 37). Conflicting results about the role of Stx in colonization have been reported. Stx-positive strains of E. coli O157:H7 colonize and persist longer than a Stx-negative strain in experimentally inoculated weaned calves (9), but the strains used in this study were not isogenic so that differences in colonization may have been due to traits other than Stx production. Robinson et al. showed that the presence of stx2-converting phage increased adherence of EHEC to epithelial cells, suggesting a role of Stx2 in colonization (54). In contrast, Woodward et al. demonstrated that an Stx-negative E. coli O157:H7 persists in weaned sheep, suggesting Shiga toxins are not important in colonization (67). Also, purified Stx1 can inhibit the activation and proliferation of certain bovine lymphocytes (18, 43), which may facilitate colonization of E. coli O157:H7 at the bovine RAJ mucosa by modulation of mucosal immune responses. Our findings with 905 and its isogenic Stx– mutant 905 stx2 showed that that Stx2 did not influence adherence and colonization at the bovine terminal rectal mucosa. Similarly, although nearly all E. coli O157:H7 isolated contain the ehxA gene and bovine STEC epidemiological data suggest that ehxA may be associated with colonization in cattle (1, 3), our findings did not support this conclusion.
Theoretically, bacterial persistence requires the rate of bacterial growth to be at least equal to the rate at which bacteria are flushed from the system by intestinal digesta/fecal passage and/or replication and sloughing of intestinal epithelial cells (21). In a previous study, we suggested that persistence of E. coli O157:H7 in the GIT is dependent on the balance between bacterial replication and the rate of epithelial cell proliferation (38). There is no information about the rate of epithelial cell proliferation and sloughing at the terminal rectal mucosa of cattle. In the mouse intestine, complete turnover of cells in intestinal crypts is complete in 2 to 5 days (17). The pattern of an increase in bacterial numbers between days 1 and 4 postapplication suggests that bovine epithelial cell replacement at this bovine location occurs in a similar time frame (days).
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ACKNOWLEDGMENTS |
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This work was supported, in part, by the Idaho Agriculture Experiment Station, the National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service, grant numbers 99-35201-8539 and 04-04562, Public Health Service grants U54-AI-57141, P20-RR16454, NO1-HD-0-3309, and P20-RR15587 from the National Institutes of Health, and grants from the United Dairymen of Idaho and the Idaho Beef Council.
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FOOTNOTES |
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