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Heterogeneity in enterohemorrhagic Escherichia coli O157:H7 fecal shedding in cattle is related to Escherichia coli O157:H7 colonization of the small and large intestine Danica Baines, Byron Lee, and Tim McAllister

Abstract: In the last decade, Escherichia coli O157:H7 have emerged as important pathogens of the gastrointestinal tract of humans. Healthy cattle have been identified as the primary reservoir, however, the factors affecting heterogeneous E. coli O157:H7 fecal shedding are not fully understood. The aim of this study was to investigate the contribution of E. coli O157:H7 colonization of small and large intestinal sites to the heterogeneity of fecal shedding in cattle. There was a dose-dependant E. coli O157:H7 E318N colonization of duodenum, jejunum, ileum, cecum, ascending colon, spiral colon, descending colon, and the rectoanal junction in vitro with no difference in E. coli O157:H7 colonization of the rectoanal junction and other intestinal sites. There were 10–100 times greater E. coli O157:H7 colonization of intestinal sites from persistent shedding cattle compared with nonpersistent shedding cattle. Novel pathologies were associated with E. coli O157:H7 colonization sites in the small and large intestine. The first pathology, focal petechiae, was present throughout the intestinal tract of cattle that ceased shedding E. coli O157:H7 for 5–12 weeks or in the jejunum, ileum, cecum, and ascending colon of cattle shedding E. coli O157:H7 for 4–5 months. The second pathology, mucosal hemorrhages, was present in the same sites as the focal petechiae in cattle shedding for 5 months and these hemorrhages were in the final stages of repair. Several features of these hemorrhages support this conclusion including the brown appearance, low amount of classic E. coli O157:H7 induced A/E lesions, flattened epithelium, and blunted villi. Although mucosal hemorrhages were present in the jejunum, ileum, cecum, and ascending colon in cattle shedding for 4 months, many other pathologies were also present that were indicative of hemorrhagic enteritis as evidenced by the blood red appearance of hemorrhages, severe edema, and dark red erythema. Escherichia coli O157:H7 were associated with both pathologies suggesting it is the causative agent. The current study supports a relationship between the amount of E. coli O157:H7 colonization in intestinal sites and heterogeneous fecal shedding by cattle. Key words: Escherichia coli O157:H7, cattle, intestine, pathology, colonization. Re´sume´ : Au cours de la dernie`re de´cennie, il est apparu qu’Escherichia coli O157:H7 e´tait un pathoge`ne important du syste`me gastro-intestinal chez l’humain. Les boivins sain a e´te´ identifie´ comme re´servoir primaire de la bacte´rie, cependant, les facteurs qui affectent la libe´ration d’E. coli O157:H7 dans les fe`ces ne sont pas parfaitement compris. Le but de cette e´tude e´tait d’examiner la contribution de la colonisation de certains sites du petit et du gros intestin par E. coli O157:H7 au processus de libe´ration fe´cale chez les boivins. Nous avons observe´ une colonisation du duode´num, du je´junum, de l’ile´on, du caecum, du coˆlon ascendant, du coˆlon spiral, du coˆlon descendant et de la jonction recto-anale in vitro par E. coli O157:H7 E318N de fac¸on de´pendante de la concentration, sans diffe´rence de colonisation de la jonction rectoanale et des autres sites intestinaux par E. coli O157:H7. La colonisation par E. coli O157:H7 des sites intestinaux des boivins libe´rant la bacte´rie de fac¸on persistante e´tait de 10 a` 100 fois plus e´leve´e comparativement aux boivins libe´rant la bacte´rie de fac¸on non-persistante. De nouvelles pathologies ont e´te´ associe´es aux sites de colonisation du petit ou du gros intestin par E. coli O157:H7. La premie`re pathologie, les pe´te´chies focales, e´tait pre´sente tout au long du tractus intestinal des boivins ayant cesse´ de libe´rer E. coli O157:H7 pendant 5 a` 12 semaines ou dans le je´junum, l’ile´on, le caecum et le coˆlon ascendant des boivins libe´rant E. coli O157:H7 depuis 4 a` 5 mois. La deuxie`me pathologie consistait en he´morragies muqueuses qui e´taient pre´sentes aux meˆmes sites que les pe´te´chies focales chez les boivins libe´rant la bacte´rie depuis 5 mois et ces he´morragies en e´taient aux stades finaux de gue´rison. Plusieurs caracte´ristiques de ces he´morragies ap-

Received 2 June 2008. Accepted 21 August 2008. Published on the NRC Research Press Web site at cjm.nrc.ca on 4 December 2008. Abbreviations: Stx, Shiga toxin; PBS, phosphate-buffered saline; LB, Luria-Bertani; SMAC, sorbitol-MacConkey; A/E, attaching and effacing; IMS, immunomagnetic separation. D. Baines,1 B. Lee, and T. McAllister. Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1, Canada. 1Corresponding

author (e-mail: [email protected]).

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doi:10.1139/W08-090

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985 puyaient cette conclusion, dont l’apparence brunaˆtre, la faible abondance le´sions A/E induites par E. coli O157:H7 classique, la pre´sence d’un e´pithe´lium pavimenteux et de villosite´s atrophie´es. Quoique les he´morragies muqueuses e´taient pre´sentes dans le je´junum, l’ile´on, le caecum et le coˆlon ascendant des boivins libe´rant des bacte´ries depuis 4 mois, plusieurs autres pathologies indicatrices d’une ente´rite he´morragique e´taient aussi pre´sentes tel que de´montre´ par l’apparence rouge sang des he´morragies, l’œde`me se´ve`re et la pre´sence d’un e´rythe`me rouge fonce´. E. coli O157:H7 e´tant associe´e aux deux pathologies, ceci sugge`re qu’elle en soit l’agent causal. La pre´sente e´tude appuie la relation entre le niveau de colonisation des sites intestinaux par E. coli O157:H7 et l’he´te´roge´ne´ite´ de la libe´ration fe´cale par les boivins. Mots-cle´s : Escherichia coli O157:H7, boivins, intestin, pathologie, colonisation. [Traduit par la Re´daction]

______________________________________________________________________________________ Introduction Mature cattle act as a reservoir for Stx-producing Escherichia coli O157:H7, a bacterium that causes disease outbreaks in humans through the consumption of contaminated food products (Duffy 2003). To reduce the incidence of disease in humans, it is necessary to develop strategies to reduce carriage of E. coli O157:H7 in cattle through understanding the factors affecting cattle susceptibility to E. coli O157:H7 infection. Escherichia coli O157:H7 challenged cattle respond with heterogeneous fecal-shedding periods, the duration and magnitude of which are related to exposure dose (Besser et al. 2001; Sheng et al. 2004), and age (Cray and Moon 1995). Neonatal calves are the susceptible age class for E. coli O157:H7 related disease and symptoms include diarrhea, hyperemia, fibrinous exudates, focal petechiae, and enteritis (mucosal infiltrates) (Dean-Nystrom et al. 1997; Dean-Nystrom et al.1998). The characteristic patchy E. coli O157:H7 A/E lesions are found in the jejunum, ileum, cecum, colon, and rectum in neonatal calves. Older calves can have transient watery diarrhea but are not seriously affected by E. coli O157:H7 infection (Brown et al. 1997; Cray and Moon 1995). These calves have E. coli O157:H7 associated with the same tissues as neonatal calves, but A/E lesions were not detected. In contrast, E. coli O157:H7 challenged cattle are asymptomatic carriers of E. coli O157:H7 with the bacteria associated with fewer small (ileum) and large (cecum, colon, and rectum) intestinal sites (Cray and Moon 1995; Grauke et al. 2002; Van Baale et al. 2004). As for older calves, A/E lesions were not detected. Escherichia coli O157:H7 can adhere to and form A/E lesions in intestinal tissue from mature cattle in vitro (Baehler and Moxley 2000; Cobbold and Desmarchelier 2004; Dibb-Fuller et al. 2001; Phillips et al. 2000), however, A/E lesions have only been identified in one site in natural and experimental challenged animals, the lymphoid follicle-associated epithelium in the rectoanal junction (Lim et al. 2007; Low et al. 2005; Naylor et al. 2003; Naylor et al. 2005). As a result, the rectoanal junction is considered the primary E. coli O157:H7 colonization site responsible for persistent shedding in cattle. This model was derived from studies that involved random sampling of intestinal tissues from cattle without any information on E. coli O157:H7 colonization status. In addition, several other factors have not been fully investigated in cattle and may influence the extent of E. coli O157:H7 colonization after a challenge including repeated exposure to single or multiple strains, bacterial cytotoxin expression (Robinson et al. 2006), and co-infection with other patho-

gens (Vitovec et al. 2001). For these reasons, the contribution of different intestinal sites to E. coli O157:H7 fecal shedding by cattle may be underestimated. As a first step towards understanding the E. coli O157:H7 colonization process in cattle, we examined whether nonpersistent (14 days), moderately persistent (30 days), and persistent (77 days to 5 months) E. coli O157:H7 shedding cattle represented differences in E. coli O157:H7 colonization. We also examined intestinal tissues from these cattle 5–12 weeks after shedding ceased for residual pathology related to chronic bacterial infection. We confirmed the significance of any identified pathology to the E. coli O157:H7 colonization process in cattle by examining intestinal tissues in persistent-shedding cattle that were shedding E. coli O157:H7 for 4 or 5 months after the initial challenge.

Materials and methods Experimental animals In the first year, 32 Hereford  Angus yearling steers (~490 kg) were randomly assigned to 4 sheltered outdoor pens that were spaced to prevent nose-to-nose contact between animals in different pens. Each pen had its own water bowl and feed bunk. The steers used in this study were cared for in accordance with guidelines set by the Canadian Council on Animal Care (CCAC) (1993). The animals were prescreened and IMS negative for E. coli O157:H7. Three additional sets of 32 cattle were set up in a similar manner over 3 consecutive years. A 1010 CFU mixture of 4 E. coli O157:H7 strains, E318N (human origin), E32511N (human origin), R508N (bovine origin), and H4420N (bovine origin) resistant to 50 mg/mL of nalidixic acid was prepared and orally administered to the steers as described previously (Bach et al. 2005). The fecal samples were collected and analyzed for the presence of nalidixic acid resistant nonsorbitol fermenting E. coli O157:H7 bacteria as described previously (Bach et al. 2005). Generally, fecal samples were collected over a 77-day period and analyzed for the presence of E. coli O157:H7 by plating serial dilutions onto nalidixic acid selective CT-SMACnal agar (Dalynn Biologicals, Calgary, Alberta) or when this method no longer detected E. coli O157:H7, an enrichment process was performed on a sample and an aliquot analyzed with IMS using Dynabeads anti-E. coli O157:H7 (Dynal, Lake Success, New York). The final material was plated onto CT-SMACnal agar and examined for colonies not fermenting sorbitol (colourless colonies). After the 77-day shedding behavior data was collected, we chose animals #

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representing the 3 characteristic shedding periods described for cattle (Grauke et al. 2002): animals that shed for about 7 days (nonpersistent), ~30 days (moderately persistent), and for several months to a year (persistent; Fig. 1). Nonpersistent shedding cattle were sampled within a week, while moderately persistent and persistent shedding cattle were sampled once fecal samples were E. coli O157:H7 negative for 5 weeks. Grauke et al. (2002) hypothesized that the 3 shedding periods were related to the amount of E. coli O157:H7 colonization and, to date, this hypothesis has yet to be confirmed. To test this hypothesis, 3 animals in the first group of cattle were chosen representing each shedding category to evaluate both the E. coli O157:H7 colonization potential and any associated residual pathology as cattle had ceased shedding for 5–12 weeks. Because a novel residual pathology was documented in the first group of cattle, we also chose similar animals from 2 additional E. coli O157:H7 challenge studies. A total of 10 nonpersistent, 3 moderately persistent, and 11 persistent shedding cattle were evaluated. The second group of cattle provided a unique opportunity to examine intestinal tissues for the reduction or loss of E. coli O157:H7 associated pathology as the majority of animals except 1 persistent shedder had ceased shedding for ~1–5 months. The third group of cattle was assessed at the same time as the first group of cattle. To link the novel pathology with an active E. coli O157:H7 infection, intestinal tissues from 2 cattle were also selected from a third group of cattle, and these animals were shedding E. coli O157:H7 5 months after the initial challenge. Finally, to confirm that these persistent-shedding cattle have a sequential resolution of novel pathologies associated with active E. coli O157:H7 infections, an additional steer was chosen from a fourth group of cattle, and this animal was shedding E. coli O157:H7 4 months after the initial challenge. Intestinal tissue At the end of the experimental period, necropsy samples were obtained from animals using standard methods (Van Baale et al. 2004). The 15-cm tissue loops were obtained from the intestinal tract within 10 min after release from the carcass. For the first group of cattle, a sample was removed from the duodenum, jejunum (plus Peyer’s patches), ileum, cecum, ascending colon, spiral colon, descending colon, and rectoanal junction. For the second and third group of cattle, samples were taken from the jejunum (30 cm), descending colon (60 cm), and rectum (full tissue), as these tissues provided a representative view of the novel pathology in all cattle shedding categories. Each piece was placed in 200 mL of 4 8C Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Hyclone, Logan, Utah) supplemented with 5% albumin and transported back to the laboratory. The tissue was cut open, washed in 4 8C PBS and eight 2.5-cm2 pieces cut out. Excess fat or connective tissue was removed and the whole organ piece (mucosa plus muscle) was used to maintain mucosal structure. Care was taken while cutting not to press down on the tissue and distort size. The tissue pieces were placed in 4 8C PBS until all sites were processed (~20 min/animal). Experiments were started within 2 h of collecting the tissue sample. Intestinal pathology Each 15-cm tissue loop from cattle that had ceased shed-

Can. J. Microbiol. Vol. 54, 2008 Fig. 1. Nonpersistent (NP), moderately persistent (MP), and persistent (P) Escherichia coli O157:H7 shedding patterns for challenged cattle.

ding for 5–12 weeks was washed and examined for mild residual pathology associated with bacterial infections, focal petechiae, and enteritis. The tissue was scored as positive if the indicator was present and negative if not. The distribution of the pathology was also scored as uniform or localized. The data was expressed as the proportion of positive intestinal sites for each animal. Gross pathology pictures were taken using a standard digital camera (Nikon Canada, Mississauga, Ontario). To determine the relevance of the novel pathology to the E. coli O157:H7 infection process, intestinal tissues were also examined in cattle shedding E. coli O157:H7 for 4 or 5 months after the initial challenge. Jejunum, ileum, cecum, colon, and rectum from 2 persistent-shedding cattle that had positive fecal samples 5 months after the initial challenge (IMS using Dynabeads anti-E. coli O157:H7) were examined. The entire intestinal tract from 1 persistent-shedding steer that had positive fecal samples 4 months after the initial challenge (IMS using Dynabeads anti-E. coli O157:H7) was also examined. All pathologies were recorded and the areas preserved for processing using standard techniques for light microscopy (LM) and transmission electron microscopy (TEM). For LM and TEM, tissues were fixed in Safefix (Fisher Scientific, Ottawa, Ontario) in 0.1 mol/L sodium phosphate buffer for 24 h at 4 8C. The tissue was washed 3 times for 10 min with sodium phosphate buffer and put though a dehydration series — 30%, 50%, 70%, 85%, and 95% ethyl alcohol (Fisher Scientific, Ottawa, Ontario) at 10 min each. The tissue was finally treated with 3 changes of 100% alcohol at 10 min each. These preparations were then embedded in resin as follows: Spurrs resin – alcohol (1:3) for 4 h, then Spurrs resin – alcohol (1:1) overnight, then 100% Spurrs resin for 8 h, followed by 100% Spurrs resin overnight, then 100% Spurrs resin for 8 h, and then the embedded material was poured into rubber molds to polymerize for 8 h at 70 8C. For LM, sections were cut at a thickness of 4 mm and stained for 2 min with 2% toluidine blue O or basic fuschin. For TEM, sections were cut at a thickness of 80 mmm, stained with 5% uranyl acetate for 7 min, washed in distilled water, poststained with 2% lead citrate for 5 min, and finally washed in distilled water. The cattle used in this study were challenged with a mix#

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ture of E. coli O157:H7 strains that were nalidixic acid resistant. This is not a common genetic trait for pathogens isolated from cattle (Lanz et al. 2003) and, as such, is a useful tool for monitoring E. coli O157:H7 fecal-shedding behavior. Near the end of the shedding process for cattle, small numbers of challenge E. coli O157:H7 were detected in the manure after enrichment culture followed by IMS using Dynabeads anti-E. coli O157:H7 (McAllister et al. 2006). In the current study, we took advantage of this resistance trait to establish the presence of the original challenge E. coli O157:H7 with the intestinal tissues. A 2.5-cm2 intact tissue was incubated with 1% Triton X-100 overnight, and the released bacteria present in the solution were plated on CTSMACnal agar to determine the number of nalidixic acid resistant non-sorbitol fermenting bacterial colonies. The identified colonies were confirmed as O157 using the Oxoid Dryspot O157 test (Fisher Scientific). To ensure that this approach was providing an accurate reflection of E. coli O157:H7 colonization, bacterial colonies isolated from the intestinal tissues of shedding and nonshedding cattle were tested for E. coli O157:H7 identity using the RIM E. coli O157:H7 test (REMEL, Fisher Scientific). In vitro organ culture (IVOC) E. coli O157:H7 adherence assay Escherichia coli O157:H7 E318N was used to inoculate intestinal organ pieces of cattle in the IVOC adherence assay. Bacteria were routinely stored at –20 8C in 15% glycerol – 85% LB. Prior to adherence studies the strain was subcultured in LB broth and incubated aerobically overnight at 37 8C without agitation. The bacterial cells were diluted to the appropriate concentrations in PBS and bacterial cell counts determined by plating on SMAC agar (Dalynn Biologicals). The IVOC adherence assay has been used extensively to evaluate E. coli O157:H7 colonization in hosts and is considered a representative model for in vivo colonization (Baehler and Moxley 2000; Cobbold and Desmarchelier 2004; Dibb-Fuller et al. 2001; Phillips et al. 2000). The IVOC adherence assay was performed as described previously except that a 4 h incubation time was used to ensure consistent E. coli O157:H7 E318N attachment. Generally, each organ piece was drained using a paper towel and placed in about 3 mL of DMEM at 15 8C adjusted to just cover the surface of the mucosa, which was essential to maintaining tissue viability. A 100 mL aliquot of E. coli O157:H7 E318N was applied to the mucosal surface providing a final dose range of 3  108, 1.1  108, 1.6  107, 3.4  106, 6.7  105, 2  105, and 6  104 bacteria per well for each intestinal site. The organ cultures were incubated for 4 h at 37 8C, 95% humidity, and 5% CO2. A 100 mL aliquot of PBS was used as the control treatment and detected the amount of challenge E. coli O157:H7 associated with the tissues. The medium was replaced for the highest 2 doses after 2 h to maintain pH and tissue viability. After incubation, each organ piece was washed 4–6 times with 10 mL of PBS to remove any unattached bacteria. The tissue was then turned mucosa-side down in 3–4 mL of PBS supplemented with 1% Triton X-100. The tissue pieces were incubated at 4 8C overnight and the released bacteria were quantified by plating the solution on CT-SMACnal agar.

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Additional tissue samples were maintained in a similar manner and used to assess tissue viability before and after each experiment using 0.1% trypan blue for 15 min. Statistical analysis The SYSTAT statistical package was used to compare data for significance using an ANOVA that included both a blocking factor (cow) and treatment factor (intestinal site). Intestinal sites were not significantly different so the data was pooled for the small and large intestines and the ANOVA performed on the small or large intestine data with a blocking factor (animal) and treatment factors (dose or shedding category). A post hoc Tukey’s test was performed to separate the mean effects. Proportional data as those generated for the intestinal site pathology was arcsin-square root transformed to normalize the data prior to analysis.

Results Intestinal pathology There were focal petechiae and no enteritis in the washed excised 15-cm sections of intestine in the first group of cattle that had ceased shedding for 5–12 weeks (Fig. 2). The proportion of intestinal sites with focal petechiae significantly increased from nonpersistent- to persistent-shedding animals (p = 0.001, n = 3), and the proportion of sites affected was inversely proportional to the amount of time elapsed since the completion of shedding. For nonpersistentshedding cattle (animal Nos. = 121, 125, and 149) that ceased shedding in 2 weeks and were sampled at ~3 months postchallenge, the focal petechiae were limited to the rectoanal junction (2 of 3 animals). For moderately persistent shedding cattle (animal Nos. = 122, 136, and 144) that ceased shedding at 1 month and were sampled at ~3 months postchallenge, the focal petechiae were found in the jejunum (2 of 3 animals), ileum (3 of 3 animals), descending colon (2 of 3 animals), and rectoanal junction (3 of 3 animals). For persistent-shedding cattle (animal Nos. = 143, 128, and 134) that ceased shedding at ~3 months and were sampled at ~4 months postchallenge, focal petechiae were in all 8 intestinal sites (3 of 3 animals). This pathology was uniformly distributed in the small intestine and much more localized to the regions extending deepest into the lumen of the large intestine (Fig. 2). There was also equal association of the pathology with follicle-associated epithelium of the Peyer’s patches and absorptive epithelium in the small intestine (Fig. 2). We examined the intestinal tissues from 2 other groups of E. coli O157:H7 challenged cattle for similar pathology to determine whether this pathology was consistently associated with E. coli O157:H7 infection sites. In the second group of E. coli O157:H7 challenged cattle (nonpersistent = 3 and persistent = 3), considerably more time elapsed since the completion of shedding by the animals and this provided us with the opportunity to examine the relationship between the distribution of focal petechiae and time elapsed since the completion of E. coli O157:H7 shedding. Examination of intestinal tissues from 3 nonpersistentshedding animals that had ceased shedding in 2 weeks and were sampled at ~4 months had no detectable focal petechiae. One persistent-shedding animal that had ceased shedding in ~3 months and was sampled at ~6 months had no #

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detectable focal petechiae in intestinal tissues. The second persistent-shedding animal that had ceased shedding in ~4 months and was sampled at ~6 months had focal petechiae in the jejunum and descending colon. The final persistent-shedding animal that had ceased shedding in 5 months and was sampled at ~6 months had focal petechiae in all intestinal tissues. These results suggested that once E. coli O157:H7 challenged cattle have ceased shedding, the associated pathology resolves in ~3 months, supporting a link between the status of E. coli O157:H7 infection and the presence of focal petechiae. We examined the intestinal tissues from a third group of animals at a similar time after the completion of shedding as for the first group of cattle (nonpersistent = 4 and persistent = 5) and the focal petechial distribution was the same as that recorded for the first group of cattle. We confirmed the association of E. coli O157:H7 with many of the focal petechial sites despite the cattle having ceased shedding for ~1–6 months. For nonpersistent-shedding cattle, 179.25 ± 82.8 CFU were isolated from the jejunum of 4 of 10 animals, 870 CFU from the ileum of 1 of 10 animals, 168 ± 85.2 CFU from the descending colon of 4 of

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10 animals, and 30 CFU from the rectoanal junction of 1 of 10 animals. For moderately persistent shedding cattle, 189 CFU were isolated from the descending colon of 1 of 3 animals. For persistent-shedding cattle, 90 CFU were isolated from the duodenum of 1 of 11 animals, 296.7 ± 173.2 CFU from the jejunum of 10 of 11 animals, 90 CFU from the ileum of 1 of 11 animals, 30 CFU from the cecum of 1 of 11 animals, 472 ± 108.3 CFU from the descending colon of 3 of 11 animals, and 958.5 ± 514.8 CFU from the rectoanal junction of 4 of 11 animals. Escherichia coli O157:H7 was isolated at a higher rate from the jejunum, descending colon, and rectoanal junction. To confirm that the approach used to identify E. coli O157:H7 in the challenged cattle were valid, we tested the non-sorbitol fermenting, nalidixic acid resistant colonies isolated from the third group of cattle using the RIM E. coli O157:H7 test kit. These colonies were E. coli O157:H7 positive and confirm that the methods used to identify the original challenge bacteria in the cattle are accurate. To establish a link between the focal petechiae and active E. coli O157:H7 infection in cattle, the intestinal tracts from 2 persistent-shedding animals (Nos. 892 and 894) that were shedding for ~5 months after the initial challenge were examined. All tissues were positive for focal petechiae as described for cattle that ceased shedding for ~1 month. There were also focal mucosal hemorrhages ~3.75 cm in diameter observed at regular intervals (about every 10–18 cm) in the jejunum. The spiral colon was similarly affected except the hemorrhage shape was elongated, measuring ~5 cm long  0.5 cm wide. There were no hemorrhages in the ileum. We did not observe any complete mucosal ulcerations or erosions, serosal hyperemia, erythema, or severe edema. We did observe mild to moderate forms of histopathology with bacteria visibly associated with the villi (Fig. 3a) and in clusters along the villi (Fig. 3b). The bacteria formed classic actin pedestals or A/E lesions (Fig. 3c) and grew in cupshaped colonization sites (Fig. 3d) characteristic of E. coli O157:H7 infections. The hemorrhaged areas were not recently formed as indicated by the fibrin deposition in the mucosal hemorrhage (Fig. 4a). There was evidence of superficial mucosal necrosis (Fig. 4b), focal mucosal necrosis (Fig. 4c) showing crypt necrosis plus loss of villi, and regenerating epithelium indicated by the flattened epithelium and blunted villi (Fig. 4d). Although not present in all sections, crypt necrosis (Fig. 5a) and marked architectural crypt distortions (Fig. 5b) were also suggestive of a chronic E. coli O157:H7 infection. As in human infections (Lamps 2007), the deep crypts were not affected. If there was a different E. coli O157:H7 infection process in cattle than for humans (Shigeno et al. 2002), we would have expected not to find any serious forms of pathologies upstream from the rectoanal junction and this was not the case. The pathologies were more extensive in the small and large intestines of a steer shedding E. coli O157:H7 for 4 months after the initial challenge. The types of pathologies present suggest a diagnosis of E. coli O157:H7-induced hemorrhagic enteritis at a grade 3 level for the jejunum, a grade 2/3 for the ileum, cecum, ascending colon and a grade 1 level for all other sections of the intestine (Shigeno et al. 2002). Focal petechiae were not present from the transverse #

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Fig. 3. Light and transmission micrographs of focal hemorrhaged areas in the jejunum of cattle shedding Escherichia coli O157:H7 5 months after the initial challenge. (a) Bacteria associated with the villi (arrow), (b) clustered bacterial colonization, (c) classic A/E lesion for E. coli O157:H7 (arrow), and (d) classic cup-shaped colonization sites formed by E. coli O157:H7 (arrow).

colon to the rectoanal junction, but were evident in the jejunum, ileum, cecum and the ascending colon. Mild serosal hemorrhages were evident in the jejunum and ileum (Figs. 6a and 6b). Exudates were present in the jejunum and ileum (Figs. 6d and 6f). The Peyer’s patches in the jejunum and ileum were raised and reddened (Fig. 6g). Severe hemorrhages in the jejunum were visible through the serosa (Fig. 6h). There was complete loss of mucosal structure in some areas of the jejunum (Fig. 6i) and extensive mucosal hemorrhages (Figs. 6j–6m), dark red erythema and edema (Figs. 6j, 6k, and 6m). The anterior ileum had edema and dark red erythema (Fig. 6e), whereas the posterior region had patchy edema and erythema (Fig. 6d). The jejunum was the most severely affected intestinal region. Non-sorbitol fermenting bacterial colonies were isolated from the different pathology regions and confirmed as E. coli O157:H7 (RIM E. coli O157:H7 test kit). These bacteria were associated with thinned mucosa in the jejunum (300 CFU), hemorrhages in the jejunum (1080 CFU), mucosa with exudate in the jejunum (160 CFU), Peyer’s patches in the jejunum (95 CFU), hemorrhages in the ileum (360 CFU),

thickened ileum (400 CFU), Peyer’s patches in the ileum (200 CFU), and hemorrhages in the ascending colon (25 CFU). Escherichia coli O157:H7 E318N adherence to intestinal tissue There was a significant difference in E coli O157:H7 adherence to intestinal tissues from different animals (p = 0.001, n = 56). The nonpersistent-shedding cattle were significantly different from the persistent-shedding cattle, whereas the moderately persistent cattle were composed of animals that were either similar to nonpersistent- or persistent-shedding cattle. It is unclear which factors may be contributing to the lower or higher than expected E. coli O157:H7 colonization in the moderately persistent shedding cattle. The adherence of E. coli O157:H7 to the rectoanal junction did not differ from other intestinal sites (p = 0.324, n = 63). The data were therefore pooled for the small (3 sites) and large (5 sites) intestines for each animal. There was a dose-dependent adherence of E. coli O157:H7 to intestinal tissues (p = 0.001, n = 72; Fig. 7). There was a sig#

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Fig. 4. Light micrograph of focal hemorrhaged areas in the jejunum of cattle shedding Escherichia coli O157:H7 5 months after the initial challenge. (a) Mucosal hemorrhage with fibrin deposition, (b) superficial mucosal necrosis displaying ragged villi (arrow), (c) mucosal necrosis displaying focal crypt necrosis (arrow) and loss of villi (arrow), and (d) regenerating epithelium displaying blunted villi (thick arrow) and flattened epithelium (thin arrow).

nificant difference in E. coli O157:H7 adherence to intestinal tissues from nonpersistent- to persistent-shedding cattle (p = 0.005, n = 56). There was greater E. coli O157:H7 adherence to small intestinal sites of persistent-shedding cattle than for nonpersistent-shedding cattle (p = 0.001, n = 21; Fig. 7a). There was greater E. coli O157:H7 adherence to large intestinal sites of persistent-shedding cattle than for nonpersistent-shedding cattle (p = 0.001, n = 35; Fig. 7b). The threshold dose for E. coli O157:H7 E318N adherence to small intestines from nonpersistent-shedding cattle was 3.4  106 bacteria, and the maximum adherence dose was 1.6  107 bacteria. The theshold dose for E. coli O157:H7 E318N adherence to large intestines from nonpersistentshedding cattle was 1.6  107 bacteria, and the maximum adherence dose was 1.1  108 bacteria. The threshold dose for E. coli O157:H7 E318N adherence to small intestines from persistent-shedding cattle was 2  105 bacteria, and the maximum adherence dose was 6.7  105 bacteria. The threshold dose for E. coli O157:H7 E318N adherence to large intestines from persistent-shedding cattle was 6.7  105 bacteria, and the maximum dose was 1.6  107 bacteria. These results confirm a greater sensitivity of the intestine of persistent-shedding cattle to E. coli O157:H7 colonization compared with nonpersistent-shedding cattle. This translates into about a 10–100 times greater E. coli O157:H7 adherence to intestinal tissues at environmental

doses for persistent-shedding cattle compared with nonpersistent-shedding cattle. There was no significant difference in E. coli O157:H7 adherence to follicle-associated epithelium (log adherence = 7.4 ± 0.1 for the jejunum and log adherence = 7.7 ± 0.1 for the ileum) compared with absorptive epithelium (log adherence = 7.3 ± 0.1 for the jejunum) in the small intestine (p = 0.124, n = 6). The lack of tissue specificity is in agreement with the pathology results that showed damage evenly distributed across Peyer’s patches and absorptive epithelium (Figs. 2 and 6).

Discussion No pathologies have been reported for intestinal tissues of natural or experimental challenged cattle (Brown et al. 1997; Cray and Moon 1995; Grauke et al. 2002; Lim et al. 2007; Low et al. 2005; Naylor et al. 2003, 2005; Van Baale et al. 2004). Because cattle are asymptomatic even when exposed to high challenge doses (1010 CFU), and random selection of shedding cattle has shown no apparent intestinal pathology, it has been assumed that intestinal tissues were not affected by E. coli O157:H7 colonization. The current study has identified mild to severe forms of pathology associated with E. coli O157:H7 infections in experimental challenged cattle that shed for >4 months and a residual pathology present in #

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Baines et al. Fig. 5. Light micrograph of focal hemorrhaged areas in the jejunum of cattle shedding Escherichia coli O157:H7 5 months after the initial challenge. (a) Crypt necrosis and (b) architectural crypt distortions.

the intestinal tracts of cattle that ceased shedding for 5– 12 weeks. Unlike disease-susceptible systems, cattle did not present with the greatest damage in the cecum and ascending colon (Kelly et al. 1990; Shigeno et al. 2002), instead the jejunum had the greatest damage. There are a range of symptoms associated with E. coli O157:H7 induced pathology in the human colon that are related to the progression and resolution of the infection. Common features of E. coli O157:H7 infections and chronic bacterial infections include mucosal/submucosal edema, mucosal/submucosal hemorrhage, mucosal microvascular thombi, mucosal ulceration, submucosal vascular thombosis, submucosal fibrin deposition, mucosal inflammation, and erythema (Griffin et al. 1990; Kelly et al. 1990; Lamps 2007; Li 2000; Murray and Patterson 2000; Shigeno et al. 2002). In the current study, we have shown for the first time that E. coli O157:H7 colonization of intestinal tissues in cattle affects the mucosa of small and large intestinal sites. The jejunum, ileum, cecum, and ascending colon all displayed hemorrhages, but only the jejunum and ileum had extensive dark red erythema and edema. The types of damage observed in persistent-shedding cattle was consistent with those described for grade 2 and grade 3 hemorrhagic colitis mediated by E. coli O157:H7 infections in humans. The hierarchy for the amount of intestinal pathology in cattle was jejunum >> ileum > cecum, ascending colon >> duodenum, transverse colon, descending colon, sigmoid colon, rectum, and rectoanal junction. In contrast, the hierarchy for the amount of intestinal pathology in humans was cecum, ascending colon > transverse colon, descending colon > sigmoid colon > ileum, and rectum.

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The tissue pathology and IVOC adherence assays demonstrated that there was no tissue tropism for cattle as described for E. coli O157:H7 infections in humans. Fitzhenry et al. (2006) determined that this colonization resulted in humans when type 1 long polar fimbriae mutants did not express these fimbriae suggesting that E. coli O157:H7 colonization is not mediated by fimbriae in cattle. The decline in pathology from 4 to 5 months in the persistentshedding cattle also suggests that E. coli O157:H7 infection involves both the small and large intestines in cattle, but because of the location of the jejunum, this site is more critical to maintaining infections and persistent shedding. Cattle resolve the E. coli O157:H7 infection on their own without any apparent serious impacts (Lamps 2007). Evidence for this comes from crypt distortions indicative of severe infection and regenerative mucosal changes including flat regenerating mucosa and blunted villi. It is unclear which E. coli O157:H7 virulence factors may be responsible for the mucosal pathology or whether other pathogens are involved, however, these results do support earlier studies that have shown that even if the mucosa were affected in cattle, the lack of vascular Stx receptors would prevent the onset of serious disease (Pruimboom-Brees et al. 2000). The focal petechiae were also a common feature in the mucosa of the E. coli O157:H7 challenged cattle even after the cattle had ceased shedding. Focal petechiae are small, pinpoint hemorrhages that are also a common feature in E. coli O157:H7 colonization of the intestine in challenged neonatal calves (Dean-Nystrom et al. 1998). This pathology results from the activities of hemorrhagic disease agents that cause leakiness in the minute vessels of affected tissues (Humble 1949). We have shown for the first time that focal petechiae result from E. coli O157:H7 challenges in mature cattle, and the distribution of focal petechiae in the intestinal tract can predict the status of E. coli O157:H7 infections. Escherichia coli O157:H7 was isolated from the focal petechial sites and classic E. coli O157:H7 colonization features were identified in the damaged mucosa supporting a role for E. coli O157:H7 in causing these pathologies in cattle. The longer time period required for all pathologies to resolve in persistent-shedding cattle suggests that a higher amount of E. coli O157:H7 colonization had occurred in these animals compared with other cattle. This conclusion is supported by the results from the IVOC E. coli O157:H7 adherence assays. The change in the distribution of focal petechiae in intestinal sites for the challenged cattle together with the sequential loss of mucosal pathologies in the small and large intestines of persistent-shedding cattle also implicates a sequence for intestinal E. coli O157:H7 infection that reflects the requirement for small and large intestinal sites. Challenged cattle are initially colonized to various degrees in all intestinal sites; however, the jejunum, ileum, descending colon, and rectoanal junction have longer resolution times. The location of the jejunum and ileum in the digestive tract also suggest that E. coli O157:H7 colonization of these sites would play a larger role in maintaining infections in cattle. In contrast, the location and function of the rectoanal junction suggests that it does not contribute to maintaining persistent E. coli O157:H7 shedding as bacteria that detach from this site are shed with the feces. The current model for E. coli O157:H7 infection in cattle suggests that it is the #

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Fig. 6. Intestinal pathology in a steer shedding Escherichia coli O157:H7 4 months after the initial challenge. (a) Serosal paintbrush hemorrhage in the jejunum, (b) serosal paintbrush hemorrhage in the ileum, (c) focal hemorrhage and erythema in the ascending colon, (d) patchy edema and erythema in the posterior ileum, (e) edema and dark red erythema in the anterior ileum, (f) exudate overlying the mucosa in the jejunum, (g) raised and reddened Peyer’s patches in the jejunum, (h) mucosal hemorrhage visible from the serosal surface in the jejunum, (i) loss of normal mucosal structure and dark red erythema in the jejunum, (j) focal hemorrhage with mucosal erosion and dark red erythema and edema in the jejunum, (k) focal longitudinal hemorrhage in the jejunum, (l) focal longitudinal hemorrhage in the jejunum, and (m) focal hemorrhages and dark red erythema in the jejunum.

colonization of the rectoanal junction that promotes longterm or persistent shedding (Lim et al. 2007; Low et al. 2005; Naylor et al. 2003, 2005). The current study has shown that the amount of E. coli O157:H7 colonization of the rectoanal junction did not differ from other small and large intestinal sites in vitro. In addition, the sequential clearance of focal petechiae from the intestinal system and the mucosal pathologies observed in the intestine of persistent-shedding cattle also suggest that the rectoanal junction is not an important site for maintaining E. coli O157:H7 infections in cattle. Instead, the position of the rectoanal junction in the intestinal tract together with its function to store contaminated stool may serve to act as a barometer for the status of E. coli O157:H7 infection in cattle. A similar conclusion has been drawn for the rectum in other animal model systems (Ritchie et al. 2003). Future studies should focus on determining the underlying mechanisms responsi-

ble for the novel pathologies associated with E. coli O157:H7 colonization of cattle. Mature cattle respond to an E. coli O157:H7 challenge with heterogeneous shedding periods, and it was proposed that this response was linked to the degree of intestinal colonization by E. coli O157:H7 (Grauke et al. 2002). In the current study, a dose-dependent E. coli O157:H7 colonization of intestinal tissues was observed for all cattle confirming that exposure dose affects the amount of E. coli O157:H7 colonization in intestinal tissues (Besser et al. 2001; Sheng et al. 2004). In addition, the 10–100 time increase in E. coli O157:H7 adherence to intestinal tissues from nonpersistent- to persistent-shedding cattle confirms the hypothesis of Grauke et al. (2002) that shedding period is related to the amount of E. coli O157:H7 colonization. Because these results were obtained in vitro without any contribution of the innate defensive system, an undefined #

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Baines et al. Fig. 7. (a) Dose-dependent adherence of Escherichia coli O157:H7 E318N to small intestine of nonpersistent( NP)- and persistent (P)shedding cattle, in vitro (p = 0.001, n = 21). (b) Dose-dependent adherence of E. coli O157:H7 E318N to large intestine of NP- and P-shedding cattle, in vitro (p = 0.001, n = 35).

mucosal factor is likely responsible for higher E. coli O157:H7 colonization in persistent-shedding cattle. Two membrane proteins, nucleolin and b-1 integrin, expressed by enterocytes of neonatal calves, can act as non-TIR based colonization sites for E. coli O157:H7 (Sinclair et al. 2006). It is possible that the expression of non-TIR based colonization sites in the intestine could account for the differences in E. coli O157:H7 colonization and shedding by cattle. Future research should focus on determining if a membrane protein(s) in the intestinal tract of persistent-shedding cattle is responsible for higher levels of E. coli O157:H7 colonization. Escherichia coli O157:H7 virulence factors affect the outcome of E. coli O157:H7 interactions with intestinal tissues in nonruminant systems (Wales et al. 2005). One E. coli O157:H7 virulence factor, Stx, has both lethal (Bauer and Welch 1996; Ferens and Hovde 2000; Figueiredo et al. 2007; Paton et al. 1995; Schuller et al. 2004) and sublethal (Ferens and Hovde 2007; Menge et al. 2004; Robinson et al. 2006) effects on cells that promote colonization by pathogens through a reduction in host defenses, providing crevi-

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ces in the mucosa for replication and increasing the availability of nonintimate attachment sites. For cattle, there is no cytotoxicity reported for colon enterocytes exposed to Stx1 and, in addition, Stx1 is rapidly degraded in the crypts (Hoey et al. 2002, 2003). This suggests that the observed mucosal changes in intestinal tissues of shedding cattle may be attributable to other cytotoxins produced by E. coli O157:H7. We recently addressed this question and determined that E. coli O157:H7 secreted cytotoxins are toxic to enterocytes isolated from the jejunum and descending colon of cattle (Baines et al. 2008a, 2008b) supporting a role for E. coli O157:H7 secreted cytotoxins in mediating the mucosal damage recorded in the current study. A second sublethal effect, an increase in the expression of non-TIR based E. coli O157:H7 colonization sites, has been reported for Stx2 in a mouse model system used to evaluate E. coli O157:H7 interactions with ruminants (Robinson et al. 2006). This study showed that infections with a Stx2producing E. coli O157:H7 strain had greater colonization and a longer duration of shedding than a Stx2-negative E. coli O157:H7 strain. Further, when both strains were inoculated together, there was greater colonization of tissues and a longer duration of shedding. This change reflected equivalent Stx-positive and Stx-negative E. coli O157:H7 strain colonization. The benefit was derived from Stx2 increasing the expression of a non-TIR-based colonization site, nucleolin. In the current study, E. coli O157:H7 E318N has the most potent cytotoxin composition and has the highest colonization potential in cattle compared with the other strains used to challenge the cattle (Baines et al. 2008a, 2008b). If a similar mechanism exists in cattle, the 4 bacterial strains used in the current study should equally colonize cattle and this has been shown in the 77-day fecal samples (McAllister et al. 2006). Similarly, the inclusion of cytotoxins from a high colonizing strain should enhance E. coli O157:H7 colonization, and this was confirmed using the IVOC adherence assay (Baines et al. 2008b). Finally, Stx2 should enhance E. coli O157:H7 colonization with the biggest benefit derived for a strain that produces less active cytotoxins, and this was confirmed using the IVOC adherence assay (Baines et al. 2008c). Together, these results strongly support a role for Stx2 in enhancing E. coli O157:H7 colonization in cattle. There is one study comparing the colonization potential of Stx2-negative and Stx2-positive E. coli O157:H7 strains in cattle (Sheng et al. 2004), but there was no difference in the colonization of the rectoanal junction. In contrast, oralchallenged cattle using single vs. multiple E. coli O157:H7 with at least 1 Stx2 positive strain does increase the duration of shedding (Cray and Moon 1995; Besser et al. 2001; McAllister et al. 2006). This difference can be attributed to the route used for the challenge dose. Since the current study suggests that other intestinal sites upstream from the rectoanal junction are critical for maintaining E. coli O157:H7 infections in cattle, the earlier study reflects the behavior of the rectoanal junction alone and not the influence of the rest of the intestinal tract. Future research should focus on the role of Stx2 in promoting E. coli O157:H7 infections in cattle. There is debate over which challenge method for cattle is a better model for E. coli O157:H7 infection, natural or experimental. Escherichia coli O157:H7 have been isolated #

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from the small and large intestines of experimental E. coli O157:H7 challenged cattle without the presence of classic A/E lesions (Cray and Moon 1995; Grauke et al. 2002; Van Baale et al. 2004). In contrast, E. coli O157:H7 have been isolated from the rectoanal junction of natural E. coli O157:H7 challenged cattle with the presence of A/E lesions (Low et al. 2005; Naylor et al. 2003, 2005). A few factors may contribute to this discrepancy between natural and experimental challenged cattle. Given that the majority of natural E. coli O157:H7 challenged cattle are low carriers (90%–96%) (Grauke et al. 2002; Low et al. 2005), this discrepancy may represent the study of low-colonized cattle as opposed to the so called ‘‘super shedders’’ that are thought to be mainly responsible for the dissemination of E. coli O157:H7 within the feedlot (Matthews et al. 2006). In addition, the function, location, and size of the rectoanal junction may predispose this site to easier isolation of A/E lesions than other intestinal sites. Experimental E. coli O157:H7 challenged cattle have several advantages over natural E. coli O157:H7 challenged cattle for pathology studies in that they receive a higher challenge dose than in the natural environment (Duffy 2003) and individual E. coli O157:H7 shedding characteristics are monitored. This provides an opportunity for selecting animals with known E. coli O157:H7 exposure dose, potential colonization level, and shedding behavior. In addition, the high E. coli O157:H7 challenge dose may elicit pathology not easily detected under natural E. coli O157:H7 challenge conditions. In the current study, the ability to effectively select highcolonizing cattle facilitated a better measurement and understanding of the E. coli O157:H7 colonization process and the potential intestinal factors responsible for prolonging E. coli O157:H7 infections in cattle. We conclude that the heterogeneity of E. coli O157:H7 shedding in cattle is related to E. coli O157:H7 colonization of the small and large intestines.

Acknowledgments This work was supported by a grant from the Beef Cattle Research Council of Canada and Agriculture and Agri-Food Canada (MII fund). We thank Lorna Selinger, Ruth Barbieri, and Stephanie Erb for technical assistance.

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2008 NRC Canada