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1,2,3,4Department of Food and Environmental Safety1, Animal Services Unit2, TSE Molecular Biology Unit3 and Department of Pathology4, Veterinary Laboratories Agency (Weybridge), Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK 5Department of Clinical Veterinary Science, University of Bristol Veterinary School, Langford, Bristol BS40 5DU, UK
Correspondence M. J. Woodward m.j.woodward{at}vla.defra.gsi.gov.uk
Received September 17, 2004
Accepted December 28, 2004
Enterohaemorrhagic Escherichia coli O157 : H7 infections of man have been associated with consumption of unpasteurized goat's milk and direct contact with kid goats on petting farms, yet little is known about colonization of goats with this organism. To assess the contribution of flagella and intimin of E. coli O157 : H7 in colonization of the goat, 8-week-old conventionally reared goats were inoculated orally in separate experiments with 1x1010 c.f.u. of a non-verotoxigenic strain of E. coli O157 : H7 (strain NCTC 12900 Nalr), an aflagellate derivative (DMB1) and an intimin-deficient derivative (DMB2). At 24 h after inoculation, the three E. coli O157 : H7 strains were shed at approximately 5x104 c.f.u. (g faeces)–1 from all animals. Significantly fewer intimin-deficient bacteria were shed only on days 2 (P = 0.003) and 4 (P = 0.014), whereas from day 7 to 29 there were no differences. Tissues from three animals inoculated with wild-type E. coli O157 : H7 strain NCTC 12900 Nalr were sampled at 24, 48 and 96 h after inoculation and the organism was cultured from the large intestine of all three animals and from the duodenum and ileum of the animal examined at 96 h. Tissues were examined histologically but attaching-effacing (AE) lesions were not observed at any intestinal site of the animals examined at 24 or 48 h. However, the animal examined at 96 h, which had uniquely shed approximately 1x107 E. coli O157 : H7 (g faeces)–1 for the preceding 3 days, showed a heavy, diffuse infection with cryptosporidia and abundant, multifocal AE lesions in the distal colon, rectum and at the recto-anal junction. These AE lesions were confirmed by immunohistochemistry to be associated with E. coli O157 : H7.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Transmission of E. coli O157 : H7 is faecal–oral (Pepin et al., 1997; Locking et al., 2001), with cattle considered as the primary reservoir (Griffin & Tauxe, 1991) and sheep also recognized as a significant reservoir (Chapman et al., 1997; Heuvelink et al., 1998; Meng et al., 1998; Fegan & Desmarchelier, 1999). Other potential sources of these organisms have been described, including rabbits and seagulls amongst other species (Griffin & Tauxe, 1991; Pritchard et al., 2001).
Several recent reports cite goats as potential sources of E. coli O157 : H7 infection. Shukla et al. (1995) reported four human cases of bloody diarrhoea affecting one adult and three children aged 2–4 years, which were traced to a farm visitor centre in Leicestershire, UK. Strains of E. coli O157 : H7 phage type 2, all with identical restriction fragment length polymorphism patterns, were isolated from the cases and from four cattle and six goats. Three other incidents in the UK have implicated goats on visitor farms as potential sources of infection (Chapman et al., 2000; Pritchard et al., 2000; Payne et al., 2003). In continental Europe, Heuvelink et al. (2002) showed that two of 11 petting zoos had goats that were positive for E. coli O157 : H7. In Bohemia, four cases of haemolytic-uraemic syndrome in children were reported and the source was identified as unpasteurized goat's milk, with E. coli O157 : H7 phage type 2 isolated from a goat and an asymptomatic human carrier who drank goat's milk (Bielaszewska et al., 1997). An outbreak of E. coli O157 : H7 associated with unpasteurized goat's milk has also been described in Canada (McIntyre et al., 2002). Foschino et al. (2002) reported a 1.7 % incidence rate of E. coli O157 : H7 in samples of raw goat's milk intended for cheese making in the Bergamo region of Italy, although it was not associated with infection in man. Despite these incidents involving the goat as the primary source, the biology of E. coli O157 : H7 in this host is not understood. Little is known of the prevalence of E. coli O157 : H7 in goats, with only a few reports suggesting low prevalence rates in the region of 0.2 % (Blanco et al., 2003; Dontorou et al., 2004).
Attaching-effacing (AE) E. coli belonging to non-O157 serogroups are found frequently in diarrhoeic goats (Duhamel et al., 1992; Drolet et al., 1994) and putative AE E. coli have been reported in healthy goats (Cid et al., 2001; de la Fuente et al., 2002; Orden et al., 2003a, b). Furthermore, E. coli belonging to serogroup O145 induced AE lesions in an adult goat (Barlow et al., 2004). Collectively, these observations suggest that goats may be a permissive host for AE E. coli including E. coli O157 : H7. Recently, it was shown that 6-day-old conventional kids were colonized by E. coli O157 : H7 and that small AE lesions in the mucosa of the ileum and large intestine were observed following oral inoculation (Wales et al., 2005). These data suggest that goats are susceptible to colonization by E. coli O157 : H7, probably in an intimin-dependent manner, but whether this is the case for older goats remains untested. In this study, we wished to assess the contribution of two bacterial factors, intimin and flagella, in colonization. Intimin is considered to play a role as a key effector in the induction of AE lesions, whereas flagella have been shown to contribute to persistence, particularly of commensal E. coli, in many species. E. coli O157 : H7 strain NCTC 12900 Nalr, which has been shown to colonize small ruminants, and isogenic intimin- and flagella-deficient mutants were selected for this study. Weaned, conventional, 8-week-old goats were orally inoculated with the test strains and, to assess colonization, faecal shedding was monitored and serial post-mortem examinations were performed to locate the organism within the gastrointestinal tract.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Strain NCTC 12900 Nalr and isogenic derivatives were stored in heart infusion broth medium supplemented with 30 % (w/v) glycerol on beads at –80 °C and working stocks were stored at room temperature on Dorset's egg medium.
NCTC 12900 Nalr and isogenic derivatives were streaked from Dorset's egg medium on to sorbitol MacConkey agar (SMAC) plates containing nalidixic acid (15 µg ml–1) and well-isolated colonies were inoculated separately into 100 ml aliquots of LB broth in 250 ml conical flasks. After incubation for 16 h at 37 °C with gentle agitation, the bacterial cells were harvested by centrifugation (3000 g for 10 min) and resuspended in PBS (pH 7.4). The bacterial suspensions contained approximately 1x109 c.f.u. ml–1 as determined by serial dilution and plating.
Animals.
Post-partum, 20 neonatal goat kids were allowed to suckle and after weaning at about 6 weeks of age were separated from their respective nannies. The animals were selected randomly into three groups comprising eight (A), six (B) and six (C) animals with each group penned separately. Each animal was identified with duplicate ear tags encoding a unique four-digit identification number and housed indoors for a further 2 weeks and provided with standard rations and water ad libitum.
Prior to inoculation with E. coli O157 : H7, faeces were taken from the rectum of each animal and cultured for E. coli O157, as described below. All animals in each of the three groups were inoculated orally with one challenge strain; group A was challenged with E. coli O157 : H7 NCTC 12900 Nalr, group B with DMB2 and group C with DMB1. The challenge doses (10 ml) were administered orally using a worming gun, ensuring delivery of the inoculum to the pharynx. Rectal faecal samples were taken from each animal on days 1, 2, 4, 7, 8, 10, 11, 15, 18, 22, 25 and 29 after challenge. Animals were observed at least twice daily to monitor clinical status.
Necropsy procedures.
Necropsy procedures were as described previously (Wales et al., 2005). Briefly, from group A, goats were selected at random at 24 (animal 1019), 48 (animal 1001) and 96 (animal 1003) h after challenge and tissue samples were collected immediately after euthanasia with barbiturate overdose. Samples were collected from the rumen, duodenum, jejunum, ileum, caecum, ascending colon, spiral colon and from six sites excised at approximately 2 cm apart measuring from the recto-anal junction (RAJ), along the rectum toward the distal colon. Samples were used for bacteriological and histological examination and electron microscopy as described below. Clips and plastic bags were used to enclose the cut ends of the intestinal tract as dissection proceeded, as described previously in sheep (Wales et al., 2001a), to prevent cross-contamination of samples.
Bacteriological examination.
Faecal samples taken prior to oral inoculation were examined for the presence of E. coli O157 following previously described methods (Wales et al., 2001a, b, 2002; Woodward et al., 2003). Faeces (1 g) were resuspended in 9 ml buffered peptone water (BPW), incubated at 37 °C for 6 h and O157 : H7 organisms were recovered by O157-specific immunomagnetic separation and plated on to cefixime-tellurite SMAC plates.
To detect the strains used to inoculate the animals orally, previously described methods were followed (Wales et al., 2001a, b, 2002; Woodward et al., 2003). Faeces (1 g) were resuspended in 9 ml BPW by vortexing and serial dilutions were plated directly on to SMAC plates containing nalidixic acid (15 µg ml–1). Additionally, dilutions were retained overnight at 4 °C and, if there were no direct counts of confirmed O157 organisms, the dilutions were incubated at 37 °C for 6 h and samples plated on to SMAC plates containing nalidixic acid (15 µg ml–1). The serogroup of bacteria recovered by these processes was checked by O157-specific latex agglutination (Oxoid). Tissue samples collected from kids for bacteriological examination were homogenized in BPW (1 g in 9 ml) and processed as described above.
Pathological studies
Light microscopy. Tissues were placed immediately into 10 % neutral buffered formalin at room temperature and left to fix for at least 24 h. Trimmed tissues were processed routinely to paraffin wax and 4 µm sections were stained with haematoxylin and eosin (H&E) and observed using an Olympus CX41 microscope.
Immunohistochemistry. Tissue blocks were fixed in 10 % neutral buffered formalin, processed to wax and sectioned at 4 µm on a rotary microtome in preparation for immunohistochemistry. Tissue sections were dewaxed in xylene and dehydrated in absolute alcohol before immersion in freshly prepared 3 % hydrogen peroxide/methanol for 10 min to inhibit endogenous peroxidase activity.
Sections were rehydrated in running tap water prior to assembly into the Shandon Sequenza staining system. The slides were washed with 2x sodium chloride/Tris-buffered saline (NaCl/TBS: 0.005 M TBS, pH 7.6, 1.7 % NaCl) for 5 min before incubation at room temperature with a normal goat serum (Vector Laboratories) for 20 min. E. coli O157-specific polyclonal antibody, raised in rabbits (Veterinary Laboratories Agency, Weybridge, UK), was then applied (diluted 1 : 1000 and 1 : 5000 in 2x NaCl/TBS supplemented with 5 % normal rabbit serum) to the sections for 1 h at room temperature (Wales et al., 2001b).
E. coli antigens were visualized following incubation with biotinylated goat anti-rabbit IgG (diluted 1 : 200, with normal goat serum diluted 1 : 66 in 2x NaCl/TBS supplemented with 3 % normal sheep serum) for 30 min at room temperature, an avidin–biotin–peroxidase conjugate (Vector Laboratories) for 30 min at room temperature and citrate-buffered diaminobenzidine for 10 min at room temperature. The sections were then counterstained in Meyer's haematoxylin before being dehydrated in absolute alcohol, cleared in xylene and coverslipped using DPX mountant. The stained tissue sections were then examined by light microscopy.
Electron microscopy. After removal from the animal, tissues were fixed in 3 % glutaraldehyde prepared in 0.1 M phosphate buffer. Following microscopic examination of the H&E sections, at sites where lesions were identified, corresponding glutaraldehyde-fixed tissues were cut to 1–2 mm thickness for TEM examination. The tissues were then washed in 0.1 M phosphate buffer, post-fixed in 1 % osmium tetroxide, dehydrated by immersion in a series of alcohol solutions increasing to 100 % alcohol and placed in propylene oxide prior to embedding in araldite resin. The resin was polymerized at 60 °C for 48 h. One micrometre sections were stained with toluidine blue for light microscopic examination. Ultrathin sections at 70–90 nm thickness were then prepared on to copper grids using a diamond knife and stained with uranyl acetate and lead citrate prior to examination using a Phillips CM10 TEM.
Statistical analyses.
The sensitivity of detection by direct plating was approximately 500 organisms (g faeces)–1. Samples positive by enrichment were considered to have up to 500 organisms (g faeces)–1 and those samples in which no organisms were detected were given an arbitrary value of 1 to avoid the issue of a zero value giving results to infinity. The non-parametric Kruskal–Wallis test was used to compare the mean group counts for each occasion. When this was significant at P 
0.05, the individual group means were compared by a multiple comparison test. The percentages of animals positive were also compared by Fisher's exact test at each time point.
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Faecal shedding of E. coli O157 : H7
All faeces samples collected from the animals prior to the experimental procedure were negative for E. coli O157 as assessed by immunomagnetic separation. The oral inoculation doses were determined to be 1 x 1010 c.f.u. for each kid in each of the three groups.
The faecal shedding data are shown in Fig. 1. At 24 h after challenge, all animals in all groups were shedding the inoculated strain [range 2 x 104–9.2 x 106 c.f.u. (g faeces)–1], with no statistically significant differences between the geometric mean values of the three groups at this time.
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On days 2 and 4 after inoculation, differences were observed. On these days, all animals in groups A and C dosed with E. coli O157 : H7 strain NCTC 12900 Nalr and DMB1 (isogenic aflagellate derivative), respectively, shed the inoculated strain at about the same numbers as on day 1, whereas DMB2 (isogenic intimin-deficient derivative) was detected in four and three of the six animals in group B on days 2 and 4 after challenge, respectively. The only statistically significant difference in numbers of animals shedding was at day 4 (P = 0.021). On days 2 and 4, the geometric mean shedding scores in groups A and C [
105 c.f.u. (g faeces)–1] were significantly higher than the geometric mean shedding score for group B animals [5 x 101 and 1 x 102 c.f.u. (g faeces)–1; day 2, P = 0.003; day 4, P = 0.014]. The geometric mean counts from group B animals were lower than those from the other two groups on days 7 and 8, but these differences were not significant (P = 0.057 and P = 0.062, respectively).
From day 10 onwards, only a few animals shed O157 organisms in each group and there were no statistically significant differences between groups or strains. However, it was noted that E. coli O157 : H7 strain NCTC 12900 Nalr was not detected in faecal samples tested on days 22, 25 and 29, whereas both DMB1 and DMB2 were detected, albeit in small numbers, on these days.
Bacteriological findings at necropsy
Three animals in group A, inoculated with E. coli O157 : H7 strain NCTC 12900 Nalr, were selected at random for post-mortem examination on days 1 (animal 1019), 2 (animal 1001) and 4 (animal 1003). On the day of post-mortem examination, faeces were taken and examined for E. coli O157 : H7. Animal 1019 had 2 x 104 c.f.u. (g faeces)–1 and animal 1001 had 2 x 104 c.f.u. (g faeces)–1 on day 1 and 7 x 105 c.f.u. (g faeces)–1 on day 2. By contrast, animal 1003 had 9.2 x 106 c.f.u. (g faeces)–1 on day 1, 1.7 x 107 c.f.u. (g faeces)–1 on day 2 and 3.5 x 107 c.f.u. (g faeces)–1 on day 4.
There were differences in the numbers of E. coli O157 : H7 strain NCTC 12900 Nalr recovered from tissues in individual animals (Fig. 2). E. coli O157 : H7 was not detected in the small intestine of animal 1019. However, E. coli O157 : H7 was detected in the jejunum of animal 1001 and the duodenum, jejunum and ileum of animal 1003. In the large intestine, by contrast, E. coli O157 : H7 was detected in most sites in all three animals. In the case of animal 1003, large numbers of E. coli O157 : H7 were found at all intestinal sites examined, with up to 106 c.f.u. E. coli O157 : H7 (g tissue)–1 in the mid-section of the rectum. The rumen was negative for all three animals.
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Histopathological findings
Significant changes were confined to the distal colon, rectum and RAJ of animal 1003, examined on day 4. The most prominent feature was the presence of large numbers of cryptosporidium organisms that were identified attached to the mucosa, along with scattered, focal, AE lesions associated with adherent bacteria (Fig. 3a). Cryptosporidia and associated bacteria appeared to be co-located on the mucosal surface. Associated with these lesions, the lamina propria was infiltrated with moderate numbers of mixed lymphoid cells and polymorphonuclear leukocytes. The remaining large intestine samples (ascending colon and spiral colon) were negative for cryptosporidia and attached bacteria.
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Sections of small intestine examined were less well preserved than the large intestine. The epithelium in the majority of samples was still intact, although some artefactual separation of the underlying lamina propria had occurred. Small numbers of coccidia were identified in the ileum, predominantly in the crypt epithelium of animal 1001, examined post-mortem at day 2. Cryptosporidia and attached bacteria were not identified. Abnormalities were not detected in the rumen.
Attempts to isolate cryptosporidia from animal 1003 could not be made as the samples had been disposed of by the time the results from the histological examination were known.
Immunohistochemistry
Multifocal, variable-sized colonies of E. coli O157 : H7 organisms were identified at all sites of the distal colon, rectum and RAJ in animal 1003 (Fig. 3b). These tended to cover from one or two to several adjacent epithelial cells. In addition, lesions identified in the mucosa at the RAJ were found on both lymphoid-associated epithelium and non-lymphoid-associated epithelium.
TEM
Cryptosporidia and AE lesions associated with bacteria were confirmed on the intestinal mucosa (Fig. 4). Both organisms were frequently co-located on the same cell (Fig. 4b), confirming the light microscopic findings.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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E. coli O157 : H7 AE lesions were observed in one animal in this study, coincidentally in association with cryptosporidia. Thus, 8-week-old conventionally reared goats are susceptible to colonization by enterohaemorrhagic E. coli O157 : H7 by intimin-dependent mechanisms (Nataro & Kaper, 1998). However, whether intimin contributes to colonization in this model by acting as an adhesin, independent from the mechanisms for AE lesion formation, as suggested by Frankel et al. (1996) and Sinclair & O'Brien (2002), is questionable. The data from these experiments do not support a substantive role for H7 flagella in colonization in this model.
AE lesions induced by O157 : H7 organisms were not detected in either of the goats examined 1 and 2 days after oral inoculation. It is possible that the number of organisms at the intestinal sites examined may have been insufficient to induce AE lesions, since it has been suggested that there may be a minimum density of organisms to trigger AE lesion formation (Dean-Nystrom et al., 1999). In our previous oral inoculation studies, E. coli O157 : H7 induced AE lesions that were rare and sparse in neonatal lambs (Wales et al., 2001b), weaned lambs (Woodward et al., 2003) and neonatal kids (Wales et al., 2005). Therefore, the failure to detect AE lesions in these two goats probably related to the number of tissue sections examined microscopically relative to the overall size of the intestinal tract.
Recent studies in cattle reported by Naylor et al. (2003) suggested a preferred site of colonization and AE lesion formation by E. coli O157 : H7 at the RAJ. Sheng et al. (2004) confirmed these findings in cattle but did not cite any data on colonization of sheep following rectal administration of the inoculum. In the studies reported here, the RAJ and six other sites along the rectum to the distal colon of the goats were examined. Lesions were not detected in this region of the animals examined 1 and 2 days after oral inoculation but were present in the animal examined on day 4 (animal 1003). This animal shed large numbers of E. coli O157 : H7, in the region of 106–107 c.f.u. (g faeces)–1, and had approaching 106 organisms (g tissue)–1 in the mid-rectum. This high density of E. coli O157 : H7 may have been sufficient to induce the AE lesions. However, the AE lesions were not associated specifically with lymphoid tissue in the RAJ, as has been described for cattle (Naylor et al., 2003). The close association of AE lesions with cryptosporidia is an interesting observation.
In natural and experimental infections in goats, Cryptosporidium parvum causes severe clinical disease, often with high morbidity and mortality (Koudela & Bokova, 1997; Johnson et al., 1999), and severe lesions are induced, often in the posterior jejunum and ileum (Koudela & Jiri, 1997). However, asymptomatic carriage of cryptosporidia has been described in adult goats (Noordeen et al., 2002). In the study reported here, the affected goat was asymptomatic and yet had cryptosporidia associated with the mucosa of the distal colon, rectum and RAJ, a region of the intestine at which cryptosporidia have been observed in the later stages of infection in calves (Tzipori et al., 1983). The observation of concurrent infections with two or more enteropathogens, including cryptosporidia in association with E. coli, has been made previously in diarrhoeic calves (Moon et al., 1978; Janke et al., 1990; de la Fuente et al., 1999; Gunning et al., 2001). Based on these recorded concurrent infections and data from the experiments reported in this study, the question arises as to the possible significance of the presence of cryptosporidium infection upon colonization and shedding of E. coli O157 : H7. It is possible that either the cryptosporidia were incidental or that prior infection with cryptosporidia may predispose the host to colonization by AE E. coli. Without further experimentation aimed at addressing this, it is not possible to draw any firm conclusions.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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of enterohemorrhagic Escherichia coli O157 : H7. J Biol Chem 277, 2876–2885.This article has been cited by other articles:
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  I. Aktan, R. M. La Ragione, and M. J. Woodward Colonization, Persistence, and Tissue Tropism of Escherichia coli O26 in Conventionally Reared Weaned Lambs Appl. Envir. Microbiol., February 1, 2007; 73(3): 691 - 698. [Abstract] [Full Text] [PDF]
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 R. M. L. Ragione, A. Best, D. Clifford, U. Weyer, L. Johnson, R. N. Marshall, J. Marshall, W. A. Cooley, S. Farrelly, G. R. Pearson, et al. Influence of colostrum deprivation and concurrent Cryptosporidium parvum infection on the colonization and persistence of Escherichia coli O157 : H7 in young lambs. J. Med. Microbiol., July 1, 2006; 55(Pt 7): 819 - 828. [Abstract] [Full Text] [PDF]
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 I. Vlisidou, F. Dziva, R. M. La Ragione, A. Best, J. Garmendia, P. Hawes, P. Monaghan, S. A. Cawthraw, G. Frankel, M. J. Woodward, et al. Role of Intimin-Tir Interactions and the Tir-Cytoskeleton Coupling Protein in the Colonization of Calves and Lambs by Escherichia coli O157:H7 Infect. Immun., January 1, 2006; 74(1): 758 - 764. [Abstract] [Full Text] [PDF]
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