Attaching and effacing lesions caused by Escherichia coli O157:H7 in experimentally inoculated neonatal lambs

Four 6-day-old conventionally reared lambs were inoculated orally with a total of 10(9) cfu comprising equal numbers of four enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains. All animals remained clinically normal. Tissues were sampled under terminal anaesthesia at 12, 36, 60 and 84 h post inoculation (hpi). EHEC O157:H7 was cultured from most gastrointestinal tract sites. Small, sparse attaching and effacing (AE) lesions were found in the caecum at 12 and 36 hpi and in the terminal colon and rectum at 84 hpi. Organisms in the lesions were labelled specifically by an O157 antiserum. The results indicate that the well-characterised mechanisms for intimate attachment encoded by the locus for enterocyte effacement (LEE) of EHEC O157:H7 may contribute to the initial events. at least, of colonisation of sheep.

Variation in the persistence of Escherichia coli O157:H7 in experimentally inoculated 6-week-old conventional lambs

Six-week-old lambs were inoculated orally with 10(9) cfu of an antibiotic-resistance marked four-strain mixture of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 to investigate faecal excretion and intestinal colonisation. In the first experiment, three E. coli O157:H7 isolates were not detected in the faeces of any lambs beyond day 8 post inoculation (pi), or from any of the tissues derived from inoculated animals. One strain, 140065 Nal(r), was isolated from the caecum and colon of one lamb on day 9 pi, from the rectum of another on day 22 pi and persisted in the faeces for up to 28 days pi. All animals remained clinically normal throughout the study period and histological evidence of adhesion of E. coli O157:H7 to the intestinal mucosa was not found. In a separate experiment, four 6-week-old lambs were inoculated orally with 10(9) efu of E. coli O157:H7 strain 140065 Nal(r) alone. Faecal samples were positive for this strain until the end of the experiment (day 19 pi). This strain was also recovered from the gastrointestinal tract of lambs on days 6, 18 and 19 pi, but was not isolated at day 17 pi. When sampled separately, rectum and terminal colon contents contained higher numbers of the inoculated strain than the intestinal tissue at these sites. Animals inoculated with O157:117 strain 140065 Nal(r) alone produced soft faeces from day 5 pi onwards. Although attaching and effacing lesions were observed in the caecum, proximal colon and rectum in one animal on day 18 pi, the adherent bacteria did not stain with antiserum raised against the O157 antigen.

Production of attaching-effacing lesions in ligated large intestine loops of 6-month-old sheep by Escherichia coli O157:H7

Shiga-toxigenic Escherichia coli O157:H7 (STEC O157:H7) is associated with potentially fatal human disease, and a persistent reservoir of the organism is present in some farm animal species, especially cattle and sheep. The mechanisms of persistent colonisation of the ruminant intestine by STEC O157:H7 are poorly understood but may be associated with intimate adherence to eukaryotic cells. Intimate adherence, as evidenced by induction of attaching-effacing (AE) lesions by STEC O157, has been observed in 6-day-old conventional lambs after deliberate oral infection but not in older animals. Thus, the present study used a ligated intestinal loop technique to investigate whether STEC O157:H7 and other attaching-effacing E. coli may adhere intimately to the sheep large intestinal mucosa. To do this, four STEC O157:H7 strains, one STEC 026:K60:H11 and one Shiga toxin-negative E. coli O157:H7 strain, suspended in either phosphate-buffered saline or Dulbecco's modified Eagle's medium, were inoculated into ligated spiral colon loops of each of two lambs. The loops were removed 6 h after inoculation, fixed and examined by light and electron microscopy. AE lesions on the intestinal mucosa were produced by all the inoculated strains. However, the lesions were sparse and small, typically comprising bacterial cells intimately adhered to a single enterocyte, or a few adjacent enterocytes. There was little correlation between the extent of intimate adherence in this model and the bacterial cell density, pre-inoculation growth conditions of the bacteria or the strain tested.

Interaction with avian cells and colonisation of specific pathogen free chicks by Shiga-toxin negative Escherichia coli O157:H7 (NCTC 12900)

The prevalence of Escherichia coli O157:H7 infection in birds is low but several deliberate inoculation studies show that poultry are readily and persistently infected by this organism indicating a possible threat to public health. The mechanisms of colonisation of poultry are not understood and the aim is to establish models to study the interaction of E. coli O157:H7, at the cellular and whole animal levels. A non-toxigenic E. coli O157:H7 (NCTC 12900) was used in adherence assays with an avian epithelial cell line (Div-1) and used to inoculate 1-day-old SPF chicks. In vitro, NCTC 12900 induced micro-colonies associated with cytoskeletal arrangements and pedestal formation with intimate bacterial attachment. In the 1-day-old SPF chick, a dose of 1 x 10(5) cfu resulted in rapid and extensive colonisation of the gastrointestinal tract and transient colonisation of the liver and spleen. The number of E. coli O157:H7 organisms attained approximately 10(8) cfu/ml caecal homogenate 24 h after inoculation and approximately 10(7) cfu/ml caecal homogenate was still present at day 92. Faecal shedding persisted for 169 days, ceasing 9 days after the birds came into lay and 6% of eggs were contaminated on the eggshell. Histological analysis of tissue samples from birds dosed with 1 x 10(7) cfu gave evidence for E coli O157:H7 NCTC 12900 induced micro-colonies on the caecal mucosa, although evidence for attaching effacing lesions was equivocal. These models may be suitable to study those factors of E. coli O157:H7 that mediate persistent colonisation in avian species.

Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only nonO157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to Hep-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli 01 57:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. (C) 2004 Elsevier B.V. All rights reserved.

Mutation of toxB and a truncated version of the efa-1 gene in Escherichia coli O157 : H7 influences the expression and secretion of locus of enterocyte effacement-encoded proteins but not intestinal colonization in calves or sheep

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in humans and animals. Non-O157:H7 EHEC strains contain the gene efa-1 (referred to in previous publications as efa1), which influences adherence to cultured epithelial cells. An almost identical gene in enteropathogenic E. coli (lifA) mediates the inhibition of lymphocyte proliferation and proinflammatory cytokine synthesis. We have shown previously that significantly lower numbers of EHEC 05 and 0111 efa-1 mutants are shed in feces following experimental infection in calves and that these mutants exhibit reduced adherence to intestinal epithelia compared with isogenic wild-type strains. E. coli O157:H7 strains lack efa-1 but encode a homolog on the pO157 plasmid (toxB/l7095) and contain a truncated version of the efa-1 gene (efa-1'/z4332 in O island 122 of the EDL933 chromosome). Here we report that E. coli O157:H7 toxB and efa-1' single and double mutants exhibit reduced adherence to cultured epithelial cells and show reduced expression and secretion of proteins encoded by the locus of enterocyte effacement (LEE), which plays a key role in the host-cell interactions of EHEC. The activity of LEE1, LEE4, and LEE5 promoters was not significantly altered in E. coli O157:H7 strains harboring toxB or efa-1' mutations, indicating that the effect on the expression of LEE-encoded secreted proteins occurs at a posttranscriptional level. Despite affecting type III secretion, mutation of toxB and efa-1' did not significantly affect the course of fecal shedding of E. coli O157:H7 following experimental inoculation of 10- to 14-day-old calves or 6-week-old sheep. Mutation of tir caused a significant reduction in fecal shedding of E. coli O157:H7 in calves, indicating that the formation of AE lesions is important for colonization of the bovine intestine.

Role for flagella but not intimin in the persistent infection of the gastrointestinal tissues of specific-pathogen-free chicks by Shiga toxin-negative Escherichia coli O157:H7

Shiga toxin (Stx)-positive Escherichia coli O157:117 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative E. coli O157:117 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of intimin and flagella, which is a known factor in the persistence of non-O157 E. coli in poultry, isogenic single- and double-intimin and aflagellar mutants were constructed in E. coli O157:117 isolate NCTC12900. These mutants were used to inoculate (10(5) CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and intiminaflagellate mutants, but not the intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.

Variable and strain dependent colonisation of chickens by Escherichia coli O157

The prevalence of enterohaemorrhagic Escherichia coli (EHEC) O157 in poultry is considered minimal compared with other species, especially ruminants. However, deliberate inoculation studies have shown that poultry are readily and persistently infected by this organism but that the mechanism of colonisation is independent of intimin, a recognised factor in host-EHEC interactions in mammalian species, and may be dependent upon flagella. Few strains of EHEC O157 have been tested in poultry and here 1-day-old and 6-week-old chicks were inoculated with seven non-toxigenic E. coli O157 strains in separate experiments. Persistence was measured semi-quantitatively by bacteriological assessment of E. coli O157 cultured from cloacal swabs (shedding score). In the 1-day-old chick model that was monitored for 43 days, all seven strains established well after inoculation. In the 6-week-old chicken model, one strain established and gave consistently high shedding for the duration of the experiment (156 days). Whereas of the remaining six strains, two persisted for 113 days, two persisted for 43 days, one persisted for 22 days and one strain was never detected.

Naturally acquired attaching and effacing Escherichia coli in sheep

In a series of experiments involving the inoculation of sheep with Escherichia coli O157:H7, and subsequent detailed histopathological examination of the intestinal mucosa, attaching-effacing (AE) lesions formed by elements of the natural flora were observed in 18% of animals. These incidental AE lesions typically were small and sparse, and were not associated with clinical disease. It was possible to identify further some of the lesional bacteria, revealing that E. coli O115 had formed lesions in one of the seven affected animals, and similarly E. coli O26 had formed some of the lesions in another. As AE strains, source flocks, housing and feed sources were diverse, a common source of lesion-forming bacteria appears to be unlikely. It is postulated that subclinical AE lesions are a mechanism of persistence of AE bacteria in sheep.

Attaching-effacing lesions associated with Escherichia coli O157:H7 and other bacteria in experimentally infected conventional neonatal goats

Four conventionally reared goats aged 6 days were inoculated orally with approximately 10(10) colony-forming units (cfu) of a non-verotoxigenic strain of Escherichia coli O157:H7. All remained clinically normal. Tissues were sampled under terminal anaesthesia at 24 (two animals), 48 and 72 h post-inoculation (hpi). E. coli O157:H7 was cultured from the ileum, caecum, colon and rectum of all animals, but the number of bacteria recovered at these sites varied between animals. Attaching-effacing (AE) lesions associated with O157 organisms, as confirmed by immunolabelling, were observed in the ileum of one of the two animals examined at 24 hpi, and in the ileum, caecum and proximal colon of an animal examined at 72 hpi. E. coliO157 organisms were detected at > 105 cfu/g of tissue at these sites. In addition, A-E lesions associated with unidentified bacteria were observed at various sites in the large bowel of the same animals. Lesions containing both E. coliO157 and unidentified bacteria (non-O157) were not observed. Non-O157 AE lesions were also observed in the large bowel of one of two uninoculated control animals. This indicated that three (one control and two inoculated) animals were colonized with an unidentified AE organism before the commencement of the experiment. The O157-associated AE lesions were observed only in animals colonized by non-O157 AE organisms and this raises questions about individual host susceptibility to AE lesions and whether non-O157 AE organisms influence colonization by E. coli O157.

A comparison of Shiga-toxin negative Escherichia coli O157 aflagellate and intimin deficient mutants in porcine in vitro and in vivo models of infection

Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, experimental infection studies have demonstrated that Stx-positive E. coli O157:H7 can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months and that persistence was not dependent upon intimin. We have shown that the flagellum of Stx-negative E. coli O157:H7 does not have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on a porcine (IPI-21) cell line, porcine in vitro organ culture (IVOC) and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nal(r) and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cells. In porcine IVOC association assays, E. coli O157:H7 NCTC12900nal(r) was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants significantly adhered in greater numbers to more IVOC gastrointestinal tissues than the parent. Groups of 14-week-old pigs were dosed orally with 10(10) CFU/10 ml of either E. coli O157:H7 NCTC12900nal(r) or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon. These data suggest that colonisation and persistence of Stx-negative E. coli O157:H7 in pigs, involves mechanisms that do not require the flagellum or intimin.

Influence of colostrum deprivation and concurrent Cryptosporidium parvum infection on the colonization and persistence of Escherichia coli O157:H7 in young lambs

Escherichia coli O157: H7 and Cryptosporidium parvum infections of man have been associated with direct contact with small ruminants. Colostrum protects neonates against gastrointestinal pathogens, and orphan lambs, which are common on petting farms, may be deprived of this protection. In a recent study, it was demonstrated that high shedding of E coli O157 : H7 by an 8-week-old goat kid was associated with coincidental C. parvum infection. Furthermore, both pathogens were co-located in the distal gastrointestinal tract. It was hypothesized that colostrum deprivation and pre-infection with C. parvum predisposed young ruminants to colonization and increased shedding of E coli O157: H7. To test this, 21 lambs 5 weeks of age were divided into four groups as follows: (A) colostrum-deprived and inoculated with E coli O157: H7, (B) colostrum-deprived and inoculated with C. parvum and then E coli O157: H7, (C) conventionally reared and inoculated with E coli O157: H7, (D) conventionally reared and inoculated with C. parvum and then E coli O15 7: H7. C. parvum was detected between 8 and 12 days post-inoculation in most of the infected lambs. At 24 h post-inoculation with E coli O157: H7, all lambs were shedding between 5 x 10(4) and 5 x 10(7) c.f.u. E coli O157: H7 per gram of faeces. E coli O157: H7 was shed in higher numbers in the groups pre-inoculated with C. parvum, whether conventionally reared or colostrum-deprived. Interestingly, for the colostrum-deprived lambs on day 3, a significant difference in shedding of E coli O157: H7 was observed (P= 0-038), with the lambs inoculated with E coli alone yielding higher counts than those pre-inoculated with C. parvum. From day 15 onwards, shedding of E coli O157: H7 was highest from the colostrum-deprived C. parvum-infected lambs, then (in descending order of shedding) the colostrum-deprived lambs, the conventionally reared lambs infected with C. parvum, and the conventionally reared animals. In total, four animals were euthanized, two at 24 h and two at 96 h post inoculation with E coli 0 157: H7 (two conventionally reared and two colostrum-deprived). All animals euthanized were from groups pre-inoculated with C. parvum prior to challenge with E coli O157 : H7. On examination of tissues, in three of the four animals examined, multifocal attaching and effacing lesions were observed in the caecum, colon, rectum and at the recto-anal junction, and were confirmed by immunolhistochemistry to be associated with E coli O157: H7.

Colonization, persistence, and tissue tropism of Escherichia coli O26 in conventionally reared weaned lambs

Escherichia coli O26 is recognized as an emerging pathogen associated with disease in both ruminants and humans. Compared to those of E. coli O157:117, the shedding pattern and location of E. coli O26 in the gastrointestinal tract (GIT) of ruminants are poorly understood. In the studies reported here, an stx-negative E. coli O26 strain of ovine origin was inoculated orally into 6-week-old lambs and the shedding pattern of the O26 strain was monitored by serial bacteriological examination of feces. The location of colonization in the GIT was examined at necropsy at two time points. The numbers of O26 organisms excreted in feces declined from approximately 10(7) to 10(4) CFU per gram of feces by day 7 and continued at this level for a further 3 weeks. Beyond day 30, excretion was from few animals, intermittent, and just above the detection limit. By day 38, all fecal samples were negative, but at necropsy, O26 organisms were recovered from the upper GIT, specifically the ileum. However, no attaching-effacing (AE) lesions were observed. To identify the location of E. coli O26 within the GIT early after inoculation, two lambs were examined postmortem, 4 days postinoculation. High numbers of O26 organisms were recovered from all GIT sites examined, and similar to 10(9) CFU were recovered from 1 gram of ileal tissue from one animal. Despite high numbers of O26 organisms, AE lesions were identified on the mucosa of the ascending colon of only one animal. These data indicate that E. coli O26 readily colonizes 6-week-old lambs, but the sparseness of AE lesions suggests that O26 is well adapted to this host, and mechanisms other than those dependent upon intimin may play a role in persistence.

The TGR5 receptor mediates bile acid-induced itch and analgesia

Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucine-enkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide- and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.