Shiga-like toxin production and attaching effacing activity of Escherichia coli associated with calf diarrhea

Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s). Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins. Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli. Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I [SLT-I]). A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II). None of the isolates hybridized with the probe for the localized adherence gene. Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111. All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture. Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits. One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC). Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.     

Natural infection with an attaching and effacing Escherichia coli in the small and large intestines of a calf with diarrhoea

Attaching and effacing Escherichia coli were identified in the small and large intestine of a calf with naturally occurring diarrhoea. The organisms were associated with intestinal lesions and were identified by immunoperoxidase staining and transmission and scanning electron microscopy, but they did not produce Shiga-like toxin (verotoxin).     

Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy.

Enteric infection with an attaching and effacing Escherichia coli was diagnosed in a puppy with protracted diarrhea. Extensive colonization of the small intestinal mucosa was observed by light and scanning electron microscopy and characteristic lesions of bacterial attachment of the brush border of the enterocytes were demonstrated by transmission electron microscopy. The E. coli strain isolated from the small intestine belonged to serotype O49:H10, did not produce any known E. coli enterotoxin or cytotoxin, was not invasive, and was negative for the known fimbrial colonization factors produced by animal and human enterotoxigenic E. coli. A positive immunoperoxidase reaction was obtained on the bacteria attached to the enterocytes with an anti-E. coli O49 antiserum.     

Detection of the astA (EAST1) gene in attaching and effacing Escherichia coli from ruminants

A total of 206 attaching and effacing Escherichia coli strains from ruminants were analysed for the presence of the astA (EAST1) and bfpA genes. None of these strains was bfpA-positive. The percentage of enteropathogenic E. coli (EPEC) strains astA-positive found in healthy cattle (15.6%) suggests that this animal species may be a significant reservoir of atypical EPEC potentially pathogenic for humans.     

Scanning and transmission electron microscopy of attaching effacing Escherichia coli in weanling rabbits

A strain of an enteropathogenic Escherichia coli, originally isolated from diarrheic weaned rabbits, produced diarrhea in five-week-old New Zealand white rabbits. Sequential examination of the intestines by scanning and transmission electron microscopy revealed that the strain attaches first to the Peyer's patch dome epithelium and later to the enterocytes of distal small intestine, cecum, and colon. Colonized cells became rounded and detached. The colibacilli were intimately associated with the apical cell membrane. Both absorptive and goblet cells were affected. The strain caused effacement of the microvillous border of colonized epithelial cells. Colibacilli were regularly seen in the partially evacuated cavities of goblet cells, but not in absorptive epithelial cells.     

Verocytotoxin-producing and attaching and effacing activity of Escherichia coli isolated from diseased farm livestock

Between May 2005 and June 2008, strategically selected isolates of Escherichia coli obtained from clinical submissions to Veterinary Laboratories Agency (VLA) regional laboratories in England and Wales were serogrouped and examined by PCR for verocytotoxin (VT) production and attaching and effacing (eae) genes, both of which are zoonotic determinants. VT-encoding genes were detected in 54 (5.3 per cent) of the 1022 isolates examined. Only one isolate (0.1 per cent) was identified as verocytotoxigenic E coli (VTEC) O157. Non-O157 VTECs were present in 4.7 per cent of isolates from cattle, compared with 7.9 per cent in pigs, 2.3 per cent in sheep and 6.7 per cent in goats. The predominant serogroup identified in cattle was O26 and the predominant serogroup in pigs was O2. Attaching and effacing activity was attributed to 69 (6.8 per cent) of all isolates.     

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 non-O157 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 O157: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.     

Escherichia coli O115 forms fewer attaching and effacing lesions in the ovine colon in the presence of E. coli O157:H7

Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching-effacing (AE) lesions in the large intestine. Following previous observations of interactions between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.     

Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants

The types of the eae and espB genes of 178 attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic and healthy ruminants were investigated by PCR. Six types of the eae gene: beta (beta), gamma1 (gamma-1), gamma2 (gamma-2), epsilon (epsilon), zeta (zeta) and iota (iota), and three types of the espB gene: alpha, beta and gamma were identified in the strains studied. Moreover, three strains were negative to all the types of the eae gene tested. The types beta and gamma2 in healthy cattle, beta, gamma2 and epsilon in healthy sheep and goats, and beta in diarrhoeic calves, lambs and goat kids were the most frequent types of the eae gene among the strains studied. Although the eaebeta gene was the most prevalent among AEEC from healthy and diarrhoeic ruminants, the percentages of AEEC strains with this type found in this study in diarrhoeic animals (66.7-100%) were higher than those found in healthy animals (33.3-40.6%). Thus, these data suggest that AEEC strains with the eaebeta gene are associated with neonatal diarrhoea in ruminants. The eaegamma1, eaezeta and eaeiota genes were found in low percentages in the strains studied (4.5, 2.8 and 7.3%, respectively). All the types of the eae gene, except the type iota, showed a close correlation with the types of the espB gene: the eaebeta and eae epsilon genes with the espBbeta gene, the eaegamma2 and eaezeta genes with the espBalpha gene and the eaegamma1 gene with the espBgamma gene.     

Polymerase chain reaction analysis of eae gene subtypes present in attaching and effacing Escherichia coli isolated from pigs with diarrhea.

In this study the subtype of eae gene was determined by polymerase chain reaction for a total of 59 attaching and effacing Escherichia coli isolated from preweaned (38 isolates) and postweaned (21 isolates) pigs. The eae(beta) gene detected in 19 E. coli from preweaned pigs and 10 E. coli from postweaned pigs was found to be the most common subtype, followed by eae(gamma), eae(epsilon), and eae(zeta) genes. Subtypes were not determined for 7 E. coli isolates. No other subtype of the eae gene was detected in eae+ E. coli evaluated in this study.     

Polymerase chain reaction typing of genes of the locus of enterocyte effacement of ruminant attaching and effacing Escherichia coli

The variability of the tir, espA, and espD genes of the locus of enterocyte effacement (LEE) in 185 attaching and effacing Escherichia coli (AEEC) strains isolated from healthy and diarrheic cattle, sheep, and goats was investigated by polymerase chain reaction. Nineteen of the strains were enterohemorrhagic E. coli (EHEC); the other 166 were enteropathogenic E. coli (EPEC). The combinations of the tir and esp genes were associated with the variants of the eae gene but not with a strain’s belonging to the EPEC or EHEC group, animal species, or health status (healthy or diarrheic) of the animal. In addition, most of the strains showed the same combinations of LEE genes and serogroups as have been found in AEEC strains isolated from humans, which indicates that ruminants seem to be an EPEC reservoir for humans.