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 24h 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 1x10(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.     

Fecal Escherichia coli O157:H7 shedding patterns of orally inoculated calves.

To assess the duration of fecal shedding upon initial infection, the duration of shedding after subsequent re-infection and the effects of dietary restriction and antibiotic treatment on shedding recrudescence, four, one-week-old calves were orally inoculated on three separate occasions with 5x10(8) cfu of Escherichia coli O157:H7 strain 86-24 Nal-R. Fecal shedding was followed by serial culture three times weekly. Following the first inoculation, the calves shed E. coli O157:H7 in their feces for a mean of 30 days, with a range of 20 to 43 days. Following the second and third inoculations, the calves shed E. coli O157:H7 in their feces for 3-8 days. In each of the three inoculations, feed was withheld from the calves for 24 h after they had become fecal culture negative. Two calves resumed shedding, one for 1 day and the other for 4 days, after food was withheld after the third inoculation, but not in the first two inoculations. In the third inoculation, one calf resumed shedding for one day after treatment with oxytetracycline. No E. coli O157:H7 strain 86-24 Nal-R was found in the calves at necropsy. These calves did not exhibit persistent low-level shedding, and did not appear to be persistently colonized with E. coli O157:H7.     

Isolation of Escherichia coli O157:H7 from the gall bladder of inoculated and naturally-infected cattle

To determine if Escherichia coli O157:H7 is capable of residing in the gall bladder of cattle, inoculation studies were conducted with O157:H7 strain 86-24 in weaned Holstein calves. Strain 86-24 was isolated from the gall bladders of five calves 36 days after inoculation. Two other calves contained the inoculation strain in the distal colon but the organism was absent in their gall bladders. A second trial in which the calves were euthanized 15 days after inoculation found strain 86-24 in six of seven inoculated calves but only in colon and/or rumen samples. In a third trial that inoculated eight calves with a four-strain cocktail of O157:H7 strains, the gall bladders from all eight animals were positive 9 days after inoculation. The colon and rumen samples from these calves were also positive. E. coli O157:H7 isolates recovered from bile samples and subtyped by pulsed field gel electrophoresis found that three of the four inoculation strains were present in one or more of the calves. Thus, residence in the gall bladder is not restricted to a single strain. Additional evidence of the ability to localize in the gall bladder of cattle was provided by testing the bile from 150 gall bladders (five collection dates, 30 samples each) obtained at an abbatoir and the isolation of E. coli O157:H7 from four samples (2.7%). This study establishes that E. coli O157:H7 can reside transiently or permanently at a low level in the gall bladder of cattle.     

A longitudinal study of Shiga-toxigenic Escherichia coli (STEC) prevalence in three Australian diary herds

Over a 12 month period, 588 cattle faecal samples and 147 farm environmental samples from three dairy farms in southeast Queensland were examined for the presence of Shiga-toxigenic Escherichia coli (STEC). Samples were screened for Shiga toxin gene (stx) using PCR. Samples positive for stx were filtered onto hydrophobic grid membrane filters and STEC identified and isolated using colony hybridisation with a stx-specific DNA probe. Serotyping was performed to identify serogroups commonly associated with human infection or enterohaemorrhagic Escherichia coli (EHEC). Shiga-toxigenic Escherichia coli were isolated from 16.7% of cattle faecal samples and 4.1% of environmental samples. Of cattle STEC isolates, 10.2% serotyped as E. coli O26:H11 and 11.2% serotyped as E. coli O157:H7, and the E. coli O26:H11 and E. coli O157:H7 prevalences in the cattle samples were 1.7 and 1.9%, respectively. Prevalences for STEC and EHEC in dairy cattle faeces were similar to those derived in surveys within the northern and southern hemispheres. Calves at weaning were identified as the cattle group most likely to be shedding STEC, E. coli O26 or E. coli O157. In concurrence with previous studies, it appears that cattle, and in particular 1-14-week-old weanling calves, are the primary reservoir for STEC and EHEC on the dairy farm.     

Persistence of Escherichia coli O157:H7 in experimentally infected swine

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.     

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR = 16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR = 0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR = 0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC 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/10ml 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.     

A homolog of the O157 urease-encoding O island 48 is present in porcine O149:H10 enterotoxigenic Escherichia coli

The relationship of the urease operon in the highly virulent O149 porcine enterotoxigenic Escherichia coli (ETEC) strain Ro8 to a genomic island (GI) homologous to O island (OI) 48 of O157 enterohemorrhagic E. coli (EHEC) strain EDL933 was investigated. Eighty-four of 84 O149:H10 strains were urease positive whereas 44 of 44 O149:H43 porcine ETEC strains were urease-negative. Seventeen of 17 O149:H10 strains that were tested possessed the OI-48 homolog whereas 24 of 24 O149:H43 strains lacked this OI. Transposon insertions in lipB or guaA genes in strain Ro8 eliminated urease activity while insertions in the caiF gene increased urease activity. When the O149 ure operon was cloned on a high copy number plasmid, urease expression was increased approximately 11-fold in Ro8 and 83-fold in O157 strain EDL933 compared with that in the wild type Ro8. The O149 urease activity was expressed despite the presence of the same premature stop codon in ureD that is present in ure+ O157:H7 strains that are urease-negative. The ure operon in Ro8 consists of 4 893 nucleotides with 99% identity with the ure operons in EHEC O157:H7 strains EDL933 and Sakai, and is part of a GI similar to GI-48 of strain EDL933. This OI, designated OI-48149 , is inserted in the serX tRNA gene in strain Ro8 and contains genes for urease, tellurite resistance, iha and an AIDA-I-like adhesin. The presence of a homolog of the O157:H7 OI-48 in highly virulent O149 porcine ETEC suggests that this OI may contribute to establishment of the bacteria in the intestine.     

Effect of antibiotics in milk replacer on fecal shedding of Escherichia coli O157:H7 in calves

The objective of this study was to compare the concentration and duration of fecal shedding of Escherichia coli O157:H7 between calves fed milk replacer with or without antibiotic (oxytetracycline and neomycin) supplementation. Eighteen 1-wk-old Holstein calves were orally inoculated with a strain of E. coli O157:H7 (3.6 x 10(8) cfu/calf) made resistant to nalidixic acid (NA). Rectal samples were obtained three times weekly for 8 wk following oral inoculation. Fecal shedding of NA-resistant E. coli O157:H7 was quantified by direct plating or detected by selective enrichment procedure. Eight weeks after inoculation, calves were killed, necropsied, and tissues (tonsils, retropharyngeal and mesenteric lymph nodes, and Peyer's patches) and gut contents (rumen, omasum, abomasum, ileum, cecum, colon, and rectum) were sampled to quantify or detect NA-resistant E. coli O157:H7. The percentage of calves shedding NA-resistant E. coli O157:H7 in the feces in the antibiotic-fed group was higher (P < 0.001) early in the study period (d 6 and 10) compared with the control group fed no antibiotics. There was no difference between treatment and control groups in the concentration of E. coli O157 in feces that were positive at quantifiable concentrations. A comparison of the duration of fecal shedding between treated and untreated calves showed no significant difference between groups. At necropsy, E. coli O157:H7 was recovered from the rumen and omasum of one calf in the control group and from retropharyngeal lymph node and Peyer's patch of two calves in the antibiotic group. Supplementation of milk replacer with antibiotics may increase the probability of E. coli O157:H7 shedding in dairy calves, but the effect seems to be of low magnitude and short duration.     

Oral infection with a Shiga toxin-negative Escherichia coli O157:H7 strain elicits humoral and cellular responses but does not protect sheep from colonisation with the homologous strain

We have previously shown that rectally inoculated sheep excrete Escherichia coli O157:H7 during weeks to months without developing a clear antibody response. However, antibodies against this bacterium were observed in naturally infected sheep, which most likely became orally infected. To understand this difference, sheep were orally inoculated with the same Shiga toxin-negative E. coli O157:H7 strain that was used for the rectal inoculation. A primary oral inoculation resulted in shedding of E. coli O157:H7 in the faeces and detection of antibody responses against intimin, EspA and EspB. The antibody titres waned as shedding decreased. A secondary inoculation resulted in longer shedding, even though a booster antibody response occurred. Cellular responses followed a similar pattern as the antibody levels, albeit with a lower secondary response. The presence of antigen-specific antibody-secreting cells indicates involvement of both a systemic response in the spleen and a local immune response in the terminal rectum. These results suggest that E. coli O157:H7 has to pass the small intestine to evoke antibody responses.     

Intranasal immunisation with Stx2B-Tir-Stx1B-Zot protein leads to decreased shedding in goats after challenge with Escherichia coli O157:H7

Ruminants are an important reservoir of Escherichia coli O157:H7. To reduce E coli O157:H7 excretion by these animals could play a key role in prevention and control of human infections. In the present study, the authors used 12 three-month-old goats to evaluate the efficacy of intranasal administration of the Stx2B-Tir-Stx1B-Zot protein. These goats were inoculated on days 0 and 21 and infected with 10(10) colony-forming units (cfu) of E coli O157:H7 by oral inoculation on day 36. Faecal shedding was monitored daily for two weeks. All of six goats immunised with recombinant protein elicited significant Stx2b-Tir-Stx1b-Zot-specific serum IgG antibodies, and three of them also showed production of antigen-specific IgA in faeces. The immunised goats showed much less shedding of E coli O157:H7 after challenge. These results demonstrate the potential for the use of Stx2B-Tir-Stx1B-Zot protein in mucosal vaccine formulations to prevent colonisation and shedding of E coli O157:H7 in goats.     

Allelic types of long polar fimbriae in bovine and human Escherichia coli O157 strains

Long polar fimbriae (Lpf) are recently discovered adhesins and increasingly important genetic markers of pathogenic Escherichia coli strains. The presence and genotype diversity of Lpf operons was screened in a collection of 97 Escherichia coli O157 strains representing different pathotypes, isolated from healthy cattle (n = 43) and human patients (n = 54) in several countries. Individual structural genes of Lpf were scanned by PCR, and allelic variants were detected with a recently developed typing scheme. Ninety-five strains carried at least one whole Lpf operon (genes lpfABCD and/or lpfABCDE). The 64 enterohaemorrhagic (EHEC) and 24 enteropathogenic (EPEC) strains all carried two Lpf operons, allele 3 of lpfA1 and allele 2 of lpfA2, a combination characteristic of the O157:H7/NM serotype. Out of the 9 bovine atypical (AT; stx-, eae-) strains, 7 carried one complete Lpf operon, allele 1 of lpfA2. The atypical strains belonged to main phylogenetic groups A and B1, while the EHEC and EPEC strains were from group D. Lpf variants carried by the 72 strains of the Escherichia coli Reference Collection (ECOR) were determined with the same typing scheme. Alleles were detected in 25 strains, of which 6 were found negative for the respective Lpf operons in earlier studies. The marker value of the Lpf allelic combination for the O157:H7/NM serotype was confirmed, and further evidence was given for the presence of at least two different genetic lineages of atypical bovine E. coli O157 strains.     

Detection and characterization of verocytoxin-producing Escherichia coli types (VTEC) from different sources]

Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic E. coli (EHEC) belonging to a few serovars embracing strains of O26, O103, O111, O118, O145 and O157 serogroup, respectively. In own investigations 3.791 food specimen of animal origin were investigated by use of an enzyme-immuno-assay (EIA) and the polymerase chain reaction (PCR). All E. coli isolates (n = 459) of food as well as isolates from cattle feces (n = 440), from HUS patients (n = 50) and asymptomatic human carriers (n = 16) were investigated by means of the PCR using primer pairs for verocytotoxin genes (vtx1, vtx2, vtx2c, vtx2d, vtx2e), the E. coli attaching und effacing gene (eae), the enterohemolysin-gene (ehlyA) and the vt2 transporter protein-gene (ile X tRNA). Differences were found in respect to the eae- and the ile X tRNA genes, which could be detected in significantly higher ratios in the isolates from patients and human carriers. Furthermore vtx2d strains were exclusively analyzed to each 25% in food and cattle strains. In six food samples pathogenic strains of serovar O157 were detected whereas some of the cattle strains were estimated to belong to EHEC serovars O26:H11, O118:H- and O157:H7. The own data support the thesis that the risk for human beings is affiliated to a great extent by direct contact with ruminants, followed by person-to-person transmission. Regarding the epidemiological data the thesis that each VTEC strain ist potentially an EHEC strain can not longer be substantiated.     

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.     

Occurrence of Escherichia coli O157 in raw material and food in Czech Republic

The study was carried out to investigate the incidence of Escherichia coli O157 in raw materials, foodstuffs and the agricultural environment. Of a total of 987 samples examined, 22 strains (2.2%) were identified as E. coli O157 and 10 of them as E. coli O157:H7. Cefixime-Tellurite MacConkey sorbitol agar (CT-SMAC) agar and Biosynth culture medium (BCM) E. coli O157:7 medium were used for the isolation. The virulence factors (stx1, stx2, eae, and ehxA genes) were identified by polymerase chain reaction (PCR). Most strains were isolated from the mechanically deboned poultry meat (nine), minced meat (six) and raw milk (four). One strain was isolated from beef carcass and two strains from waste water. No strains were were found in mass for sausages, refreshment salads, swabs of pork and poultry carcasses and faeces of cattle and pigs. Ten strains from the 22 identified proved to be positive for all factors of virulence. They were isolated from minced meat (four), raw milk (four), waste water (one) and swab from beef carcass (one). Sensitivity to the antimicrobial drugs ampicillin (AMS), ampicillin-sublactam (SAM), tetracycline (TET), ofloxacine (OFL), cefuroxime (CRX), chloramphenicol (CPM), gentamicine (GEN), colistin (COL), cephalozine (CLZ), cefoxitin (CXT), aztreonam (AZT), and sulphamethoxazole + trimethoprim (COT) was tested using the standard dilution technique and disc diffusion test. Minimum inhibitory concentrations (MIC) characteristics (MIC(50), MIC(90), MIC range) and inhibitory zone diameter were determined for each strain. As determined by MICs, the resistance to tested antibiotics in E. coli O157 isolates was found to AMS (90.9%), CLZ (81.8%), CRX (63.6%), CXT (72.7%), CPM (72.7%), TET (81.8%), SAM (59.1%), COT (9.1%), COL (63.61%), AZT (9%) and GEN (4.5%). The similar results were obtained using the disc diffusion method. The differences were found relating to SAM, CXT, CMO and TET. Resistance against one or more antibiotics was found in 95.4% of E. coli O157. Only one strain was susceptible to all tested antibiotics. Most of the strains were resistant to ampicillin and cephalozine. Eight different resistance phenotypes were demonstrated in E. coli O157.     

Escherichia coli O157:H7 and other Shiga toxin-producing E. coli in white veal calves

The aims of the study were to determine the prevalence of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) and other Shiga toxin-producing E. coli (STEC) in feces of white veal calves in an operation in Ontario, to evaluate exposure of the calves to EHEC O157, and to investigate the milk replacer diet and antimicrobial resistance as factors that might influence the prevalence of EHEC O157. Feces from three cohorts of 20-21 calves were collected weekly for 20 weeks and processed for isolation of EHEC O157:H7 and detection of STEC by an ELISA. Exposure to EHEC O157 was also investigated by measuring IgG and IgM antibodies to the O157 lipopolysaccharide (O157 Ab) in sera by ELISA. The prevalences of EHEC O157 were 0.17% of 1151 fecal samples and 3.2% of 62 calves, and for STEC were 68% of 1005 fecal samples and 100% of 62 calves. Seroconversion to active IgG and IgM O157 Ab responses in some calves was not associated with isolation of EHEC O157. The milk replacer contained low levels of antibodies to EHEC antigens and without antimicrobial drugs, it did not inhibit the growth of EHEC O157 in vitro. Two E. coli O157:H7 that were isolated were totally drug sensitive whereas 60 commensal E. coli isolates that were examined were highly resistant. Antibodies in milk replacer that might be protective in vivo, and susceptibility to antimicrobial agents in the milk replacer may contribute to the low prevalence of EHEC O157 in white veal calves.     

出血性大肠杆菌O157 Eae-Stx1/2B融合蛋白的免疫学特性

为了研究重构融合蛋白Eae-Stx1/2B免疫特性及对出血性大肠杆菌感染的免疫保护作用,进行了细胞粘附抑制试验和免疫保护试验。结果证明Eae-Stx1/2B融合蛋白免疫血清在体外能降低出血性大肠杆菌对HEp-2细胞的粘附作用,这一特性在出血性大肠杆菌疫苗设计中具有重要价值。以纯化的融合蛋白Eae-Stx1/2B为抗原对小鼠进行腹腔注射免疫和滴鼻免疫,ELISA检测结果显示,2次免疫后7 d,免疫组抗Eae-Stx1/2B融合蛋白和抗出血性大肠杆菌O157血清IgG抗体显著高于未免疫组;用出血性大肠杆菌O157强毒株EDL933攻击免疫小鼠,免疫组小鼠排菌时间显著缩短,免疫组小鼠存活率均高于未免疫组,免疫组小鼠体质量恢复增长较快。上述试验结果表明,Eae-Stx1/2B融合蛋白对出血性大肠杆菌感染有保护作用。     

Longitudinal prevalence study of diarrheagenic Escherichia coli in dairy calves

A longitudinal study (cohort study) elaborating 1,224 rectal swabs from 221 calves aging between 1 and 12 weeks was conducted on 11 dairy farms (i) to ascertain associations between diarrhea and shedding of diarrheagenic E. coli and (ii) to facilitate the zoonotic potential assessment of E. coli strains shed by young calves. Calves were screened weekly by PCR of swab cultures for shedding of enterotoxigenic E coli [ETEC; by detection of heat stable (est) and heat labile enterotoxin genes (elt)], diffusely adhering E. coli [DAEC; diffuse adhesion (daa)], typical enteropathogenic E. coli [EPEC; bundle-forming pili (bfpA) and intimin (eae)] as well as enterohemorrhagic E. coli [EHEC, intimin (eae) and Shiga toxin (stx)]. In addition, EHEC-hemolysin- (Hly(EHEC)) and alpha-hemolysin- (alpha-Hly) producing E. coli were detected by inoculation of blood agar plates. Within the 221 calves, prevalences were 69.7% (25.2% of the 1,224 samples) for Hly(EHEC)-producing E. coli, 55.3% (19.3%) for eae, and 18.2% (4.5%) for stx. E. coli strains exhibiting an alpha-Hly phenotype were detected in 66.5% of the calves and 21.9% of fecal samples. The est gene was detectable in 31.7% of the calves from only 9 of 11 herds and in 7.8% of the samples. Calves shedding DAEC or typical EPEC were not identified. The detection frequency of virulence traits significantly depended on the calves' age and shedding dynamics differed between the traits. A significant correlation between calf diarrhea and shedding of EHEC virulence traits was determined for several postnatal periods (1 week: Hly(EHEC); 1st & 10th week: eae; 4th week stx). Shedding of ETEC (est) was associated with diarrhea in newborn calves (1st week) only. Hly(EHEC)- and alpha-Hly-producing E. coli were shed significantly more frequently by diarrheic calves in 1st and 8th week of life, respectively. The knowledge gained in this study highlights the high prevalence of zoonotic E. coli already in calves.The age-dependent shedding dynamic of the various E. coli pathovars has to be considered regarding prophylaxis as well as planning intervention studies, both for calves and humans.     

Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -(eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin beta being present in 51.5% of the strains, followed by intimins theta (23.7%), epsilon (6.2%), kappa (5.2%), zeta (5.2%), alpha, eta and iota (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.     

Serotypes and Shiga toxin genotypes among Escherichia coli isolated from animals and food in Argentina and Brazil

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from animals and food in Argentina (n=44) and Brazil (n=20) were examined and compared in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. The clonal relatedness of STEC O157 isolates (n=22) was established by phage typing (PT) and pulsed-field gel electrophoresis (PFGE). All O157 strains studied carried eae and enterohemorrhagic E. coli (EHEC)-hly sequences. In Argentina, these strains occurred both in cattle and meat, and 50% of them carried stx2/stx2vh-a genes, whereas in Brazil the O157 strains were isolated from animals, and most harbored the stx2vh-a sequence. At least 13 different O:H serotypes were identified among the non-O157 strains studied, with serotype O113:H21 being found in both countries. All but one non-O157 strains did not carry eae gene, but EHEC-hlyA gene was found in 85.7% of them, and the stx2 genotype was also more prevalent in Argentina than in Brazil (P<0.01), where stx1 alone or in association was most common (68.8%). One STEC strain isolated from a calf in Brazil harbored the new variant referred to as stx2-NV206. PFGE analysis showed that STEC O157 strains were grouped in four clusters. One Brazilian strain was considered possibly related (> or =80%) to Argentinean strains of cluster I. Differences in the pathogenic potential, especially in regard to serotypes and stx genotypes, were observed among the STEC strains recovered from animals and food in both countries.