Escherichia coli O157:H7 in the gallbladders of experimentally infected calves.

Fifteen weaned calves (age 89-141 days) were treated with dexamethasone (0.25 mg/kg, IV) for 3 days before, the day of, and the day after inoculation with 10 colony-forming units of either Escherichia coli O157:H7 (strain 86-24, which produces Shiga toxin 2 and intimin; n = 13) or nonpathogenic E. coli (strain 123, which does not produce Shiga toxin or intimin; n = 2). All calves were necropsied 4 days after inoculation. Histologic lesions of attaching and effacing bacteria were observed in the large intestine (12/13) and in the gallbladder mucosa (5/13) of calves inoculated with E. coli 86-24. Cholecystitis was present in 12 of 13 calves that received E. coli 86-24. Inoculum bacteria were recovered from the distal colons or feces (13/13) and gallbladders (3/4) of calves inoculated with 86-24.     

Attaching and effacing bacteria in the intestines of calves and cats with diarrhea

Histopathologic and electron microscopic examination of intestines of three calves and two cats revealed attaching effacing bacteria characteristic of enteropathogenic Escherichia coli (EPEC) in ileum, cecum, and colon. The attaching effacing bacteria in one of the calves contained bacteriophages, and an E. coli isolate from that calf was shown to produce Shiga-like toxin. These findings contribute to emerging evidence that attaching effacing intestinal bacteria are globally distributed pathogens in a variety of host species and that bacteriophage-mediated production of Shiga-like toxin is related to the virulence of such bacteria.     

O-serogroups, eae gene and EAF plasmid in Escherichia coli isolates from cases of bovine mastitis in Brazil

Mastitis has been recognized for some time as the most costly disease in dairy herds. From March 1997 to August 1998, 2144 samples of bovine mastitic milk were collected, from which 182 Escherichia coli isolates were made, and from which 141 isolates had the somatic antigen (serogroup) determined. Twelve different serogroups were isolated from mastitic milk, and among them were O26, O55, O111 and O119, all of them classic enteropathogenic E. coli (EPEC) serogroups. These represented 40.0% of the isolates. The 20 of 57 isolates tested had plasmids and in dot blot hybridization, nine isolates were positive for an EaeA probe and an EPEC adherence factor (EAF) probe while two isolates were negative for EaeA probe but positive for the EAF probe. The nine isolates were characterized as attaching and effacing (A/E) E. coli (AEEC) isolates.     

Attaching and effacing Escherichia coli infections in calves, pigs, lambs, and dogs

Attaching and effacing Escherichia coli (AEEC) adhere to mucosal epithelium in both small and large intestine and induce a distinctive lesion characterized by an irregular scalloped appearance of the epithelial layer. Infection with attaching and effacing E. coli was detected in 14 calves, 7 pigs, 2 lambs, and 3 dogs. Affected animals were from farms and kennels in South Dakota, Minnesota, Iowa, Nebraska, and Wisconsin. Ages of affected animals were calves, 2 days to 4 months; pigs, 1-6 weeks; lambs, 1 week; and dogs, 7-8 weeks. Clinical signs included diarrhea in all animals, but other nonenteric disease problems were present in some animals. Concurrent infection with other enteropathogens was detected in 9 calves and 5 pigs. Infection with AEEC appeared to be the sole cause of illness and death in some animals. There was evidence of intestinal hemorrhage in 5 of the calves and in all 3 dogs. Attaching and effacing lesions varied from small scattered foci to widespread involvement of large areas of intestinal mucosa. Verotoxin was produced by E. coli strains isolated from 9 calves, but not by strains from pigs, lambs, or dogs.     

Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.     

Attaching and effacing and enterotoxigenic Escherichia coli associated with enteric colibacillosis in the dog.

The objective of this study was to describe observations from cases of enteric colibacillosis in the dog. Thirteen cases of canine enteric colibacillosis were diagnosed from routine necropsy submissions to our diagnostic laboratory from 1980 to 1992. In all cases there was a clinical history of gastrointestinal disease associated with histological and bacteriological evidence of either attaching and effacing Escherichia coli (AEEC) or enterotoxigenic Escherichia coli (ETEC) infection. Of these 13 cases of enteric colibacillosis, 12 were associated with AEEC and one with ETEC. Eight of the 12 AEEC isolates were available for study. They were of various serogroups, non-hemolytic, and negative for the genes coding for fimbrial antigens F4, F5, F6, F41 and F165; enterotoxins STap, STb and LT; and verotoxins VT1 and VT2. These eight isolates were EAE-positive (E. coli attaching and effacing) by colony hybridization; six of these were also EAF-positive (EPEC adherence factor), and six were BFP-positive (bundle-forming pilus). The ETEC isolate was negative for the EAE, EAF and BFP determinants and for the fimbrial antigens tested but was positive for the STap and STb genes. Most of the dogs affected with enteric colibacillosis originated from kennels and pet shops and were aged between 1.5 and 3 months. Coinfection with other enteric pathogens was identified in eight of these 13 cases. This study showed that Escherichia coli should be considered of causal significance when investigating diarrheal disease in dogs, particularly in puppies.     

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.     

Cytotoxin activity on Vero cells among Escherichia coli strains associated with diarrhea in cats

The role of Escherichia coli as a causative agent of diarrhea in cats was investigated. Isolates of E coli from healthy and diarrheic cats were serotyped and investigated for their biochemical characters, production of cytotoxin activity on Vero cells, heat-labile enterotoxin, heat-stable enterotoxin, and hemagglutination of erythrocytes from other animal species. None of 48 investigated strains produced heat-labile enterotoxin or heat-stable enterotoxin, nor did they agglutinate erythrocytes. Most strains were hemolytic and belonged to O-serotypes 2 and 6. Cytotoxin activity on Vero cells was significantly more common and produced in greater amounts among E coli strains isolated from diarrheic cats, and was neutralized by anti-Shiga-like toxin I serum.     

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.     

Prevalence and characteristics of attaching and effacing strains of Escherichia coil isolated from diarrheic and healthy sheep and goats

OBJECTIVE: To determine the prevalence and characteristics of attaching and effacing Escherichia coli (AEEC) in diarrheic and healthy small ruminants. ANIMALS: 502 lambs and kids with diarrhea and 511 healthy sheep and goats. PROCEDURE: Fecal samples from diarrheic and healthy sheep and goats were screened for the eae gene. In addition, E coli isolates with positive results for the eae gene (E coli eae+) were analyzed for the espB gene, production of verotoxins (VT), and serogroup. RESULTS: A significantly higher prevalence of healthy lambs and kids were infected with AEEC, compared with diarrheic lambs and kids and healthy adult sheep and goats. Some differences in the characteristics of E coli eae strains isolated from diarrheic and healthy animals were detected. Thus, the espB gene was detected more frequently among E coli eae+ strains isolated from healthy animals than in those isolated from diarrheic animals, and VT production was only detected in E coli eae+ strains isolated from healthy lambs and kids. The E coli eae+ isolates belonged to several O serogroups. However, 17 of 40 (42.5%) isolates from diarrheic lambs and only 4 of 168 (2.4%) isolates from healthy sheep belonged to serogroup 026. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that E coli eae+ 026 strains may play a role in diarrheal disease in lambs, whereas E coli eae+ strains that also had VT production and eae+ strains that had positive results for the espB gene did not appear to be associated with diarrhea in small ruminants.     

犊牛腹泻主要病原菌多重PCR方法的建立

产毒性大肠埃希菌、A/E大肠埃希菌和沙门菌是造成犊牛腹泻的主要病原菌。选取产毒性大肠埃希菌的st和lt基因、A/E大肠埃希菌的eae基因和沙门菌的invA基因作为扩增靶基因序列,通过优化反应条件建立了四重PCR检测体系。对PCR产物回收、测序,验证PCR。通过特异性试验和敏感性试验证明该PCR体系特异性强、敏感性高,可有效地检测犊牛腹泻病原菌。使用该四重PCR检测22份临床样品,发现其中9份携带相关基因,并分离得到了相关致病菌。     

The prevalence of Shiga toxin-producing Escherichia coli in cattle and sheep in the lower North Island, New Zealand

AIM: To genotype Escherichia coli cultured from the faeces of healthy cattle and sheep in the lower North Island, in order to investigate the possible role of ruminants as a reservoir for Shiga toxin-producing E. coli (STEC) in New Zealand. METHODS: A total of 952 strains of E. coli were isolated on selective media, from faecal swabs from 319 animals (187 cattle and 132 sheep) from four sites in the Manawatu and Rangitikei regions of New Zealand. A multiplex polymerase chain reaction (PCR) was used to genotype the E. coli isolates, using amplification of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae). RESULTS: Isolates of E. coli were cultured from swabs from 178/187 (95.2%) cattle and all 132 (100%) sheep. Ninety-nine (10.4%) of the isolates were stx1 only, 83 (8.7%) stx2 only, 33 (3.5%) stx1 and stx2, 23 (2.4%) stx1 and eae, one (0.1%) stx2 and eae, and 115 (12.1%) were eae only. Overall, 51 (27.3%) cattle and 87 (65.9%) sheep were stx-positive, whereas 69 (36.9%) cattle and 36 (27.3%) sheep were eae-positive. CONCLUSIONS: Both healthy cattle and sheep are asymptomatic reservoirs of STEC in New Zealand. Direct contact with cattle and sheep or consumption of water or foodstuffs contaminated with cattle of sheep faeces may represent a significant source of infection for humans.     

Antimicrobial susceptibility of pathogenic Escherichia coli isolated from pigs in Korea

The in vitro susceptibilities of 285 isolates of Escherichia coli from preweaned and postweaned pigs with diarrhea and edema disease were tested with the 15 commonly used antimicrobial drugs by an agar dilution minimal inhibitory concentration procedure according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. All E. coli isolates tested in this study belonged to enterotoxigenic E. coli, attaching and effacing E. coli, or Shiga toxin-producing E. coli. Field isolates had low MIC90 for ceftiofur (1 microg/ml). No correlation in antimicrobial resistance was found in three types of E. coli.     

The possible influence of LuxS in the in vivo virulence of rabbit enteropathogenic Escherichia coli

Attaching and effacing (A/E) organisms, such as rabbit enteropathogenic Escherichia coli (EPEC), human EPEC or enterohemorrhagic E. coli (EHEC) share attaching and effacing phenotype and LEE pathogenicity island responsible for A/E. The present study was undertaken to investigate the impact of the LuxS quorum sensing (QS) signaling system in vitro and in vivo pathogenicity of A/E organisms using rabbit EPEC (rEPEC) strain E22 (O103:H2). Analysis of the bioluminescence indicated abolished production of the QS signal AI-2 by luxS mutant (E22DeltaluxS). Strain E22Deltalux also exhibited impaired expression of several normally secreted proteins and reduced adherence to cultured HeLa cells. Complementation of the intact luxS gene to E22DeltaluxS restored secreted protein expression comparable to the WT type but not adherence to HeLa cells. In experimentally infected rabbits, the isogenic luxS mutant induced clinical illness and intimate adherence to the intestinal mucosa, albeit to a less extent, comparable to that seen with the parent virulent strain. It is worth noting that reduced fecal bacterial shedding, mucosal adherence and improved cumulative weight gain were seen for the mutant strain complemented with luxS when compared to the WT. It appears that the luxS gene is not essential for in vivo pathogenicity by rEPEC where exogenous QS signals are present in the gut. The impact of AI-2 provided by multicopy plasmid on bacterial virulence is discussed.     

Attaching and effacing Escherichia coli infection as a cause of diarrhea in young calves

A form of enteric Escherichia coli infection was identified in 60 calves from 59 farming operations. The E coli responsible for these infections principally colonized the colon, inducing a distinctive lesion described as attaching and effacing. Hemorrhagic enterocolitis or blood in the feces was observed on 40% of the farms. Of affected calves, 86.6% were dairy calves (average age, 11.8 days). Forty-four calves were infected concurrently with other enteropathogens (cryptosporidia, rotavirus, coronavirus, enterotoxigenic E coli, bovine viral diarrhea virus, coccidia). Verotoxin-producing E coli was recovered from 31 calves; 8 were serotype O111:NM isolates, 3 were serotype O5:NM, and 1 was serotype O26:NM.     

Serotypes and analysis of distribution of Shiga toxin producing Escherichia coli from cattle and sheep in the lower North Island, New Zealand

AIMS: To serotype a subset of Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep to determine whether any corresponding serotypes have been implicated in human diarrhoeal disease, both in New Zealand and worldwide, and to examine the distribution of STEC and enteropathogenic Escherichia coli (EPEC) amongst cattle (calves, heifers and dairy) and sheep (lambs, rams and ewes), to assess whether carriage of identified bacterial genotypes may be associated with a particular age of animal. METHODS: Recto-anal mucosal swabs (RAMS) were taken from 91 calves, 24 heifers and 72 dairy cattle, and 46 lambs, 50 ewes and 36 rams, from four sites in the Manawatu and Rangitikei regions of New Zealand. Strains of E. coli selected from primary isolation plates were subjected to a multiplex polymerase chain reaction (PCR), to determine the presence of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae). RESULTS: Overall, 186/319 (58.3%) animals sampled were positive for stx1, stx2, or eae isolates. More sheep (43.9%) were stx1-positive than cattle (2.7%; p = 0.036), and amongst sheep more lambs and ewes were stx1-positive than rams (p = 0.036). Amongst cattle, more calves and heifers were eae-positive than dairy cows (p = 0.030). Two or more different STEC were isolated from at least 28 (9%) animals (three cattle and 25 sheep), based on their stx/eae genotype. Enterohaemolysin genes were found in 39/51 (76%) isolates serotyped. Twenty-one different serotypes were detected, including O5:H-, O9:H51, O26:H11, O84:H-/H2 and O149:H8 from cattle, and O26:H11, O65:H-, O75:H8, O84:H-, O91:H-, O128:H2 and O174:H8 from sheep; O84:H-, O26:H11, O5:H-, O91:H- and O128:H2 serotypes have been associated with human disease. CONCLUSIONS: If nationally representative, this study confirms that cattle and sheep in New Zealand may be a major reservoir of STEC serotypes that have been recognised as causative agents of diarrhoeal disease in humans. Distribution of STEC and EPEC in cattle and sheep indicates that direct contact with, in particular, calves or their faeces, or exposure to environments cross contaminated with ruminant faeces, may represent an increased risk factor for human disease in New Zealand.     

Characterization of eae+ Escherichia coli isolated from healthy and diarrheic calves.

Strains of Escherichia coli from 101 healthy and 114 diarrheic calves were screened by PCR for the eae (intimin) gene and Shiga toxin genes (stx). Each eae+ and eae/stx+ strain was examined for antimicrobial susceptibility, enterohemolysin activity, and the somatic O antigen was determined. An immunoassay was used to detect Shiga toxin antigens for the eae/stx+ E. coli. Significantly more (p = 0.005) of the healthy calves carried eae+ and eae/stx+ E. coli in their feces when compared to strains from diarrheic calves. Moreover, Shiga toxin antigens were detected significantly more (p = 0.001) often among the eae/stx+ strains from healthy calves when compared to eae/stx+ strains from diarrheic calves. However, significantly more (p = 0.001) of the eae+ and eae/stx+ strains from diarrheic calves were resistant to at least one of the antimicrobials tested, and the strains from diarrheic calves had a significantly (p = 0.05) higher rate of antimicrobial resistance to at least two different antimicrobial classes. No significant difference (p> or =0.05) was detected among the eae+ and eae/stx+ strains from healthy and diarrheic calves for enterohemolysin production. Serogroups O-negative, O5, O26, and O111 were predominate among both healthy and diarrheic calves.     

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.     

Infections with enteropathogenic Escherichia coli (EPEC) in cattle. 1. Comparison of different animal models and a cell culture system for the establishment of a detection system for "attaching and effacing" (AE) lesions]

In several animals species, enteropathogenic Escherichia coli (EPEC) were described as agents causing diarrhea. The histopathogenic pattern of EPEC is due to a typical adherence to enterocytes, called "attaching and effacing" (AE). This lesions are characterized by the formation of pedestals, cups and a marked loss of microvilli on enterocytes. In view of using an "in vitro system" (HeLa-cell culture) to test the adherence of EPEC, we first tested bovine EPEC in several laboratory animals. Various strains of mice, one day chicks (peroral) and a three day old calf (ligated intestinal loops) were inoculated with a bovine pathogenic EPEC (S 102-9). The adherence of EPEC "in vivo" was histologically, electron microscopically and bacteriologically investigated and compared to adherence to HeLa cell cultures. AE-lesions were found on calf enterocytes as well as on HeLa-cells, no lesions were seen in mice and chicks. The ligated intestinal loop test seems to be a useful model to compare "in vivo" to "in vitro" adherence.     

Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil

In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3?%) from diarrhoeic animals and 60 (85.7?%) from healthy calves. The virulence markers identified were: Stx1 (82.8?%), eae (24.3?%), F41 (11.4?%), F5 (10?%), STa (4.28?%) and Stx2 (4?%). In diarrhoeic animals, Stx1 (70?%) and F41 (30?%) were identified, while Stx1 (83.3?%), eae (28.3?%), F41 (8.3?%), F5 (11.6?%), STa (5?%) and Stx2 (1.6?%) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26?% of all calves and on 75?% of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.