Escherichia coli producing the attachment-effacement (AE) lesion (EPEC) and/or Shiga toxins (STEC) cause enteritis and (bloody) diarrhoea in young calves and in humans, and are also present in the intestines of healthy cattle. Besides the O157:H7 serotype, which is the main serotype causing STEC outbreaks in the world EPEC and STEC can belong to dozens of O serogroups. Of them, 9 have been frequently identified worldwide: O5, O26, O103, O104, O111, O118, O121, O145 and O165. The aim of this study is to identify the virulotypes and the O serotypes of EPEC and STEC isolated from healthy cattle at slaughterhouses in Wallonia. A total of 245 faeces (216 <1year-old bulls, 25 cows and 4 heifers) were sampled between April and June 2014 in 2 slaughterhouses in Wallonia and grown overnight at 37°C in Lauryl sulfate Enterobacteriaceae selective broth. The enrichment broths were assayed with an stx1, stx2 (Shiga toxin) and eae (AE lesion) triplex PCR and positive broths were inoculated onto 4 agar media: McConkey’s, Chromagar ES, Chromagar ES with tellurite and Chromagar STEC. Up to ten colonies per plate were picked up, sub-cultured and tested by the colony hybridization assay with gene probes targeting the stx1, stx2 and eae genes. The triplex PCR was again performed on all probe-positive isolates. The PCR-positive E. coli were subsequently assayed with two pentaplex PCR targeting the specific genes coding for the ten O serogroups listed above. Of the 2563 sub-cultured isolates, 744 isolates (29%) from 62 animals (25%) tested positive with the colony hybridization assay. Of them, 687 isolates (92%) from 59 animals were positive with the triplex PCR and the results of both tests were in agreement for 617 isolates (83%). One to 29 isolates per animal were probe- and PCR-positive. The positive isolates grew on Chromagar STEC (379; 55%), on Chromagar ES with tellurite (189; 28%), on Chromagar ES (62; 9%) or on McConkey’s agar (57; 8%). The most frequent virulotypes were eae+ (EPEC: 372 isolates; 54%), eae+stx1+ (AE_STEC: ...