Epidemiol. Infect. (1997), 119, 245���250. Printed in the United Kingdom # 1997 Cambridge University Press A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry P. A. CHAPMAN*, C. A. SIDDONS, A. T. CERDAN MALO ��������� M. A. HARKIN Public Health Laboratory, Herries Road, Sheffield S5 7BQ (Accepted 21 May 1997) SUMMARY Samples of rectal faeces were collected immediately after slaughter from 400 cattle each month for a 1-year period and from 1000 each of sheep, pigs and poultry over the same period. Samples were examined for Escherichia coli O157 by enrichment culture in buffered peptone water with vancomycin, cefixime and cefsulodin followed by immunomagnetic separation and culture of magnetic particles onto cefixime tellurite sorbitol MacConkey agar. E. coli O157 was isolated from 752 (15���7%) of 4800 cattle, 22 (2���2%) of 1000 sheep and from 4 (0���4%) of 1000 pigs, but not from any of 1000 chickens. Of the cattle sampled, 1840 (38���4%) were prime beef animals, 1661 (34���6%) were dairy animals being culled and the status could not be determined for the other 1299 (27%) animals. E. coli O157 was found in 246 (13���4%) of the 1840 beef cattle and 268 (16���1%) of the 1661 dairy cattle. The monthly prevalence of E. coli O157 in cattle was 4���8���36���8% and was at its highest in spring and late summer. Seventeen of the 22 isolates from sheep were also made over the summer period. All E. coli O157 isolates from sheep and 749 (99���6%) of the 752 E. coli O157 isolates from cattle were verocytotoxigenic as determined by Vero cell assay and DNA hybridization, eaeA gene positive, contained a 92 kb plasmid and were thus typical of strains causing infections in man. In contrast isolates from pigs were non-toxigenic, eaeA gene negative and did not contain a 92 kb plasmid and would, therefore, be unlikely to be a source of infection for man. INTRODUCTION Verocytotoxin-producing (VT+) E. coli (VTEC) cause haemorrhagic colitis (HC), the haemolytic-uraemic syndrome (HUS) and occasionally mild non-bloody diarrhoea in man, although some infections may be asymptomatic. In the UK, VT+ E. coli O157, the most common serogroup associated with illness in man, has been isolated from cattle [1���4] and beef, beef products, milk and milk products have been identified as sources of human infection [2, 3, 5, 6]. A seasonal prevalence of E. coli O157 has been reported in dairy herds in the USA [7] and in one study of a dairy herd in the UK the seasonal prevalence was 1���11% [8] it is not * Author for correspondence. known whether there is a seasonal prevalence of E. coli O157 in cattle at slaughter. At present there is very limited information on the prevalence of E. coli O157 in food animals other than cattle. The aims of this study were to examine samples of rectal faeces taken immediately after slaughter from sufficient cattle to determine any monthly variation in prevalence and from sufficient pigs, sheep and poultry to detect a prevalence rate as low as 0���25%. MATERIALS AND METHODS Sample collection Swabs (Transwabs, Medical Wire Co.) of rectal faeces were taken from cattle, pigs, sheep and chickens

246 P. A. Chapman and others immediately after slaughter, placed in transport medium supplied by the swab manufacturer, and were stored at 4 C and transported to the laboratory the same day. All samples from cattle, sheep and pigs were collected at the same abattoir and all samples from chicken from the same poultry processing plant. Samples were collected from April 1995 to March 1996. Each month for a year, 400 samples were collected from cattle to allow detection of monthly variation in prevalence from 1% (95% confidence limits, 0���28���2���6%) to 5% (95% confidence limits, 3���1���7���6%). Ear tag numbers of cattle were recorded in case they were needed to trace E. coli O157-positive animals. Whether the cattle were prime beef animals or cull dairy cows was also recorded whenever possible. One thousand samples were collected evenly over the same 1-year period from each of pigs, sheep and poultry this number was insufficient to detect monthly variation in prevalence within statistically acceptable confidence limits, but would detect a minimum prevalence rate of 0���25% (95% confidence 0���01���0���25%). Samples were collected on 4 days per week (Monday to Thursday) as examination of recent records at the abattoir suggested that this would give the widest possible geographical source of animals. Isolation of E. coli O157 E. coli O157 was isolated by an immunomagnetic separation technique [9, 10] and culture of magnetic beads on to cefixime tellurite sorbitol MacConkey agar [11]. Swabs were placed in 5 ml of buffered peptone water (Oxoid ��� CM509) supplemented with vancomycin 8 mg l-", cefixime 0���05 mg l-", and cef- sulodin 10 mg l-" (BPW���VCC) [2] and faecal material was suspended in the medium by vigorous vortex mixing for 20���30 sec. Suspensions were incubated at 37 C for 6 h and 1 ml of broth was added to 20 ��l of magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli O157, Dynal, Oslo) in a 1���5 ml microcentrifuge tube. The beads were suspended, mixed, separated in a magnetic particle concentrator (MPC-10, Dynal, Oslo) and washed as described previously [9]. After the final wash and separation the beads were resuspended in c. 25 ��l of nutrient broth, inoculated on to CT-SMAC medium and incubated overnight at 37 C. Colonies not fermenting sorbitol from CT-SMAC were tested for agglutination with a latex test kit (Oxoid ��� DR622) for detecting E. coli O157. Isolates that gave positive results were confirmed as E. coli by biochemical tests and as serogroup O157 or serotype O157:H7 by agglutination to titre with antiserum to E. coli O157 (Laboratory for Microbiological Reagents, Central Public Health Laboratory, Colindale, London) [1] or antiserum to E. coli H7 (Difco) [8]. Characterization of isolates Verocytotoxin production Verocytotoxigenicity was determined by Vero cell culture assay [1] and toxin type by specific hybrid- ization with DNA probes for the VT " and VT # genes. Presence of the eaeA gene was also determined by DNA hybridization. From published sequence data [12, 13] DNA probes specific for the A cistrons of the VT " and VT # genes, and for the eaeA gene, were prepared and labelled with digoxigenin-11-dUTP by the polymerase chain reaction and used in colony hybridization reactions [2, 14]. Known VT+, " VT+, # VT-, eaeA+ and eaeA- strains were included as controls in each batch of tests. Plasmid analysis Plasmids were extracted by an alkaline detergent method [15], separated by submerged gel electro- phoresis in Tris-acetate-EDTA buffer with agarose 1%, stained by ethidium bromide and visualised on an ultraviolet transilluminator. A control E. coli K-12 strain (NCTC 50192-39R861) carrying plasmids of 148, 63���4, 36 and 6���9 kb was included with each batch of tests so the size (kb) of the plasmid could be estimated. Phage typing All E. coli O157 isolates were phage typed by the Laboratory for Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London. RESULTS Of the 4800 cattle sampled, 1840 (38���4%) were prime beef animals, 1661 (34���6%) were cull dairy animals and the status could not be determined for the other 1299 (27%) animals. E. coli O157 was found in 246 (13���4%) of the 1840 beef cattle and 268 (16���1%) of the 1661 dairy cattle. Overall, E. coli O157 was isolated from 752 (15���7%) of the 4800 cattle with a monthly