Veterinary significance of verocytotoxin-producing Escherichia coli

Veterinary significance of verocytotoxin-producing Escherichia coli

World Journal of Microbiology & Biotechnology 16: 725±732, 2000. Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands. 725 Veterinary sign...

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World Journal of Microbiology & Biotechnology 16: 725±732, 2000.

Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.

725

Veterinary signi®cance of verocytotoxin-producing Escherichia coli O157 Barti A. Synge SAC Veterinary Science Division, Drummondhill, Stratherrick Road, Inverness IV2 4JZ, UK Tel.: +44-1463-243030, Fax: +44-1463-711103, E-mail: [email protected] Received and accepted 12 October 2000

Keywords: Cattle, epidemiology, E. coli O157, sheep, verocytotoxin

Summary Verocytotoxin-producing Escherichia coli O157 is a serious pathogen in man that is carried by ruminants and has been isolated from some other animal species. Except in the very young of certain species and in greyhounds, the organism is not associated with disease in animals. Humans may be infected by ingestion of the organism through direct animal contact, from contaminated food or water or from the environment. Great e€orts have been made to improve hygienic food production and handling, to protect water supplies and to give adequate advice to people handling animals. It is also essential to try to reduce the numbers of organisms shed by animals and, to do this, a clear understanding of the ecology of the organism is required. Introduction Verocytotoxin-producing Escherichia coli (VTEC) O157:H7, or its immotile form (H)), is a serious pathogen in man (H. Chart in this issue). Apart from a condition described in greyhounds and in some very young animals, VTEC O157 is not a signi®cant animal pathogen. However, E. coli O157 has enormous veterinary public health signi®cance because the agent is a potential zoonosis, and may be transmitted to man through foods, water or by direct contact. Since ground beef was ®rst implicated as a source of infection, in the form of improperly cooked hamburgers, interest has focussed upon animal reservoirs. It is considered that by studying the epidemiology in animals, it may be possible to devise methods of control to reduce the possibility for direct infection or contamination of meat, dairy products, vegetables and water supplies that can all be contaminated by animal faeces. Sharp et al. (1995), when giving an epidemiological overview of E. coli O157 in Scotland 1984±1994, commented on the importance of interdisciplinary collaboration, in particular the close working relationship among microbiologists, public health physicians, veterinarians and epidemiologists. Great progress has been made and especially in countries where such teamwork exists. Recently, thanks to the generosity of the Wellcome Trust, an exciting new International Partnership in Veterinary Epidemiology has been formed that adds mathematical modellers, evolutionary and molecular biologists, and ecologists to a team trying to unravel the mysteries of Enterobacteriaceae. The interest in this subject over the last two decades has been so immense that this review cannot be

comprehensive but it seeks to cover areas of veterinary importance with which the author is most familiar. Non-O157 VTEC are not covered here but have been reviewed elsewhere (Bettelheim 2000). VTEC O157 in ruminants Soon after VTEC O157 was ®rst associated with haemolytic uraemic syndrome (HUS) in man (Riley et al. 1983), cattle were identi®ed as carriers in the USA (Martin et al. 1986) and Canada (Borczyk et al. 1987). Smith et al. (1987) also pointed out the relationship of this organism with haemorrhagic colitis in man in England and Wales, while Chapman et al. (1989) made the ®rst English isolations and Synge & Hopkins (1992) detected the organism in diarrhoeic calves in Scotland. However Orskov et al. (1987), who established the somatic antigen O157 in 1972, detected VTEC O157 in 1±3-week-old calves with colibacillosis on four farms in Argentina, sampled in 1977. These authors did not ®nd this strain in any other animal isolates at this time. Montenegro et al. (1990) isolated VTEC O157 from healthy cattle in Germany and the organism has been found in cattle in many countries, for example Australia (Cobbold & Desmarchelier 2000), Japan (Itoh et al. 1999), Switzerland (Stephan et al. 2000) and Norway (Vold et al. 1998). Following the independent identi®cation in Australia, Great Britain and the USA that sheep meat could also be a source of VTEC O157 for man, Chapman & Siddons (1996) isolated the organism from sheep rectal swabs taken immediately post slaughter. The organism

726 has been isolated from goats in follow-up studies of human infection (Smith et al. 1998). Wild deer sharing rangeland with cattle were found to be carriers in the USA (Rice et al. 1995) and, in the UK, Chapman & Ackroyd (1997) found the organism in farmed deer. This particular farm had not kept other types of livestock for the last 20 years. VTEC O157 in other animal species VTEC O157 has been isolated from pigs (Chapman 2000) during on-farm investigations. Surveys of pigs however do not suggest it as a major reservoir of infection. E. coli O157 isolates from pigs are frequently not verocytotoxin producers (Chapman et al. 1997) but they can be enterotoxigenic possessing the F4 (K88) adhesin (Wray et al. 1993). VTEC that cause oedema disease in pigs carry the gene encoding a speci®c verocytotoxin VT2e and this is not associated with E. coli O157. However, Heuvelink et al. (1999b) have isolated potentially pathogenic VTEC O157 from Dutch slaughter pigs. It is perhaps only a matter of time before more pigs are found to carry this organism. VTEC O157 has been isolated from other domestic animals, for example, horses (Trevena et al. 1996; Chalmers et al. 1997) and dogs (Trevena et al. 1996), especially greyhounds (Cowan et al. 1997). The organism was isolated from zoo animals including primates (Bauwens et al. 2000). Natural infection of poultry has not been reported, but the organism was isolated from domestic geese by the Scottish Agricultural College in a human follow-up study (Smith et al. 1998), from turkeys in Holland (Heuvelink et al. 1999b) and from seagulls (Wallace et al. 1997). House ¯ies (Hancock et al. 1998b; Iwasa et al. 1999) and fruit ¯ies (Janisiewicz et al. 1999) have been shown to carry the organism. Pathogenicity of E. coli O157 and animals as models for human infection E. coli O157 is the agent most commonly associated with HUS in man, especially children. Virulence is attributed to the production of verocytotoxins and its ability to intimately adhere to the enterocyte cell membrane of the host by an attaching and e€acing mechanism. The genes encoding the verocytotoxins are described as VT1 and VT2, while the eae gene encodes the mechanisms for attachment and e€acement. E. coli strains that contain either VT gene plus eae are known as enterohaemorrhagic E. coli (EHEC) and those that contain eae but are not VTEC are termed enteropathogenic E. coli (EPEC). Studies in Scotland suggest that the vast majority of VTEC O157 also have eae and so are EHEC. Synge (2000) has looked at VTEC in general in animals. There is little ®eld evidence that E. coli O157 causes clinical disease in animals. One study (Synge & Hopkins 1994) identi®ed scouring calves shedding the organism

B.A. Synge but other pathogens were present and Richards et al. (1998) found the organism most frequently in animals without enteric disease. Occasional ®eld cases of enteritis have been associated with other VTEC, for example serotype O26 was isolated from two-week-old calves with dysentery (Anon 2000) and attachment and e€acement was shown with the same serotype in an eightmonth-old heifer (Pearson et al. 1999). Greyhounds are susceptible to a naturally occurring disease, cutaneous and renal glomerular vasculopathy (CRGV), where thrombotic vascular lesions occur in the skin and kidneys. The glomerular lesions and clinical pathology of CRGV closely resemble HUS in man (Cowan et al. 1997). Experimental studies have attempted to determine if the organism colonises and is pathogenic for cattle. Dean-Nystrom et al. (1997) inoculated calves less than 36 hours old with VTEC O157 and produced diarrhoea associated with attaching and e€acing lesions in both the small and large intestine. In other studies where older calves were infected arti®cially (Cray & Moon 1995; Woodward et al. 1999), attaching and e€acing lesions were produced but without clinical e€ects. Wray et al. (2000) described experimental infection in naturally infected cattle and found no evidence of adherence to the mucosa but in three out of six animals there was an increase in IgG after challenge. Neonatal calves are not useful models for the study of the disease in man because of the diculties involved in reproducing the condition. There is no evidence of pathogenicity in other ruminants. The infant rabbit (5±10-days-old) was the ®rst model of E. coli O157 induced diarrhoea to be developed, and very extensive studies have been carried out, including the use of vaccines to protect against the uptake of verocytotoxins. Mice have also been used to study the e€ects of parenteral injection of verocytotoxin. A gnotobiotic pig model developed by Francis et al. (1986), and Tzipori et al. (1995), demonstrated that intimate bacterial attachment was not a prerequisite for VT2 transfer from the gut lumen to the circulation. Oneday-old chicks inoculated in the crop with VTEC O157 developed disease, and attachment and e€acement was detected in the caecal mucosa. Under experimental conditions hens have become chronic shedders (Schoeni & Doyle 1994). An overview of animal models is given by Moxley & Francis (1998). The prevalence of VTEC O157 in livestock Studies of prevalence are important in helping to ascertain the risk of human infection but because of di€erent methodologies great care is required when comparing ®gures from di€erent studies. As E. coli O157 is usually present as a minority organism and in low numbers, the detection method is of great importance. In early studies, direct plating on to sorbitol MacConkey agar was used. Synge & Hopkins (1996) found only

Veterinary signi®cance of VTEC O157 0.25% positive out of more than 5000 bovine samples submitted to veterinary investigation laboratories throughout Scotland, whilst Chapman et al. (1993), by a similar method, found 4% cattle to be carriers at an abattoir in Sheeld, England. A study of diagnostic samples in England and Wales, similar to the Scottish study but using immuno-magnetic separation (IMS), found 0.83% bovines positive and a later study in the Sheeld abattoir, again using IMS, identi®ed 15.7% of cattle as carriers. In both studies using IMS the prevalence increased by fourfold and is likely to be the result of using a more sensitive technique rather than an increase of prevalence with time (Chapman et al. 1994). A major prevalence study in Scottish beef cattle, aged between 12 and 30 months, has just been ®nished and the preliminary analysis suggests that 24% of herds had at least one animal shedding and 8.6% of animals were positive (Synge & Paiba 2000). The studies in England and Wales described in the same publication show up to 50% herds may be shedding the organism but this was in a wider sampling frame. Studies in the USA have shown similar trends with improved techniques. Hancock et al. (1994), using direct plating to sorbitol MacConkey, isolated E. coli O157 from 8% of dairy herds and 16% of pastured beef herds. A later study by the same group, using broth enrichment but not IMS, showed nine out of 14 herds to be positive (Hancock et al. 1997). In a Dutch study, 10 dairy farms were visited and all the cattle on the farm were sampled and tested by IMS. Seven out of 10 farms were positive for VTEC O157, with the proportion of positive animals varying from 0.8 to 22.4% (Heuvelink et al. 1998). Thus it must be assumed that in many countries the majority, but not necessarily all, farms have some animals shedding the organism. It remains unclear if there are countries where the organism is not present. The prevalence of VTEC O157 in animal meats has also been examined. Heuvelink et al. (1999a), in the Netherlands, found VTEC O157 in 1% of raw minced beef and 1% of raw pork products, while raw products from poultry, lamb and wild animals were all negative. In the UK, 1.4% of 5000 beef and lamb products were contaminated (Chapman et al. 2000). Factors a€ecting the prevalence of E. coli O157 shedding Age has an e€ect. Synge & Hopkins (1996) found more calves less than 2 months-old shedding than older calves, which in turn had less shedding than adults. Hancock et al. (1997) found a higher prevalence (1.8%) in weaned heifers than in unweaned calves (0.9%) and adults (0.4%). Seasonal e€ects have also been described and in Canada and the USA higher prevalences are reported in the summer months (Clarke et al. 1994; Hancock et al. 1997). In a longitudinal study of a single dairy herd in England, Mechie et al. (1997) found a peak of shedding in the ®rst month of lactation with a peak between May and July and a smaller peak in November.

727 Synge (1999) found a similar pattern in a beef suckler herd in Orkney, Scotland. In a large study of 953 groups of ®nishing beef cattle and a longitudinal study of 32 beef suckler herds, the preliminary results suggest shedding is higher in the spring and autumn than in the winter or summer (Synge et al. 2000). These studies are attempting to elucidate interactions with housing, feeding, calving and weaning, etc. that might in¯uence shedding of the organism. However, to date the e€ects of these interactions are unclear. In the study of a dairy herd by Mechie et al. (1997), E. coli O157 persisted in the herd for at least 15 months, even though there was no detectable shedding of the organism for 5 months during the winter. Only one cow out of 73 shed the organism more than 2 weeks consecutively. Synge (2000) found similar results in Orkney but, in a separate group of intensively managed dairy calves, the organism was consistently isolated for 7 months. In a Canadian study of eight dairy herds, Rahn et al. (1997) concluded that shedding by dairy cattle was transient. In an experimental study, inoculated calves shed the organism intermittently up to 58 days and cows up to 44 days, but on the majority of sampling days the organism was not detected (Wray et al. 2000). Brown et al. (1997) showed that, following inoculation, E. coli O157 persists in the rumen, reticulum, omasum and parts of the large colon during the period of faecal shedding. A de®nitive site of mucosal colonisation was not found. It has been postulated that the persistence of E. coli O157 may be increased by its ability to survive within protozoa in the soil and possibly the rumen (Barker et al. 1999). Nutritional e€ects on the shedding of VTEC O157 It has been suggested that the diet, abrupt changes in diet, or fasting may in¯uence the shedding of E. coli O157 but many of the results are contradictory. Kudva et al. (1997) investigated these hypotheses with experimentally inoculated sheep as a ruminant model. Two diets were investigated; a concentrate that consisted of a mixture of corn and pelleted alfalfa, high in protein, digestible energy and low in ®bre; and a grass hay diet, low in protein and digestible energy but high in ®bre. Hay fed sheep shed the organism for twice as long and in higher numbers than sheep that had been fed the concentrate. The number of culture positive animals increased when the diet was abruptly changed from concentrate to hay and decreased with the opposite change. A total of 24 h fasting did not in¯uence E. coli O157 shedding. In cattle, Diez-Gonzalez et al. (1998) showed that animals, which were fed mostly grain, had a lower colonic pH and more acid resistant E. coli than cattle that were fed only hay. A brief period of hay feeding decreased the acid resistant count substantially. E. coli O157 tend to be more acid resistant than many other E. coli. However, Hovde et al. (1999) suggested that hay fed cattle shed E. coli O157 for longer than

728 grain fed animals. Russell et al. (2000) concluded that feeding hay for a period of up to 7 days decreases the number of cattle shedding E. coli O157. The fate of VTEC O157 in faeces and the environment Knowledge of the ability of the organism to survive in faeces, on pasture and associated water systems has implications for its spread to crops, which may be a source of infection for man or indeed for animals. Maule (2000) found that the organism survived longest in soil cores containing rooted grass where levels of 108 per gram declined to 106 per gram after 130 days. The organism was detectable in cattle faeces at high levels for more than 50 days. The organism survived much less readily in slurry or river water. The decline in E. coli numbers increases with temperature. Himathongkham et al. (1999) showed more rapid destruction at 37 °C than at 20 °C or 4 °C. In manure, reduction times varied between 6 days and 3 weeks, while in slurry the times ranged from 2 days to 5 weeks. Beuchat (1999) demonstrated survival of the organism on lettuce contaminated by cattle faeces for at least 15 days. Fenlon et al. (2000) related the fall o€ in E. coli numbers in soil to penetration to deeper soil layers and to run o€ into drains following rain. Experimental studies showed that E. coli O157 on grass, which was subsequently ensiled in conditions allowing aerobic spoilage, could multiply to numbers exceeding 106 per gram in the silage. Sources of VTEC O157 for livestock Cattle can undoubtedly pass E. coli O157 from one to another. This is likely to happen in both housed and grazing animals. Recent studies by Synge et al. (2000) found more housed animals shedding the organism. Buying in replacements rather than home rearing was also a factor that increased the likelihood of a farm being positive. The possibility of infection from poorly made silage has also been demonstrated (Fenlon et al. 2000). In the USA, replication of E. coli O157 in wet grains and silage/corn mixes has been observed (Lynn et al. 1998). Cattle or sheep may also acquire infection from wild animals, e.g. deer grazing the same pasture (Rice et al. 1995). Wild birds, especially seagulls and other scavengers, may spread infection from farm to farm, from human sewage or refuse tips to pasture (Wallace et al. 1997). Routes of infection for man Major VTEC O157 outbreaks in man are most usually associated with food. For example, in the USA (Grin 1998), food was the most common source ± 67%, followed by person to person contact, usually in child care centres ± 22%, swimming water ± 8% and drinking

B.A. Synge water ± 2%. O'Brien (2000) reviewed the sources of outbreaks in the UK. These included hamburgers, yoghurt, unpasteurised milk, milk sold as pasteurised, animal contact, various meat products in the large central Scotland outbreaks, cheese made from unpasteurised milk, contaminated mud at a pop festival, a private water supply and a contaminated beach. In the case of outbreaks associated with meat products, it is rarely possible to trace the contamination back to farm level but with the other types of food such as milk or where direct contact has occurred, trace backs have allowed an animal source to be identi®ed. Synge & Hopkins (1996) isolated indistinguishable VTEC O157 from cattle and humans in 10 out of 19 incidents investigated. Pulsed-®eld gel electrophoresis was proved to be an enormous bene®t in identifying the source. In Scotland, where there have been consistently higher rates of human infection than almost any country, the majority of cases are sporadic rather than related to outbreaks (Reilly et al. 2000), although Scotland has also su€ered some major outbreaks. A case control study in Scotland showed contact with animal faeces, likely contact with animal faeces and animal contact to be highly associated risk factors. In all cases this contact was with animals other than pets (Reilly et al. 2000). Direct contact with animals is a well-established risk factor; for example with calves (Renwick et al. 1993; Synge et al. 1993) or lambing ewes (Allison et al. 1997). Outbreaks have also been associated with contaminated fruit or vegetable products; for example unpasteurised apple juice (Cody et al. 1999). Contaminated water has lead to several outbreaks (Chalmers et al. 2000). A waterborne outbreak, due to the contamination by sheep of a private water supply from a hillside in a remote area of the Scottish Highlands (Synge 2000), highlights the ubiquitous nature of the organism. The prevention of VTEC O157 infection from animals There is always a potential risk when people have contact with animal faeces. Persons working with animals must be made aware of the risks to themselves and their families, and adequate protective clothing which is not taken home, and hand washing facilities must be provided. The risk appears to be higher on open farms visited by members of the public who are not familiar with animals. The Health and Safety Executive in the UK have issued advice to farmers and schoolteachers (HSE 2000). Much attention has focussed upon the need to produce meat that has a low level of contamination. It is generally considered that there must be farm to fork strategies to reduce the risk of infection, including control strategies at farm level together with hygienic food handling systems and adequate cooking. Gannon (1999) discussed the control of VTEC O157 in cattle at slaughter and advocated the use of a quality control system such as hazard analysis and critical control points (HACCP).

Veterinary signi®cance of VTEC O157 However, VTEC O157 presents a particularly dicult problem since it is considered to have a very low infectious dose, and is also able to withstand low pH. Fortunately some intervention strategies have been devised to improve the safety of beef. Examples include whole carcase treatment with hot water or pressurised steam followed by a wash using hot lactic acid or acetic acid solution. Alternatively, low to medium dose irradiation of packaged beef products can be used. Pasteurisation of milk e€ectively eliminates the hazard from milk and dairy products, but instances have occurred where there has been pasteurisation failure (Upton & Coia 1994) and there are still many unpasteurised products marketed. This makes the production of clean milk, i.e. with low E. coli counts, of great importance. Environmental contamination can be reduced by not allowing animals to graze on grass to which the public will have considerable access. Water supplies can be protected by add-in chlorine (Kaneko 1998), though there are many private supplies in many parts of the world and chlorination may not always be practical. It is important also to reduce the suspended solids to less than 5 mg/l for the process to be e€ective. Spray treatments of contaminated lettuce using 20 ppm chlorine, followed by rinsing later, were e€ective in removing VTEC O157 (Beuchat 1999). Similar methods can be used for apples, etc. The control of VTEC O157 in animals There is still a great need to attempt to reduce the faecal shedding from animals as it is dicult to assess the risk of contamination, impractical to disinfect every food product and impossible to exclude people from contaminated environments. Hancock et al. (1998a) discussed six possible control strategies for cattle:

729 many cattle are clipped but this is a dangerous operation without the use of a good set of stocks. Commonly, food is withheld before transport to avoid punctures of a full gut and to reduce defecation during transport, but there is evidence that fasting animals may actually increase the shedding of VTEC O157 (Duncan et al. 2000). Vaccination This is dependent upon VTEC O157 colonising and initiating a predictable immune response. Since neither of these happen consistently, it is unlikely that a means of vaccination will be found in the near future. Competitive inhibition The theory is that competitive organisms will inhibit the build up of VTEC O157, especially in cattle moved into feedlots. Probiotics have been used in young calves with some success in a small trial (Zhao et al. 1998) but this method needs to be converted to ®eld trials before it can be adequately assessed. Niche engineering This was de®ned as `Modifying the environment to make an ecosystem less susceptible to sustaining a particular agent'. Hancock et al. (1998a) believe that, by the management of feed and water troughs, the multiplication of VTEC O157 may be reduced. Duncan et al. (2000) have also reviewed the potential for the control in farm animals. These authors believe that, by identifying animal husbandry methods or feed additives, proliferation of the organism in the gut may be avoided. The e€ects of some antibacterial plant compounds on E. coli could lead to the development of novel control methods.

Traceback and eradication

The dynamics of VTEC O157

Since VTEC O157 is probably on most farms at some time this would be impractical.

Recent studies in Scotland have shown a steady increase, in the last decade, of the phage type 21/28 that was very rare in the early 90s. The proportion of VTEC O157 producing the di€erent verocytotoxins varies geographically. Table 1 shows the di€erences between Scottish and Washington studies, for example (Rice et al. 1999). It is fascinating to conjecture that, if the prevalence in cattle is so high and there is frequent human exposure, why are more humans not a€ected. There has been a suggestion that there are two distinct lineages of VTEC

Pre-slaughter testing of all cattle This is ¯awed because the organisms are likely to survive on the hide. It would be more logical to test groups of animals and reject the whole group if any positives are found. It is likely, however, that too many groups would be rejected to make this a practical proposition. In the UK, at least one supermarket has used pre-slaughter testing, but the only action taken was to slaughter the positive groups of animals at the end of each day.

Table 1. Percentage of samples with genes encoding the verocytotoxins in two studies.

Other pre-slaughter measures E€orts to reduce hide soiling are worthwhile and can be achieved by providing adequate bedding. In the UK,

Scotland Washington

VT1

VT2

VT1 + VT2

0.2 0.5

94.9 33

4.9 66.5

730 O157 in the USA. Jaehyoung (1999) has described an octamer-based genome scanning system that identi®es a population of VTEC O157 found in cattle, but not found in man. Recent studies in Scotland have shown a signi®cant decline in prevalence of VTEC O157 across three seasons. Care must be taken not to extrapolate these ®ndings without evidence, but it is possible that VTEC O157 will one day disappear. Conclusion It is likely that di€erent populations of VTEC O157 exist in di€erent parts of the world. Because climates vary, and animal husbandry systems and human eating practices change so much from place to place, concerted activity is necessary all around the world to unravel the ecology and combat this infection. Acknowledgement SAC receives ®nancial assistance from the Scottish Executive Rural A€airs Department and from the Ministry of Agriculture, Fisheries and Food. References Allison, L.J., Thomson-Carter, F.M., Gray, D., Rusbridge, S.M. & MacLennan, M. 1997 Human cases of E. coli O157:H7 infection associated with exposure to lambing ewes. In Association of Veterinary Teachers & Research Workers 51st Scienti®c Meeting, Scarborough, ed. Spence, J.A., pp. 28. Pentland Science Park, Penicuik, Midlothian, Scotland: AVT&RW. Anon. 2000 Veterinary laboratories agency disease surveillance report. Veterinary Record 146, 388±389. Barker, J., Humphrey, T.J. & Brown, M.W.R. 1999 Survival of Escherichia coli O157 in a soil protozoan: implications for disease. FEMS Microbiology Letters 173, 291±295. Bauwens, L., De Meurichy, W. & Vercammen, F. 2000 Isolation of Escherichia coli O157 from zoo animals. Vlaams Diergeneeskundig Tijdschrift 69, 76±79. Bettelheim, K.A. 2000 Role of non-O157 VTEC. Journal of Applied Microbiology 88, 38S±50S. Beuchat, L.R. 1999 Survival of enterohemorrhagic Escherichia coli O157:H7 in bovine feces applied to lettuce and the e€ectiveness of chlorinated water as a disinfectant. Journal of Food Protection 62, 845±849. Borczyk, A.A., Karmali, M.A., Lior, H. & Duncan, L.M.C. 1987 Bovine reservoir for verotoxin-producing Escherichia coli O157:H7. Lancet 1, 98±98. Brown, C.A., Harmon, B.G., Zhao, T. & Doyle, M.P. 1997 Experimental Escherichia coli O157:H7 carriage in calves. Applied and Environmental Microbiology 63, 27±32. Chalmers, R.M., Aird, H. & Bolton, F.J. 2000 Waterborne Escherichia coli O157. Journal of Applied Microbiology 88, 124S±132S. Chalmers, R.M., Salmon, R.L., Willshaw, G.A., Cheasty, T., Looker, N., Davies, I. & Wray, C. 1997 Vero-cytotoxin-producing Escherichia coli O157 in a farmer handling horses. Lancet 349, 1816±1816. Chapman, P.A. 2000 Sources of Escherichia coli O157 and experiences over the past 15 years in Sheeld, UK. Journal of Applied Microbiology 88, 51S±60S.

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