Serotypes, virulence genes and intimin types of verotoxin-producing Escherichia coli and enteropathogenic E. coli isolated from healthy dairy goats in Spain

Faecal samples from 222 healthy dairy goats on 12 farms in Spain, as well as bulk tank milk samples of these farms, were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and enteropathogenic E. coli (EPEC). VTEC and EPEC were isolated in 47.7 and 7.7% of the animals, respectively. VTEC were isolated more frequently from adults and replacement animals than from goat kids. In contrast, EPEC were detected more frequently from goat kids than from replacement animals and adults. VTEC or EPEC strains were not detected in the bulk tank milk samples. Although a selective enrichment protocol was used, the serotype O157:H7 was not detected. The most frequent serotypes among the 106 VTEC strains isolated from goats were O5:H-, O76:H19, O126:H8, O146:H21, ONT:H- and ONT:H21. None VTEC strain was eae-positive. The absence of the eae gene in the VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, 16% of VTEC strains isolated from healthy goats belonged to serotypes associated with haemolytic uraemic syndrome in humans. The ehxA gene was detected in 84.9 and 52.9% of the VTEC and EPEC from goats, respectively. The beta1, theta/gamma2 and zeta were the most frequent intimin types among the 17 EPEC strains studied and the most prevalent serotypes of these strains were O156:H25 and O177:H11. Our data show that in Spain healthy goats are an important reservoir of VTEC and EPEC, and a potential source of infection for humans.     

Verocytotoxin-producing Escherichia coli O157 on a farm open to the public: outbreak investigation and longitudinal bacteriological study

Verocytotoxin-producing Escherichia coli (VTEC) O157 phage type 2 (PT2) was isolated from three calves and two goats on a farm open to the public. Phenotypic and DNA-based typing showed that the strains were identical or very closely related to those obtained from an outbreak of VTEC O157 infection in two separate family groups who visited the farm. No VTEC O157 PT2 was isolated again from the farm during a 12-month longitudinal bacteriological study undertaken after the infected animals had been removed. However, phenotypically and genotypically indistinguishable VTEC O157 PT2/28 strains were detected in two of 474 faecal samples collected at monthly visits from 15 species of animals of various ages. The two isolates were obtained from calves from different sources sampled 146 days apart, suggesting that the infection had persisted on the farm although it was not detected in the other species. The same strain was subsequently isolated from another calf housed in the same pen as one of the infected calves. The longest period during which the organism was excreted was seven days. No VTEC O157 was isolated either from 204 replacement animals (including 138 orphan lambs and 10 calves) brought in from various sources, and sampled while they were kept in isolation for two weeks before being introduced to the farm, or from environmental samples. During the study a visitor became ill with VTEC O157 PT2. However, the isolate was distinct from those recovered from the farm and there was no evidence to suggest that the visit was the source of the infection.     

Surveillance of dairy production holdings supplying raw milk to the farmhouse cheese sector for Escherichia coli O157, O26 and O111

Clinically healthy domestic animals can harbour Escherichia coli O157 and other verocytotoxigenic E. coli (VTEC) strains in their faeces. Milk filters can be used to microbiologically monitor direct milk secretion and environmental contamination for these pathogens. The aim of this study was to establish baseline data on the prevalence and characteristics of VTEC organisms in lactating animals (bovine, ovine and caprine) supplying milk to the farmhouse cheese sector, with particular emphasis on serogroups O157, O111 and O26. Fifty-six bovine, 13 caprine and 5 ovine herds/flocks, the majority of which supplying milk for farmhouse cheese production, were surveyed from May 2004 to July 2005. Milk filters were analysed by immunomagnetic separation followed by PCR, on a serogroup-specific basis for E. coli O157, O26 and O111. Positive isolates were examined using a multiplex PCR protocol, for their potential to produce verocytotoxins (vt1/vt2), the haemolysin-encoding gene (hlyA) and the gene encoding attaching and effacement (eae). Five verocytotoxigenic and 22 non-virulent E. coli O157 isolates were detected. Seventeen E. coli O26 isolates were also detected, four of which were verocytotoxigenic, seven isolates contained the eae gene only and six isolates were devoid of any of the virulence factors. The VTEC O157 and O26 isolates contained the hlyA and eae genes along with the verocytotoxin genes. No E. coli O111 isolates were detected. Some of the herds were positive on more than one occasion and multiple E. coli serogroups were isolated from the same milk filter sample. Although all food products tested were VTEC negative, routine surveillance for such pathogens in raw milk/raw milk products is of public health importance. Herd-level surveillance along with subsequent risk management action may be a cost-effective component of risk reduction strategies for food production, drinking water supplies and the protection of public health.     

Longitudinal study to investigate VTEC O157 shedding patterns in young cattle

Individual animal samples were collected from ten VTEC O157 positive farms approximately monthly over 11 months to investigate the shedding of VTEC O157 by youngstock. VTEC O157 was isolated from 7.7% of the 6266 samples and 28.9% of the 1383 animals. On six of the farms VTEC O157 was isolated at multiple visits from several animals, whereas the remaining four farms had one or two positive animals at any one visit, with VTEC isolated from a maximum of four visits. A total of 92 animals were positive more than once (up to four sampling occasions) with a maximum of four negative samples between positive isolations. The results reveal a large variation in individual animal shedding patterns; the proportion of shedding animals on positive farms; and over time within the same farm. The lack of consistent shedding restricts the ability to target potential interventions to specific positive animals/groups or herds.     

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.     

Serotypes and virulence genes of bovine Shigatoxigenic Escherichia coli (STEC) isolated from a feedlot in Argentina

Grazing-fed cattle were previously demonstrated to be reservoir of non-O157 Shigatoxigenic Escherichia coli (STEC) serotypes in Argentina. The acid-resistance of some STEC strains makes it reasonable to assume the presence in feedlot of particular STEC serotypes. Fifty-nine animals were sampled every 2 weeks during 6 months by rectal swabs. Twenty-seven of 59 animals (45.8%) were shown to be Stx2(+); 3/59 (5.1%) carried Stx1(+) and 7/59 (11.9%) were Stx1(+) Stx2(+). Among 44 STEC isolates, 31 isolates were associated to 10 O serogroups (O2, O15, O25, O103, O145, O146, O157, O171, O174, O175) and 13 were considered non-typable (NT). Six H antigens (H2, H7, H8, H19, H21, H25) were distributed in 21 isolates whereas 23 were non-mobile (H-). Seventeen of 44 strains (38.6%) were eaeA(+) and 14 (31.8%) harbored the 60MDa plasmid. The megaplasmid (Mp) and eaeA gene were simultaneously found in a limited number of serotypes belonging to the enterohaemorrhagic E. coli (EHEC). E. coli O157:H7 strains, isolated from four (6.8%) animals, corresponded to the Stx2(+), eaeA(+), Mp(+) pattern. Three O157:H7 strains belonged to phage type 4 and the other strain was atypical. Many serotypes isolated from grain-fed cattle (O2:H25, O15:H21, O25:H19, O145:H-, O146:H-, O146:H21, O157:H7, O175:H8) also differed from those isolated by us previously from grazing animals. The serotypes O15:H21, O25:H19 and O175:H8 had not been identified at present as belonging to STEC. This work provides new data for the understanding of the ecology of STEC in grain-fed cattle and confirms that cattle are an important reservoir of STEC.     

Isolation of a rare Escherichia coli O157:H7 strain from farm animals in Greece

A strain of Escherichia coli O157:H7 was isolated from goat faeces during a surveillance study on the prevalence of this serotype of E. coli in farm animals in Greece. Three hundred and fifty one faecal samples were collected from goat, sheep and cattle breeding farms in the area of Epirus, Northwestern Greece. The E. coli O157:H7 isolate was nonsorbitol-fermenter, produced only VT2 and showed a beta-glucuronidase positive activity, a rather unusual biochemical feature for the E. coli O157:H7 serotype. No other strain of E. coli O157:H7 was isolated from the faecal samples of the rest farm animals examined, thus the overall prevalence of animal carriage was found to be 0.2%. The findings also indicate that goats can be a reservoir of E. coli O157:H7 and goat milk, dairy products and meat may serve as a vehicle for the pathogen transmission to humans.     

Influence of age,sex and herd characteristics on the occurrence of Verocytotoxin-producing Escherichia coli O157 in Danish dairy farms

Verocytotoxin-producing Escherichia coli (VTEC) O157 is an important emerging human pathogen. Cattle are considered to be the main reservoir for VTEC O157. The objectives of this study were to determine the prevalence of VTEC O157 in Danish dairy herds and to investigate the relationship between shedding of VTEC O157 and a number of animal and herd characteristics. Sixty dairy farms were visited once in August-October, and from each herd faecal samples from up to 50 animals were analysed for VTEC O157 by enrichment, immunomagnetic separation (IMS), and plating on selective agar. In total, 2419 animals were sampled, and 3.6% of these excreted VTEC O157. These animals were located on 10 farms (17%). On average, 21% of the sampled animals in the positive herds excreted VTEC O157. Register data, including age, sex, breed, housing conditions and herd composition, were extracted from a database. No influence of herd size or housing conditions was found. A strong effect of age was seen with 2-6-month-old calves as the high-risk age group (8.6% positive) in contrast to calves <2 months (0.7%) and cows (2.4%). There was a non-significant tendency of bull calves to have a higher prevalence than heifers within the age group of 2-6 months. Significantly, more of the herds characterised by having relatively many bull calves or many animals bought into the herd were positive for VTEC O157. Despite the low incidence of human VTEC O157 infections in Denmark, the prevalence in Danish dairy herds was found to be at a similar level as in many other countries.     

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.     

Verotoxinogenic Escherichia coli (VTEC) O157:H7--a nationwide Swedish survey of bovine faeces

In the autumn of 1995 the first outbreaks of enterohemorrhagic Escherichia coli O157:H7 including ca 100 human cases were reported in Sweden. From outbreaks in other countries it is known that cattle may carry these bacteria and in many cases is the source of infection. Therefore, the present study was performed to survey the Swedish bovine population for the presence of verotoxin-producing E. coli (VTEC) of serotype O157:H7. Individual faecal samples were collected at the 16 main Swedish abattoirs from April 1996 to August 1997. Of 3071 faecal samples, VTEC O157 were found in 37 samples indicating a prevalence of 1.2% (CI95% 0.8-1.6). All 37 isolates carried genes encoding for verotoxin (VTI and/or VT2), intimin, EHEC-haemolysin and flagellin H7 as determined by PCR. Another 3 strains were of serotype O157:H7 but did not produce verotoxins. The 37 VTEC O157:H7 strains were further characterised by phage typing and pulsed-field gel electrophoresis. The results clearly show that VTEC O157:H7 is established in the Swedish bovine population and indicate that the prevalence of cattle carrying VTEC O157:H7 is correlated to the overall geographical distribution of cattle in Sweden. Results of this study have formed the basis for specific measures recommended to Swedish cattle farmers, and furthermore, a permanent monitoring programme was launched for VTEC O157:H7 in Swedish cattle at slaughter.     

Serotypes,phage types and virulence genes of shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED: Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE: The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH: Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS: STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS: This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.     

Detection of numerous verotoxigenic E. coli serotypes, with multiple antibiotic resistance from cattle faeces and soil

Verotoxigenic E. coli (VTEC) belong to a diverse range of serotypes. Serotypes O157 and O26 are predominately identified in VTEC-associated disease in Europe, however due to difficulty in detection little is known about the epidemiology of non-O157 serotypes. This study reports the identification of 7 VTEC serotypes from cattle faeces and soil. Cattle faeces samples (n=128) were taken from animals in 6 different farms, with soil samples (n=20) obtained from 1 farm. After sample incubation in modified tryptone soy broth (mTSB) supplemented with streptomycin sulphate samples were plated onto sorbitol MacConkey (SMAC) also supplemented with streptomycin sulphate. Bacteria detected on the plates were subjected to biochemical testing, antibiotic resistance profiling, and PCR to detect typical virulence genes, beta-lactamase and class 1 integron associated genes. Serotyping was performed on isolates positive for virulence genes. E. coli was identified from 103 samples, with verotoxin genes present in 7 E. coli isolates. Of these 7 isolates, 5 were resistant to 5 or more antibiotics. The isolate resistant to 9 antimicrobials contained a class 1 integron structure. Serotyping identified 7 separate VTEC, O2:H27, O26:H11, O63:H(-), O148:H8, O149:H1, O174:H21 and ONT:H25. Six of these VTEC have been previously associated with human disease, however with the exception of O26:H11, these serotypes have been rarely reported worldwide. Increased surveillance is required to determine the prevalence of these and other non-O157 VTEC. The presence of multi-antibiotic resistance in these isolates is of concern, and the overall implications for public health must be ascertained.     

Occurrence and characterization of verocytotoxigenic Escherichia coli (VTEC) strains from dairy farms in Trinidad

A cross-sectional study was conducted to determine the prevalence and characteristics of verocytotoxigenic Escherichia coli (VTEC) on 25 dairy farms each located in Waller field and Carlsen field farming areas in Trinidad. On each selected farm, faecal samples were collected from milking cows, calves and humans; rectal swabs were obtained from pet farm dogs; bulk milk was sampled as well as effluent from the milking parlour. Escherichia coli was isolated from all sources on selective media using standard methods. Isolates of E. coli were subjected to slide agglutination test using E. coli O157 antiserum, vero cell cytotoxicity assay to detect verocytotoxin (VT) and heat labile toxin (LT) production, the polymerase chain reaction (PCR) to detect VT genes, and the dry spot test to screen for E. coli O157 and non-O157 strains. In addition, faecal samples from animal and human sources were tested for VT genes using PCR. Of a total of 933 E. coli isolates tested by the slide test, eight (0.9%) were positive for the O157 strain. The vero cell cytotoxicity assay detected VT-producing strains of E. coli in 16.6%, 14.6%, 3.2% and 7.1% of isolates from cows, calves, farm dogs and humans respectively (P < 0.05; chi(2)). For LT production, the highest frequency was detected amongst isolates of E. coli from calves (10.8%) and the lowest (0.0%) amongst isolates from humans and bulk milk (P < 0.05; chi(2)). Of the 61 VT-producing isolates by vero cell cytotoxicity assay tested by PCR, the VT, LT and eae genes were detected in 62.3%, 4.9% and 1.6% respectively (P < 0.05; chi(2)). Amongst the 45 E. coli isolates that were VT positive (vero cell) or VT-gene positive by PCR, 2.2%, 2.2%, 4.4% and 6.7% belonged to non-O157 strains O91, O111, O103 and O157, respectively, as determined by the Dry spot test. Detection of VTEC strains in milk and dairy animals poses a health risk to consumers of milk originating from these farms. In addition, the demonstration of VTEC strains in humans, VT gene in faecal samples and E. coli isolates as well as non-O157 VTEC strains of E. coli are being documented for the first time in the country.     

Prevalence and concentration of Verotoxigenic Escherichia coli O157:H7 in adult sheep at slaughter from Italy

A 1-year study on the animal-level prevalence and concentration of Escherichia coli O157 in adult sheep at slaughter was performed, to collect qualitative and quantitative information on the diffusion of the pathogen in adult sheep from Italy. A total 533 samples were collected, with a similar distribution in the four seasons. For prevalence estimates, a simple random sampling technique was used. An immuno-magnetic separation technique was used for sample screening, with enumeration of the pathogen in positive samples, along with molecular and serological identification of isolates. An overall prevalence of 7.1% (38/ 533, 95% CI 4.9-9.3%) was observed for fully virulent E. coli O157. A wide interval of VTEC O157 per gram was observed (< 100 to 6 x 10(5) CFU g(-1)), with 28.9% (11/38) of positive samples > or = 1 x 10(3) CFU g(-1), set as the threshold for those animals defined 'active shedders' for the purpose of the study. Eight per cent (3/38) of animals shed > 1 x 10(4) g(-1) VTEC O157, which represents > 96% of the total VTEC O157 bacteria cultured from all animals tested. The prevalence estimate of active shedders was therefore 2.1% (95% CI 0.9-3.3%). Most (34/38, 89.5%) of the positive animals were found in summer (July-September). Prevalence and concentrations of virulent VTEC O157 obtained in this study contribute to the demonstration that adult sheep represent a relevant source of environmental contamination from virulent VTEC O157, as well as a source of VTEC O157 contamination for food of ovine origin (meat and dairy products), especially during warm months.     

Kinetics and role of antibodies against intimin β in colostrum and in serum from goat kids and longitudinal study of attaching and effacing Escherichia coli in goat kids

The presence of antibodies to the intimin β-binding region (Int280-β) of attaching and effacing Escherichia coli (AEEC) in serum from 20 goat kids from 2 herds, as well as in goat colostrum, was investigated by enzyme-linked immunosorbent assay. In addition, the onset and subsequent pattern of shedding of AEEC from the same goat kids over a 6-mo period was investigated. All the colostrum and serum samples tested contained antibodies against Int280-β. The association between the antibody titer and the isolation of AEEC suggests that antibodies to intimin β do not prevent colonization of the intestine by AEEC in goat kids. The AEEC were generally shed only transiently. Most AEEC isolated from the kids belonged to serogroup O26. Three isolates belonged to serogroup O157. These data show that goat kids may be a reservoir of AEEC that are potentially pathogenic for humans.     

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.     

Non-O157 verotoxigenic Escherichia coli and beef: a Canadian perspective

Verotoxigenic Escherichia coli (VTEC) are important foodborne pathogens in Canada. VTEC of the O157:H7 serogroup have been the focus of regulatory action and surveillance in both Canada and the USA, due to their role in a number of high profile outbreaks. However, there is increasing evidence that other VTEC serogroups cause a substantial proportion of human illness. This issue is of particular importance to the cattle industry due to the role of beef as a vehicle for VTEC transmission. In this review, the evidence for non-O157 VTEC as cause of human illness in Canada and the potential for Canadian beef and cattle to serve as a source of VTEC are presented. In addition, the available strategies for the control of VTEC in cattle and beef are discussed.     

Escherichia coli 'O' group serological responses and clinical correlations in epidemic HUS patients

This first comprehensive serological analysis of an haemolytic uraemic syndrome (HUS) outbreak in which a wide range of 'O' group Escherichia coli antibody responses in patients and controls provided a unique insight into the epidemiology of such epidemics. Possible answers to clinical aspects related to severity of disease and complications were revealed. A microagglutination assay was used to examine E. coli 'O' group serological responses in 49 serum samples of 21 children hospitalised with HUS and 14 single samples from contemporaneous age-matched controls. A total of 51 O serogroup strains were used, including those reported to be associated with cases of HUS, with six isolates from patients associated with the Adelaide outbreak, environmental verocytotoxi-genic/shiga-toxin producing E. coli (VTEC/STEC) strains and common human commensal strains. Amongst the 21 patients, there were 226 instances of seroreactivity (titre > or = 100) against 34 E. coli serogroups while six instances of seroreactivity against four serogroups occurred in controls. There were 128 instances in patients and one instance in controls in which titres > or = 400 were observed. All 21 patients were seroreactive (titre > or = 100 and <400) to one or more of the 17 HUS-associated serogroups included in the study. Titres ranged from 100 to 6,400, some of the highest in three patients were against O157, whose faeces yielded only EHEC O111, and only one developed O111 antibody. Mixed infection was demonstrated serologically by microagglutination (confirmed by western blot) and was consistent with the multiple serogroups of VTEC found in the mettwurst incriminated as the source, and suggests further strains (not found in the source or in patients' faeces) were probably involved. In HUS-associated EHEC infection, multiple strain infection may be the rule rather than the exception. Analysis of 34 of the 51 serogroup antibody responses in the HUS patients revealed clues to possible relationships with clinical severity and complications. Patients with severe renal failure tended to develop antibodies to a larger number of serogroups than those with moderate or mild impairment. The same was true for central nervous system complications. Other associations were observed. While VTEC O157 remains an important causal serogroup in HUS, non-O157 serogroups also appear to play a significant role and therefore the latter should always be sought in all future HUS cases as new insights into pathogenicity may be discovered. This study indicates that co-infection with different VTEC serogroups may affect clinical outcome.     

An investigation of factors associated with the prevalence of verocytotoxin producing Escherichia coli O157 shedding in Scottish beef cattle

The prevalence of verocytotoxin-producing Escherichia coli (VTEC) O157 in 12-30-month-old beef finishing cattle in Scotland was determined using 1g faeces samples enriched in buffered peptone water, followed by immunomagnetic separation (IMS) and isolation on sorbitol MacConkey agar with cefixime and tellurite supplement (CT-SMAC). A validated questionnaire was used to collect information that could be associated with the samples. Generalised Linear Models and Generalised Linear Mixed Models were used to identify factors associated with shedding both between and within groups. A total of 14,856 samples were collected from 952 farms, of which 1231 were positive for VTEC O157. Prevalence levels were calculated with 95% confidence intervals as follows: 7.9% (6.5%, 9.6%) of animals sampled were estimated to be shedding VTEC O157, while 22.8% (19.6%, 26.3%) of farms were estimated as having at least one animal shedding in the group sampled. The median percentage of animals shedding in positive groups was 25% (20%, 32%). An increased probability of a group containing a shedding animal was associated with larger numbers of finishing cattle, the presence of pigs on the farm, or the farm being classed as a dairy unit stocking beef animals. Farms that spread slurry on grazing land were more likely to have shedding animals, while those that spread manure were at lower risk. Groups with older animals were less likely to be identified as positive. There was no significant regional difference in group shedding probabilities, but the proportion of positive groups dropped over two successive years of the study. Higher mean levels of shedding in positive groups were associated with animals being housed rather than at pasture, and this effect was stronger in groups which had recently had a change in housing or diet. Farms with animals at pasture had lower mean prevalence where water was supplied from a natural source, as had farms with higher numbers of finishing cattle. There remained unexplained variability in mean prevalence levels on positive farms in different areas of Scotland.     

The characterisation of E. coli O157:H7 isolates from cattle faeces and feedlot environment using PFGE

The objectives of this study were to investigate the diversity of Escherichia coli O157:H7 isolates obtained over a 3-month period from a cattle feedlot in order to assess the relationship between environmental and faecal isolates and to determine the pattern of transmission of E. coli O157:H7 between groups of cattle. Faecal samples were obtained from cattle housed in four adjacent feedlot pens at monthly intervals, with environmental pen samples collected simultaneously. All E. coli O157:H7 isolates obtained were examined by pulsed field gel electrophoresis (PFGE), polymerase chain reaction (PCR) to detect eaeA, ehxA, stx1 and stx2 genes and antibiotic sensitivity profiling. Ten isolates were subjected to acid shock to imitate conditions in the acidic cattle abomasum and assess the effect on PFGE profiles. E. coli O157:H7 was isolated from 69 faecal samples and 26 environmental samples. All isolates (n=95) carried the genes for eaeA, ehxA and stx2 and were sensitive to all antibiotics tested. The PFGE profiles of all isolates differed by no more than two bands and clustered within 80% similarity following dendrogram analysis. Acid shock had no effect on the subsequent PFGE patterns. A total of 8.7% (6/69) of cattle were shedding E. coli O157:H7 in the first month with faecal shedding increasing to 52% (36/69) by the third month of the study. A single isolate of E. coli O157:H7 may be passed rapidly through cattle pens, with the environment acting as a significant reservoir for transmission. PFGE is a useful tool for tracking the direct and indirect transmission of E. coli O157:H7 isolates on the farm.