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.     

Verocytotoxin-producing Escherichia coli O157:H7 in the Swedish pig population

Verocytotoxin-producing Escherichia coli O157:H7 (VTEC O157:H7) was detected in two of 2446 individual faecal samples collected from pigs slaughtered at five Swedish slaughterhouses, indicating a prevalence of 0.08 per cent, with a 95 per cent confidence interval from 0 to 0.16 per cent Four Swedish VTEC O157:H7-positive farms which kept ruminants and pigs were studied by repeated faecal sampling; VTEC O157:H7 was isolated from the ruminants and pigs on all the farms and the same strains were present in the pigs and the ruminants. On one of the farms, the organism persisted in the pig population for 11 months. On all four farms, management practices which might have influenced the isolation rate in pigs were identified. A group of young VTEC O157:H7-positive pigs was moved from one of the VTEC O157:H7-positive farms to a fattening herd where there were no ruminants. The number of VTEC O157:H7-positive faecal samples decreased gradually and after nine weeks the pigs were all negative; at slaughter none of the pigs was VTEC O157:H7-positive.     

Survey of Campylobacter species,VTEC O157 and Salmonella species in Swedish wildlife

Samples collected from 791 wild animals (Canada geese, roe deer, hares, moose, wild boar and gulls) shot during hunting were examined for verocytotoxin-producing Escherichia coli (VTEC) O157, and thermophilic Campylobacter and Salmonella species. With the exception of one positive isolate from a wild boar, VTEC O157 was not isolated from any of the animals. Salmonella species were isolated only from the gulls, of which 4 per cent were estimated to be positive. Thermophilic Campylobacter species were commonly isolated from all the species except deer.     

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.     

Study on Seasonal Impact to the Distribution of Bovine E.coli O157: H7 in Xinjiang

To study the impact of season to the distribution of bovine E.coli O157:H7,samples of anus swabs (399),feces (68),water (29) and feed (43) were collected in the spring, summer,autumn and winter from A,B and C farms of Xinjiang. After enrichment by EC broth, SMAC and MUG selective culture were then performed. Finally,PCR was used for identification and virulence gene detection of isolated strains. A total of 5 E.coli O157:H7 strains were isolated from 539 samples from three farms (0.93%,5/539), 2 of them were from spring (1.44%,2/139),1 from autumn(0.56%,1/180),2 from winter (1.38%,2/145) and no strains were isolated from summer samples. One strain were isolated from anus swab samples in farm B (0.69%,1/145) and one were isolated from anus swabs (0.66%,1/152) and three strains were isolated from feed samples in farm C (20.00%,3/15),and no target strains were isolated from water samples. The distribution of bovine E. coli O157:H7 had obvious seasonal characteristics.One E.coli O157:H7 strain of farm B was isolated from autumn and four of farm C were from isolated spring (2 strain) and winter (2 strain),and the isolation rate of E. coli O157:H7 in spring and winter were higher than that in summer and autumn. In conclusion,under the special climate characteristics and feeding mode in Xinjiang,to prevent and control the spreading of E. coli O157:H7 of cattle,we must pay great attention on hygiene management of pens at cold season, specially avoiding the feed contaminated by feces.     

Feeding supplemental dried distiller's grains increases faecal shedding of Escherichia coli O157 in experimentally inoculated calves

Escherichia coli O157 is an important foodborne pathogen and asymptomatic cattle serve as major reservoirs for human infection. We have shown a positive association between feeding distiller's grains and E. coli O157 prevalence in feedlot cattle. The objective of this study was to determine the effect of feeding dried distiller's grain (DDG) on faecal shedding of E. coli O157 in calves experimentally inoculated with E. coli O157. Holstein calves (five per treatment group), fed steam-flaked corn-based high-grain diets supplemented with 0% (control) or 25% DDG, were orally inoculated with a five-strain mixture (6 x 10(9) CFU/calf) of nalidixic acid-resistant (NalR) E. coli O157. Faecal samples were taken three times per week for 6 weeks to determine the prevalence and concentration of Nal E. coli O157. At the end of the study (day 43), calves were euthanized and necropsied. Ruminal, caecum, colon, and rectal contents, and rectoanal mucosal swab (RAMS) samples were collected at necropsy to determine NalR E. coli O157 concentration. There was a trend for an interaction between treatment and faecal sampling day. The concentration of NalR E. coli O157 in the faeces was significantly higher in faecal samples from calves fed DDG compared with control calves on days 35, 37, 39 and 42. At necropsy, the concentration of NalR E. coli O157 was higher in the caecum (P = 0.01), colon (P = 0.03) and rectum (P = 0.01) from calves fed DDG compared with control animals. The number of sites at necropsy positive for NalR E. coli O157 was higher in calves fed DDG compared with calves in the control treatment (P < 0.001). Our results indicate that E. coli O157 gut persistence and faecal prevalence increased in calves fed DDG, which potentially have important implications for food safety.     

Campylobacter spp., Salmonella spp., verocytotoxic Escherichia coli, and antibiotic resistance in indicator organisms in wild cervids

Faecal samples were collected, as part of the National Health Surveillance Program for Cervids (HOP) in Norway, from wild red deer, roe deer, moose and reindeer during ordinary hunting seasons from 2001 to 2003. Samples from a total of 618 animals were examined for verocytotoxic E. coli (VTEC); 611 animals for Salmonella and 324 animals for Campylobacter. A total of 50 samples were cultivated from each cervid species in order to isolate the indicator bacterial species E. coli and Enterococcus faecalis / E. faecium for antibiotic resistance pattern studies. Salmonella and the potentially human pathogenic verocytotoxic E. coli were not isolated, while Campylobacter jejuni jejuni was found in one roe deer sample only. Antibiotic resistance was found in 13 (7.3%) of the 179 E. coli isolates tested, eight of these being resistant against one type of antibiotic only. The proportion of resistant E. coli isolates was higher in wild reindeer (24%) than in the other cervids (2.2%). E. faecalis or E. faecium were isolated from 19 of the samples, none of these being reindeer. All the strains isolated were resistant against one (84%) or more (16%) antibiotics. A total of 14 E. faecalis-strains were resistant to virginiamycin only. The results indicate that the cervid species studied do not constitute an important infectious reservoir for either the human pathogens or the antibiotic resistant microorganisms included in the study.     

牛源大肠杆菌O157:H7的分离鉴定

为调查新疆部分地区E.coli O157：H7的感染情况和菌株致病性,从新疆阿克苏、伊犁、塔城3个地区的牛场采集新鲜粪样564份,对E.coli O157：H7进行分离与鉴定。利用E.coli营养肉汤（EC肉汤）对样品进行增菌后,用山梨醇麦康凯培养基（SMAC）平板选择性培养,再经过4-甲基伞形酮-β-D葡萄糖醛酸苷培养基（MUG）的筛选,对疑似菌株进行生化和PCR鉴定,并将分离鉴定到的菌株进行小鼠攻毒试验。结果显示,从伊犁地区采集的样品中共分离出2株E.coli O157：H7（Y166和Y226）,其检出率为0.88%;小鼠攻毒试验中,Y166和Y226试验组小鼠在48 h内全部死亡,具有一定致病性;从阿克苏、塔城所采样品中未分离到E.coli O157：H7。     

The studies on the aetiology of diarrhoea in neonatal calves and determination of virulence gene markers of Escherichia coli strains by multiplex PCR

The purpose of this study was to determine aetiological agents of diarrhoea in neonatal calves and to investigate virulence gene markers of Escherichia coli strains isolated from calves by multiplex polymerase chain reaction (PCR). Eighty-two diarrhoeic calves and 18 healthy calves were used as subjects. Faeces were taken from the rectums of all the calves and were subjected to bacterial culture. Antigen enzyme-linked immunosorbent assay (ELISA) was performed to detect rotavirus, coronavirus and E. coli K99 in faeces of all the calves. A multiplex PCR was used to characterize E. coli strains in all the calves. Escherichia coli was isolated from 37 faeces samples, Enterococcus ssp. was isolated from 22 faeces samples and Salmonella was isolated from one faeces sample in diarrhoeic calves. Furthermore, only E. coli was isolated from all 18 faeces samples of healthy calves. Of the 37 E. coli isolated from diarrhoeic calves, K99 (18.9%), F41 (18.9%), heat-stable enterotoxin a (STa) (18.9%), Shiga toxin 1 (Stx1; 13.5%) and Shiga toxin 2 (Stx2; 5.4%) and intimin (8.1%) genes were identified by multiplex PCR. Of the 18 E. coli isolated from healthy calves, K99 (16.6%) and intimin (55.5%) genes were identified by PCR. A total of 15 rotavirus, 11 coronavirus and 11 E. coli K99 were detected in diarrhoeic calves by the antigen ELISA. As a result, this study shows that rotavirus, coronavirus, E. coli and Enterococcus ssp. were determined to play a role in the aetiology of diarrhoea in the neonatal calves. K99, F41, STa, Stx1 and Stx2 were found as the most common virulence gene markers of E. coli strains isolated from calves with diarrhoea. Multiplex PCR may be useful for characterization of E. coli isolated from calves.     

Wild rabbits (Oryctolagus cuniculus) as potential carriers of verocytotoxin-producing Escherichia coli

In summer 2001, visitors to a wildlife park in Norfolk, uk, became infected with verocytotoxin-producing Escherichia coli (vtec) O157, which was associated with wild rabbit faeces. The rabbits lived in an adjacent field together with E coli O157-positive cattle. A pilot study was therefore performed to investigate the factors affecting the association between E coli O157-positive cattle and wild rabbits. Samples of faeces were taken from 16 herds of cattle that lived close to populations of wild rabbits. Analysis by culture and pcr showed that seven of the herds were positive for E coli O157. Faeces were collected from individual rabbits at six of these farms during late winter, when there were few rabbits, and during high summer, when there were many. None of the 32 samples collected on two farms in late winter was positive for E coli O157, but eight of 97 samples collected in summer were positive on four of the six farms. pcr analysis for vtec, including non-O157, showed that 20 of the 97 samples were positive.     

A longitudinal study of verotoxin-producing Escherichia coli in two dairy goat herds

A longitudinal study was conducted on two dairy farms to investigate the pattern of shedding of verotoxin-producing Escherichia coli (VTEC) in goats. Faecal samples were taken from 20 goat kids once weekly during the first 4 weeks of life and then once every month for the next 5 months of life, and from 18 replacement animals and 15 adults once every month for 12 months. The proportion of samples containing VTEC was higher for replacement animals and adults (85.7% and 78.7%, respectively) than for goat kids (25.4%). About 90% of the VTEC colonies isolated from healthy goats belonged to five serogroups (O33, O76, O126, O146 and O166) but the most frequent serogroups of these isolates, except one, were different in the two herds studied. E. coli O157:H7 was found in three goat kids on only one occasion. None of the VTEC isolates, except the three E. coli O157:H7 isolates, was eae-positive. The patterns of shedding of VTEC in goat kids were variable, but, in contrast, most of the replacement animals and adults were persistent VTEC shedders. Our results show that isolates of VTEC O33, O76, O126, O146 and O166 are adapted for colonising the intestine of goats but that, in contrast, infection with VTEC O157:H7 in goats seems to be transient.     

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.     

Isolation of Verocytotoxin-producing Escherichia coli O157:H7 from cattle at slaughter in Italy.

Cattle arriving for slaughter at a large abattoir in northern Italy between April 1997 and January 1998 were examined for intestinal carriage of Verocytotoxin-producing Escherichia coli (VTEC) O157 using an immunomagnetic separation technique. Sixty sorbitol non-fermenting VTEC O157 strains were isolated from 59 (13.1%) of the 450 cattle examined. In particular, VTEC O157 was found in 37 (16.6%) of 223 feedlot cattle and in 22 (16.1%) of 137 dairy cull cows, but not in the 90 veal calves sampled. The isolation rate was higher during warm weather (17.5%), falling to an average of 2.9% during the winter months. VT-negative, O157 latex-agglutinating E. coli strains were isolated from 23 (5.1%) of the 450 animals. PCR analysis showed that all 60 VTEC O157 strains carried the VT2 gene and that 25 strains also carried the VT1 gene. In addition, four of the VT-negative, O157 latex-agglutinating E. coli strains carried the VT2 gene. Atypical biochemical features were observed in some VTEC O157: two strains (3.3%) showed beta-glucuronidase activity, and seven (11.7%) produced urease.     

Campylobacter spp., Salmonella spp., Verocytotoxic Escherichia coli,and Antibiotic Resistance in Indicator Organisms in Wild Cervids

Faecal samples were collected, as part of the National Health Surveillance Program for Cervids (HOP) in Norway, from wild red deer, roe deer, moose and reindeer during ordinary hunting seasons from 2001 to 2003. Samples from a total of 618 animals were examined for verocytotoxic E. coli (VTEC); 611 animals for Salmonella and 324 animals for Campylobacter. A total of 50 samples were cultivated from each cervid species in order to isolate the indicator bacterial species E. coli and Enterococcus faecalis/E. faecium for antibiotic resistance pattern studies. Salmonella and the potentially human pathogenic verocytotoxic E. coli were not isolated, while Campylobacter jejuni jejuni was found in one roe deer sample only. Antibiotic resistance was found in 13 (7.3%) of the 179 E. coli isolates tested, eight of these being resistant against one type of antibiotic only. The proportion of resistant E. coli isolates was higher in wild reindeer (24%) than in the other cervids (2.2%). E. faecalis or E. faecium were isolated from 19 of the samples, none of these being reindeer. All the strains isolated were resistant against one (84%) or more (16%) antibiotics. A total of 14 E. faecalis-strains were resistant to virginiamycin only. The results indicate that the cervid species studied do not constitute an important infectious reservoir for either the human pathogens or the antibiotic resistant microorganisms included in the study.     

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.     

Simultaneous occurrence of selected food-borne bacterial pathogens on bovine hides, carcasses and beef meat

The aim of this study was to determine the simultaneous occurence of Salmonella spp., L. monocytogenes, verotoxigenic E. coli (VTEC), and Campylobacter spp. in slaughtered cattle and in beef meat subjected for human consumption. A total of 406 bovine hides and 406 corresponding carcasses were used to collect the samples with a swab method after exsanguination and evisceration of animals, respectively. Furthermore, 362 beef meat samples were purchased in local retail shops over the same period of time as for the bovine samples. Food-borne bacterial pathogens were identified with standard ISO methods with some modification by the use of PCR for VTEC. The isolated bacteria were then molecularly speciated (Campylobacter), serotyped (L. monocytogenes) and characterized for the presence of several virulence marker genes (VTEC and Campylobacter). It was found that 49 hide (12.1%) and 3 (0.7%) carcass samples were contaminated with more than one bacterial pathogen tested. Most of the hides were positive for Campylobacter spp. and VTEC (27 samples) and Campylobacter spp. together with L. monocytogenes (12 samples). Eight bovine hides contained L. monocytogenes and VTEC while L. monocytogenes and Salmonella spp. were detected in one sample. Furthermore, 3 pathogens (Campylobacter spp., L. monocytogenes and VTEC) were simultaneously identified in one bovine hide tested. In case of bovine carcasses 2 samples contained Campylobacter spp. and VTEC whereas one carcass was positive for L. monocytogenes and VTEC. On the other hand, 10 out of 362 (2.8%) minced beef samples were contaminated with at least two pathogens tested. The majority of these samples were contaminated with L. monocytogenes and Salmonella spp. (6 samples). It was noticed that equal number of C. jejuni and C. coli were found, irrespective of the origin of the samples. Most of the strains possessed more than one pathogenic factor as identified by PCR. Molecular serotyping of L. monocytogenes revealed that the majority of the isolates (27 out of 31; 87.1%) belonged to 1/2a serogroup. It was found that most of the VTEC isolates possessed the Shiga toxin stx2 gene (12 strains) whereas only 2 strains were str1-positive. The eneterohemolysin and intimin markers were identified only in 7 and 2 isolates, respectively. PCR analysis revealed that 4 VTEC belonged to O91 serogroup, 2 strains were O145 and 1 isolate was identified as O113. None of the VTEC detected in the study was O157 serogroup.     

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.     

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.     

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.     

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.