Transfer of verocytotoxigenic Escherichia coli O157, O26, O111, O103 and O145 from fleece to carcass during sheep slaughter in an Irish export abattoir

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from fleece to dressed carcasses of 500 sheep, and to establish the virulence potential of recovered VTEC. Individual sheep were tracked and sampled (10 g fleece, full carcass swab) through the slaughter process. Samples were examined for the presence of verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay and positive samples were further screened for the presence of the above five serogroups by real-time PCR. VTEC cells were recovered from PCR positive samples by serogroup specific immunomagnetic separation and confirmed by serogroup specific latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR and isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). VTEC O26 was recovered from 5/500 (1.0%) fleece and 2/500 (0.4%) carcass samples. VTEC O157 was isolated from 4/500 (0.8%) fleece samples and 3/500 (0.6%) carcass samples. E. coli O103 was recovered from 84/500 (16.8%) fleece and 68/500 (13.6%) carcasses, but only one E. coli O103 isolate (0.2%) carried vt genes. E. coli O145 was recovered from one fleece sample, but did not carry vt genes. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from fleece to carcass was not observed with PFGE showing that VTEC O26 isolates from a matched fleece/carcass “pair” were not identical. This study shows that while VTEC O157 are being carried by sheep presented for slaughter in Ireland, other potentially clinically significant verotoxin producing strains (particularly VTEC O26) are emerging.     

Verocytotoxigenic Escherichia coli O157 in beef and sheep abattoirs in Ireland and characterisation of isolates by Pulsed-Field Gel Electrophoresis and Multi-Locus Variable Number of Tandem Repeat Analysis

This study aimed to investigate verocytotoxigenic Escherichia coli O157 in the largest beef and sheep slaughter plants in Ireland over a one-year period. Samples consisted of pooled rectal swabs (n = 407) and pooled carcass swabs (n = 407) from 5 animals belonging to the same herd or flock and minced meat (n = 91) from the same sampling date. E. coli O157 isolates were characterised using PCR for a range of genes, i.e. 16S, rfbE, fliC, vtx1, vtx2, eaeA and confirmed VTEC O157 isolates were tested for antimicrobial susceptibility and typed using Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). VTEC O157 was isolated from 7.6% and 3.9% of bovine rectal and carcass swab samples and from 5.8% and 2.9% of ovine rectal and carcass swab samples respectively. None of the bovine minced meat samples (n = 77) and only one of the 14 ovine minced meat samples was positive for VTEC O157. Following PFGE and MLVA, cross contamination from faeces to carcasses was identified. While PFGE and MLVA identified the same clusters for highly related strains, MLVA discriminated better than PFGE in addition to being more rapid and less labour intensive. Results showed that cattle and sheep presented for slaughter in Ireland harbour VTEC O157, and although the levels entering the food chain are low, this should not be overlooked as possible sources of zoonotic infection; molecular typing was able to demonstrate relationships among strains and could be used to elucidate the sources of human infection.     

Faecal carriage of Verocytotoxin-producing Escherichia coli O157 and carcass contamination in cattle at slaughter in northern Italy.

A study on the prevalence of the faecal carriage of Verocytotoxin (VT)-producing Escherichia coli (VTEC) O157 and on the rate of carcass contamination was carried out on feedlot cattle and dairy cows at slaughter in northern Italy. Between April 1998 and January 1999, 12 sampling visits were performed on different days in seven different slaughterhouses. At each visit, 5-12 animals consecutively slaughtered were selected. From each animal, faeces were collected from the rectum immediately after slaughter and surface swabs were taken from the leg region and the diaphragmatic insertion of the carcass. All samples were examined for the presence of VTEC O157 using an immunomagnetic separation technique. A total of 100 animals coming from 60 different farms were examined. In total, VTEC O157 was isolated from the intestinal content of 17, and from the carcasses of 12 of the 100 animals examined. In particular, VTEC O157 was recovered from six (35.3%) out of the 17 carcasses from which the organism had previously been isolated from rectal content and from six (7.3%) of the 82 carcasses of the stool-negative cattle. In seven carcasses, VTEC O157 was isolated from the leg area, in two carcasses from the diaphragmatic area, and in three carcasses from both areas. Major differences in the prevalence of VTEC O157 were observed in the different groups of cattle sampled. In 7 of the 12 sampling visits, all the specimens examined were negative, while 16 of the 17 positive stool samples and 11 of the 12 positive carcass swabs were collected during three of the visits, performed in June in three different abattoirs. In these three visits, the ratios between the percentage of animals carrying VTEC O157 in the stools and the percentage of contaminated carcasses were 0.33, 0.57, and 1.66, respectively; thus, confirming that slaughter practices can largely influence the rate of carcass contamination. Phage typing and PFGE analysis of VTEC O157 isolated from samples collected at the same visit suggested that both auto- and cross-contamination occurred.     

Detection of verocytotoxin-producing Escherichia coli serogroups 0157 and 026 in the cecal content and lymphatic tissue of cattle at slaughter in Italy

Verocytotoxin-producing Escherichia coli (VTEC) has emerged as a foodborne pathogen that can cause severe and potentially fatal illnesses, such as hemorrhagic colitis or the hemolytic uremic syndrome. In this study, 182 cattle at slaughter (119 dairy cows and 63 feedlot cattle) were randomly selected and tested for the presence of VTEC serogroups O26, O103, O111, O145, and O157 in their cecal content and lymphatic tissue (tonsils or mesenteric lymph nodes). A total of 364 samples were evaluated with an immunomagnetic separation technique followed by slide agglutination. Presumptive VTEC 026, O103, O111, O145, and O157 isolates were tested by Vero cell assay for verocytotoxin production and by multiplex PCR assay for the detection of vtxl, vtx2, eae, and E-hlyA genes. VTEC O157 was detected in 6 (3.3%) of 182 animals, and VTEC 026 was detected in 1 (0.5%) of 182 animals. No VTEC O103, VTEC O111, or VTEC O145 isolates were found in cattle feces, but one VTEC O91:H- vtx2+, eae-, E-hlyA+ strain nonspecifically cross-reacted with the VTEC O103 type. The prevalence of VTEC O157 in the lymphatic tissue of cattle was 1.1% in both tonsils (1 of 93 samples) and mesenteric lymph nodes (1 of 89 samples). Lymphatic tissue contamination was observed only in VTEC O157 intestinal carriers; two (33.3%) of six fecal carriers were simultaneously VTEC O157 lymphatic carriers. This finding suggests that VTEC O157 contamination of meat does not necessarily come from feces or the environment. No other VTEC serogroups were detected in the lymphatic tissue of slaughtered cattle.     

Prevalence of verotoxin-producing Escherichia coli and E. coli O157:H7 in pig carcasses from three French slaughterhouses.

Verotoxin-producing Escherichia coli (VTEC) are important food-borne pathogens in humans. Several studies have demonstrated that cattle are a major reservoir of VTEC but few data are available about the occurrence of VTEC in other species. In France, there is no data about pigs and pork meat. The main purpose of this study was to evaluate the prevalence of E. coli O157:H7 and other VTEC in pork carcasses. The second aim of the study was to get a picture of pork carcass contamination by VTEC. Pork carcasses from three French slaughterhouses (50 carcasses per slaughterhouse) were tested for the presence of VTEC and E. coli O157:H7. For each carcass, both internal and external sites were investigated (five on pig skin and three on muscles) and samples were collected by cutting out a surface of 25 cm2. A total of 1200 samples were analyzed by the polymerase chain reaction (PCR) after an enrichment step. Primers used were degenerate-sequences which allowed amplification of various types of verotoxin genes (stx). In addition, a second PCR which specifically detected E. coli O157:H7 was carried out on the stx-positive samples. The percentage of stx-positive PCR samples and carcasses was 12.7% (152/1200) and 50% (75/150), respectively. No E. coli O157:H7 was detected. The prevalence for each slaughterhouse was not significantly different. Skin samples of belly, leg and shoulder allowed detection of more than 80% of the VTEC positive carcasses.     

Methods for recovering Escherichia coli O157:H7 from cattle fecal, hide, and carcass samples: sensitivity and improvements

The Meats Research Unit (MRU) methods, developed by MRU scientists of the U.S. Meat Animal Research Center, have been used to study the prevalence of Escherichia coli O157:H7 in cattle carcass, hide, and fecal samples. The sensitivity of these methods for recovery of injured E. coli O157:H7 cells from inoculated and uninoculated samples was determined, and potential improvements to these methods were evaluated. When using the conventional MRU methods, 91% of the pre-evisceration carcass samples tested positive for E. coli O157:H7 when inoculated with 5 to 10 CFU, 100% of hide samples tested positive for E. coli O157:H7 when inoculated with 30 to 50 CFU, and 96% of the fecal samples produced positive results when inoculated with 300 to 400 CFU per 10 g. The addition of a phosphate buffer to the tryptic soy broth enrichment improved recovery of E. coli O157:H7 from feces. Using the modified enrichment, 92% of the samples were identified as positive when inoculated with 10 to 30 CFU per 10 g. Substituting a commercially available wash buffer for the phosphate-buffered saline (PBS) plus Tween 20 wash buffer during immunomagnetic separation of hide samples improved recovery of the target organism at lower inoculum concentrations. When comparing uninoculated samples, substituting a PBS buffer plus a zwitterionic detergent for PBS plus Tween 20 also had a positive effect on recovery of E. coli O157:H7 from hide samples. Data presented here indicate that the MRU methods are highly effective at recovering injured E. coli O157:H7 from fecal, hide, and beef carcass samples; however, modifications can be added to increase the sensitivity.     

Prevalence and numbers of Escherichia coli O157 on bovine hides at a beef slaughter plant

In this study, we investigated the prevalence and numbers of Escherichia coli O157 on bovine hides. Samples (n = 1,500) were collected over a 17-month period (30 samples per week) by sponge swabbing approximately 122-cm2 areas of the bovine rump of slaughtered cattle at an early stage of carcass processing (first legging). Sponge samples (n = 1,500) were stomached in buffered peptone water supplemented with novobiocin, directly plated on sorbitol MacConkey with Cefixime tellurite (SMAC-CT), enriched for 24 h, extracted by immunomagnetic separation, and plated onto SMAC-CT agar. Presumptive E. coli O157 colonies from SMAC-CT plates were confirmed by PCR for the presence of eaeA, hlyA, fliCh7, vt1, vt2, and portions of the rfb (O-antigen encoding) region of E. coli O157. Overall, E. coli O157 was recovered from 109 samples (7.3%) at concentrations ranging from less than 0.13 to 4.24 log CFU/100 cm2. PCR analysis revealed a wide diversity of genetic profiles among recovered isolates of verocytotoxigenic E. coli. Of the isolates recovered, 99 of 109 contained the attaching and effacing gene (eaeA) and the hemolysin gene (hlyA), and 78 of 109 had the flagellar H7 antigen-encoding gene (fliCh7). Only 6 of 109 isolates contained both verotoxin-producing genes (vt1 and vt2); 91 of 109 contained the vt2 gene only, whereas 1 of 109 contained the vt1 gene only. The remaining 11 of 109 contained neither vt1 nor vt2.     

Prevalence and level of Escherichia coli O157 on beef trimmings, carcasses and boned head meat at a beef slaughter plant

This study investigated the prevalence and level of Escherichia coli O157 on samples of beef trimmings (n=1351), beef carcasses (n=132) and bovine head meat (n=132) in a beef slaughter plant in Ireland. The survey also included an assessment of the prevalence of virulence genes in the E. coli O157 isolates obtained. Samples were examined for the presence of E. coli O157 by direct plating on SMAC-CT and by enrichment/immunomagnetic separation (IMS) with plating of recovered immunobeads onto SMAC-CT agar. Presumptive E. coli O157 isolates were confirmed by PCR targeting a range of genes i.e. vt1, vt2, eaeA, hlyA, fliC(h7) and portions of the rfb (O-antigen encoding) region of E. coli O157. Enterobacteriaceae on head meat samples were estimated by direct plating onto Violet Red Bile Glucose agar. E. coli O157 was recovered from 2.4% (32/1351) of beef trimmings samples, at concentrations ranging from<0.70-1.61 log10 cfu g(-1). Of the 32 positive isolates, 31 contained the eaeA and hylA genes while 30/32 contained the fliC(h7) gene and 31/32 contained vt1 or vt2, or both vt genes. E. coli O157 was recovered from 3.0% (4/132) of carcass samples, at concentrations ranging from <0.70-1.41 log10 cfu g(-1). All of the carcass isolates contained the eaeA, hylA and fliC(h7) genes. E. coli O157 was recovered from 3.0% (3/100) of head meat samples, at concentrations of 0.7-1.0 log10 cfu g(-1). All of the head meat isolates contained the eaeA, hylA, fliC(h7) and vt2 genes. No head meat isolates contained the vt1 gene. Head meat samples (n=100) contained Enterobacteriaceae, at concentrations ranging from 0.70-3.0 log10 cfu g(-1). Overall, the qualitative and quantitative data obtained for E. coli O157 on beef trimming samples in this study could be employed as part of a quantitative risk assessment model.     

An investigation of Escherichia coli O157 contamination of cattle during slaughter at an abattoir

The extent of contamination with Escherichia coli O157 was determined for 100 cattle during slaughter. Samples from 25 consecutively slaughtered cattle from four unrelated groups were collected from the oral cavity, hide, rumen, feces after evisceration, and pre- and postchill carcass. Ten random fecal samples were collected from the pen where each group of animals was held at the abattoir. E. coli O157 was detected using automated immunomagnetic separation (AIMS), and cell counts were determined using a combination of most probable number (MPN) and AIMS. E. coli O157 was isolated from 87 (14%) of the 606 samples collected, including 24% of 99 oral cavity samples, 44% of 100 hides, 10% of 68 fecal samples collected postevisceration, 6% of 100 prechill carcass swabs, and 15% of 40 fecal samples collected from holding pens. E. coli O157 was not isolated from rumen or postchill carcass samples. E. coli O157 was isolated from at least one sample from each group of cattle tested, and the prevalence in different groups ranged from less than 1 to 41%. The numbers of E. coli O157 differed among the animals groups. The group which contained the highest fecal (7.5 x 10(5) MPN/g) and hide (22 MPN/cm2) counts in any individual animal was the only group in which E. coli O157 was isolated from carcasses, suggesting a link between the numbers of E. coli O157 present and the risk of carcass contamination. Processing practices at this abattoir were adequate for minimizing contamination of carcasses, even when animals were heavily contaminated with E. coli O157.     

Seasonal prevalence of Shiga toxin-producing Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants

The seasonal prevalence of Escherichia coli O157:H7, Salmonella, non-O157 E. coli (STEC), and stx-harboring cells was monitored at three Midwestern fed-beef processing plants. Overall, E. coli O157:H7 was recovered from 5.9% of fecal samples, 60.6% of hide samples, and 26.7% of carcasses sampled before the preevisceration wash. This pathogen also was recovered from 1.2% (15 of 1,232) of carcasses sampled at chilling (postintervention) at approximate levels of <3.0 cells per 100 cm2. In one case, the E. coli O157:H7 concentration dropped from ca. 1,100 cells per 320 cm2 at the preevisceration stage to a level that was undetectable on ca. 2,500 cm2 at the postintervention stage. The prevalence of E. coli O157:H7 in feces peaked in the summer, whereas its prevalence on hide was high from the spring through the fall. Overall, Salmonella was recovered from 4.4, 71.0, and 12.7% of fecal, hide, and preevisceration carcass samples, respectively. Salmonella was recovered from one postintervention carcass (of 1,016 sampled). Salmonella prevalence peaked in feces in the summer and was highest on hide and preevisceration carcasses in the summer and the fall. Non-O157 STEC prevalence also appeared to vary by season, but the efficiency in the recovery of isolates from stx-positive samples ranged from 37.5 to 83.8% and could have influenced these results. Cells harboring stx genes were detected by PCR in 34.3, 92.0, 96.6, and 16.2% of fecal, hide, preevisceration carcass, and postintervention carcass samples, respectively. The approximate level of non-O157 STEC and stx-harboring cells on postintervention carcasses was > or = 3.0 cells per 100 cm2 for only 8 of 199 carcasses (4.0%). Overall, the prevalence of E. coli O157:H7, Salmonella, and non-O157 STEC varied by season, was higher on hides than in feces, and decreased dramatically, along with pathogen levels, during processing and during the application of antimicrobial interventions. These results demonstrate the effectiveness of the current interventions used by the industry and highlight the significance of hides as a major source of pathogens on beef carcasses.     

Super shedding of Escherichia coli O157:H7 by cattle and the impact on beef carcass contamination

Beef carcass contamination is a direct result of pathogen transfer from cattle hides harboring organisms such as enterohemorrhagic Escherichia coli. Hide contamination occurs from direct and indirect fecal contamination in cattle production and lairage environments. In each of these environments, individual animals shedding E. coli O157:H7 at high levels (> 10⁴ CFU/g of feces, hereafter referred to as “super shedders”) can have a disproportionate effect on cattle hide and subsequent carcass contamination. It is not known what criteria must be met to cause an animal to shed at levels exceeding 10⁴ CFU/g. Understanding the factors that play a role in super shedding will aid in minimizing or eliminating the super shedding population. Interventions that would prevent supershedding in the cattle population should reduce E. coli O157:H7 transmission in the production and lairage environments resulting in reduced risk of beef carcass contamination and a safer finished product.     

The occurrence of Escherichia coli O157 in/on faeces,carcasses and fresh meats from cattle

The aim of this study was to investigate whether Escherichia coli O157 is present in/on raw beef in Serbia. Correlated faecal and carcasses samples from 115 slaughtered cattle plus 26 uncorrelated carcass samples were examined. E. coli O157 detection and identification was performed using selective enrichment and immunomagnetic separation followed by selective media-plating and biochemical tests.     

Ascophyllum nodosum supplementation: a preharvest intervention for reducing Escherichia coli O157:H7 and Salmonella spp. in feedlot steers

Ascophyllum nodosum (Tasco-14) decreased the prevalence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in animals fed prior to harvest. Tasco-14 was supplemented on a 2% dry matter basis 14 days prior to harvest to determine its effects on EHEC and Salmonella spp. prevalence. Two hundred mixed crossbred steers and heifers (Bos indicus x Bos taurus), in a large commercial finishing facility, served as experimental units. Treatment (TRT, n = 100) animals received a steam-rolled corn-based diet containing 2% Tasco-14 on a dry matter basis, and control (CON, n = 100) animals received only the steam-rolled corn-based diet. Hide swabs and fecal samples were obtained for EHEC and Salmonella spp. evaluations. Animals were sampled 1 day prior to (d - 1) the feeding of Tasco-14 and immediately following exsanguinations. The prevalence of EHEC O157 on hide swabs and in fecal samples (P = 0.0001 and P < 0.0001, respectively) and the prevalence of EHEC O157:H7 on hide swabs and in fecal samples (P < 0.0001 and P < 0.0001, respectively) was reduced by 33 and 36% from d - 1 levels on TRT hide swabs and by 9 and 11% in TRT fecal samples. The prevalence of EHEC O157 and EHEC O157:H7 was reduced by 33 and 36% from d - 1 levels on TRT hide swabs and by 9 and 11% in TRT fecal samples. The prevalence of Salmonella spp. on hide swabs did not change for TRT animals (P = 0.64). CON animals showed an increase in Salmonella spp. prevalence (P < 0.0001) from d - 1 feeding levels on hide swabs. The prevalence of Salmonella spp. increased in both TRT and CON fecal samples when compared to d - 1 levels (P = 0.002). However, TRT samples exhibited a lower (P < 0.05) postfeeding prevalence of Salmonella spp. in fecal samples than did CON samples. Results from this study indicate that 2% Tasco-14 supplementation in feedlot cattle diets reduces EHEC O157 and EHEC O157:H7 prevalence on hide swabs and in fecal samples and may suppress increases in Salmonella spp.     

Zero-tolerance for faecal contamination of carcasses as a tool in the control of O157 VTEC infections.

The Dutch government, the meat producers organisation and the meat industry have recognised O157 VTEC as an important public health hazard, and agreed on the necessity to improve the hygiene in Dutch cattle- and calf-slaughtering establishments. This paper reports activities within a national action programme to achieve this objective, "Zero-tolerance for faecal contamination during slaughter of cattle and calves". The study included inspection of hygienic performances in slaughterhouses, and visual and microbiological (aerobic plate counts, Enterobacteriaceae counts and O157 VTEC presence/absence on visually clean cattle and calf carcasses) assessment of carcass cleanliness. Initial studies concluded that the hygienic performances in the Dutch cattle and calf slaughterhouses should be immediately improved. In 52% of the slaughterhouses inspected, carcasses were observed to be contaminated with hide, hair or faeces. Around 45% of the slaughterhouses had constructural deficiencies likely to lead to structural cross-contamination of carcasses, by direct carcass-carcass contact, or by indirect contacts with floors, walls or steps. In 39% of the slaughterhouses, cleaning and disinfection procedures were inadequate. Visual inspection of chilled carcasses found that in 11 of the 27 slaughterhouses visited, more than 10% of the carcasses were visibly contaminated. In 6 of the 27 slaughterhouses visited, more than 50% of the carcasses inspected were visibly contaminated. Microbiological analysis of visually clean carcasses noted contamination levels similar to those reported from other countries. O157 VTEC were not isolated during this study. Circulation of these findings lead to increased efforts by all parties to fulfil the requirements of the statutory "Zero-tolerance" programme. A follow-up study noted a significant decrease in the proportions of faecally contaminated carcasses, i.e., 7% of chilled carcasses were visibly contaminated with faeces, as opposed to 22% contamination during the initial study. The follow-up study also noted a greater awareness of the importance of good hygienic practices among slaughterhouse personnel and government meat inspectors.     

Effects of slaughter processes on pig carcass contamination by verotoxin-producing Escherichia coli and E. coli O157:H7

The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) faecal carriage of slaughtered pigs; (ii) to determine the effects of three different pig slaughtering processes on pig carcass contamination by VTEC; (iii) to characterise the VTEC strains isolated from pig and pig slaughterhouses (virulence genes and serotype); and (iv) to compare the strains isolated in the same slaughterhouse in order to identify the routes of contamination inside the slaughterhouse. Pork carcasses from three French slaughterhouses were sampled at three steps of the slaughter process and different sites in each slaughterhouse were sampled at three different times in the work day. Faecal material from each sampled carcass, potable water and scalding water were also collected. Detection of stx genes was performed by polymerase chain reaction (PCR) on a total of 1227 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2c, uidA genes associated with virulence) and pulsotyped. No E. coli O157:H7 was isolated from the three uidA-positive samples. VTEC faecal carriage was 31%. Global carcass contamination decreased with slaughter process (from 46% to 15%), whereas environmental contamination increased (from 7% to 29%). No VTEC isolates harboured eae, ehx, and uidA genes. VTEC contamination routes were not clearly identified.     

Inhibition of verocytotoxigenic Escherichia coli by antimicrobial peptides caseicin A and B and the factors affecting their antimicrobial activities

The antimic robial activities of caseicin A and B antimicrobial peptides (AMPs) were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n = 11), other bacterial pathogenic and spoilage bacteria (n = 7), using a model broth system. The ability of the AMPs to retain their antimicrobial activities against a strain of E. coli O157:H7 380-94 under various test conditions (pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competitive microflora) was assessed and the minimum inhibitory concentrations (MIC) and number of surviving E. coli O157:H7 calculated. The mean number of VTEC surviving after exposure to 2 mg/ml caseicin A and B was reduced by 4.96 and 4.19 log₁₀ cfu/ml compared to the respective controls. The susceptibility of E. coli O157:H7 to the caseicin AMPs decreased as temperature, pH, water activity and inoculum size were reduced. The presence of sodium chloride (0.5-2.5%) did not affect the activity of caseicin A (p > 0.05), however it did inhibit the activity of caseicin B. The presence of a competitive microflora cocktail did not significantly (p > 0.05) affect the activities of the AMPs for the majority of the concentrations tested. Using a quantitative PCR assay, the levels of verotoxins (vt1 and vt2) expressed by E. coli O157:H7 following exposure to a sub-inhibitory concentration (0.5 mg/ml) of caseicin A showed that the verotoxin levels did not differ from the levels produced by the control cultures. The antimicrobial activity of caseicin A against E. coli O157:H7 was also tested in a model rumen system, however concentrations of ≥ 2 mg/ml did not significantly (p > 0.05) reduce E. coli O157:H7 numbers in the model system over a 24 h period. The application of caseicin AMPs in food and/or animal production may be valuable in combination with other antimicrobials although further research is required.     

The prevalence of verocytotoxin-producingEscherichia coli(VTEC) andE. coliO157:H7 in beef in Sweden determined by PCR assays and an immuno-magnetic separation (IMS) method

The number of cases of enterohaemorrhagicEscherichia coli(EHEC) in Sweden has increased dramatically since 1995, but no source of the infections has been identified. The prevalence of verocytotoxin-producingE. coli(VTEC), includingE. coliO157:H7 in imported and domestic raw beef in Sweden, was determined by PCR assays and immuno-magnetic separation (IMS, Dynal). VTEC was detected by a multiplex PCR directed at sequences on the vt1 and vt2 genes, and the prevalence ofE. coliO157:H7 was estimated by the IMS assay and a SZ-PCR which detectsE. coliO157:H7 (and some strains of O157:NM, O55:H7 and O55:NM). VTEC was detected in 15.055% (57/368) and 1.051% (6/543) of the imported and domestic beef samples, respectively, by the multiplex PCR. E. coliO157:H7 was detected in 0.053% and 2.054% of the imported beef samples by the IMS and SZ-PCR assays, respectively. In domestic beefE. coliO157:H7 was not detected by either method, indicating that the occurrence in Swedish beef was less than 0.056% with a 95% certainty. The overall prevalence of VTEC was estimated to be 4.050%, by adjusting for the relative proportion of imported and domestic beef in Sweden and, similarly, the overall prevalence ofE. coliO157:H7 was 0.0506% (IMS) or 0.055% (SZ-PCR). We propose that a useful strategy to analyse a large number of food samples for VTEC andE. coliO157:H7 is an initial screening of samples using the multiplex VT-PCR in conjunction with enrichment and buoyant density centrifugation, followed by further analyses on the fraction of VT-positive samples using either the IMS method or SZ-PCR.     

Determining the prevalence of Escherichia coli O157 in cattle and beef from the feedlot to the cooler

Prevalence of Escherichia coli O157 on cattle entering the slaughter floor may range from 10 to > 70%. This study was conducted to determine the effect of E. coli O157 prevalence in fecal pats collected from feedlot pen floors on subsequent E. coli O157 prevalence on carcasses at various points in the slaughter process. Fecal pats from the feedlot pen floor were collected within 3 days before slaughter. During cattle processing at the slaughter facility, additional samples were collected from the hide, from the colon, and from the carcasses before and after evisceration and after final decontamination. Of 15 lots (a group of cattle from the same pen from a feedlot) sampled, 87% had at least one positive fecal pat from the feedlot floor, 47% had a positive hide sample, 73% had a positive colon/fecal sample, and 47% had a positive carcass sample preevisceration; however, only 8% of lots had a positive carcass sample postevisceration or after final intervention. Of the total samples tested (n = 1,328), 24.7, 14.7, 27.6, 10.1, 1.4, and 0.3% of fecal pats from the feedlot floor, hide, colon, preevisceration, postevisceration, and final intervention samples, respectively, were positive for E. coli O157. Pens with greater than 20% positive fecal pats from the feedlot floor had 25.5% hide, 51.4% colon, and 14.3, 2.9, and 0.7% carcass samples positive at preevisceration, at postevisceration, and after final intervention, respectively. However, fecal pats from feedlot floor samples that contained less than 20% positive fecal samples showed lower pathogen prevalence, with 5.0% hide, 7.5% colon, and 6.3, 0, and 0% carcass positive samples at preevisceration, postevisceration, and post-final intervention, respectively. Data from this study can be used as part of risk assessment processes in order to identify mitigation strategies to minimize prevalence of E. coli O157 on fresh beef carcasses.     

Effects of cutting process on pork meat contamination by verotoxin-producing Escherichia coli (VTEC) and E. coli O157:H7

The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) prevalence in pork cutting meat; (ii) to determine the effects of cutting process on pork meat contamination by VTEC; (iii) to characterise the VTEC strains isolated from pork and pork cutting plants (virulence genes and serotype); and (iv) to compare the strains isolated the same day in the same cutting plant in order to identify the routes of contamination inside the cutting plant. Pork carcasses from three French cutting plants were sampled before carcass cutting (carcass samples), after carcasses were divided into big portions (untrimmed cuts) and after preparation of primal cuts (rindless boneless cuts), and different environmental sites in each cutting plant were sampled at three different times in the work day. Potable water was also collected. PCR detection of stx genes was performed on a total of 2042 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2e, uidA and genes which are associated with virulence) and pulsotyped. No E. coli O157:H7 was detected. Meat contamination decreased from carcass (12%) and primary cuts (19%) to secondary cuts (5%), whereas environmental contamination increased after 2 h of activity (from 3% before the commencement of the work day to 25% and 20%, 2 and 6 h after commencement of cutting). No VTEC isolates harboured eae, ehx and uidA genes. VTEC contamination routes were not clearly identified.     

Contamination of bovine carcasses and abattoir environment by Escherichia coli O157:H7 in Istanbul

The aim of this study was to investigate contamination of carcasses and abattoir environment with Escherichia coli O157:H7. Five abattoirs in Istanbul were visited between January 2000 and April 2001. During visits, sampling was performed and a total of 330 cattle were selected. Cattle were examined for the presence of faeces on the hide (abdomen and legs) before slaughter. The swabs from the carcasses and environmental samples (abattoir floor, benches including conveyors, knives, aprons, saws, hooks, hands) were taken at the abattoir immediately after slaughter using sterile cotton swabs. A sample from the wash water of the abattoir was also taken. Preenrichment, immunomagnetic separation and CT-SMAC agar were used for the isolation. The reaction of the isolates with anti-O157 and H7 antisera were also analysed. Twelve strains (3.6%) of E. coli O157 were isolated from the cattle carcasses and eight (2.4%) of them gave positive reaction with anti-H:7. Six strains of E. coli O157 were isolated from the environmental samples and all strains were positive for H7. The number of E. coli O157H:7 strains isolated from the environmental samples was two from the knife, two from the hands, one from the apron and one from the floor. No E. coli O157 was isolated from the abattoir water.