Identification of Escherichia coli O157:H7-strains from pigs with postweaning diarrhoea and amplification of their virulence marker genes by PCR

Escherichia coli isolated from pigs with postweaning diarrhoea were examined by PCR for the presence of the O157 rfb gene responsible for the biosynthesis of E coli O157 lipopolysaccharide. Among the 372 isolates tested, 38 (10.2 per cent) were of the O157 serogroup, but none of these possessed the H7 determinant. Further analysis of the E coli O157 isolates revealed that seven of them had the genes responsible for the production of Shiga toxin 1 and eaeA intimin, four other strains had genes responsible for the production of Shiga toxin 2, and four other strains were positive for the enterohaemolysin gene.     

Characteristics of verotoxigenic Escherichia coli from pigs.

Porcine verotoxigenic Escherichia coli were characterized with respect to frequency of occurrence, serogroup, and association with disease, weaning, and selected properties of the bacterium. Of 668 strains of E. coli from southern Ontario pigs with enteric disease, 32 (4.8%) produced verotoxin at 10(3)-10(7) cytotoxic doses per mL of culture supernatant. Of 22 isolates which belonged to O serogroups 138, 139 and 141, 15 produced verotoxin. Among other enterotoxigenic types of E. coli, two of 57 isolates of O157:K"V17" and two of 96 isolates of O149:K91 were verotoxigenic. The remaining 13 verotoxigenic E. coli belonged to O groups 2, 107, 120, 121 and 130. An additional 21 verotoxigenic E. coli belonging to O groups 138, 139 and 141 and three to O157:K"V17" were identified in a collection of 47 E. coli recovered from weaned pigs with enteric disease. Verotoxigenic E. coli were associated with postweaning diarrhea, bloody stools, sudden death and edema disease. They were isolated at similar frequencies (14%) from healthy weaned pigs, and from weaned pigs with enteric disease. Isolation rates from neonates were low and significantly different from rates in weaned pigs. Neutralizing antibody to verotoxin was not detected in the sera of 45 pigs, which included pigs from herds with a history of edema disease. Verotoxin was not associated with production of colicin, hemolysin, or enterotoxins or with any of 23 biochemical properties of the organisms. The serological data indicate that porcine verotoxigenic E. coli are not a common source of verotoxigenic E. coli for humans. Porcine verotoxin may play a role in postweaning diarrhea and absence of detectable neutralizing antibody in serum may be an important aspect of pathogenesis.     

Prevalence of serogroups and virulence genes in Escherichia coli associated with postweaning diarrhoea and edema disease in pigs and a comparison of diagnostic approaches

Identification of Escherichia coli causing porcine postweaning diarrhoea (PWD) or edema disease (ED) requires knowledge regarding the prevalent pathotypes within a given region. This study was undertaken to determine the present distribution of serogroups, hemolytic activity and virulence factor gene profiles among porcine pathogenic E. coli isolates in Denmark and to compare detection of these characteristics as diagnostic approaches. Five hundred and sixty-three E. coli were serogrouped using E. coli O-antisera and investigated for hemolytic activity. Of these, 219 isolates were further characterized using a 5'-nuclease PCR assay detecting genes for adhesion factors, enterotoxins and verocytotoxin 2e (VT2e). Forty-two different serogroups were found. The most prevalent serogroup was O149 accounting for 49.9% of all isolates, followed by O138 (14.9%), O139 (6.9%), O141 (4.1%) and O8 (3.7%). Hemolytic activity was detected in 87.7% of all isolates. Virulence factor genes detected were F4 (44.7%), F18 (39.3%), intimin (1.4%), F6 (0.9%), STb (77.6%), EAST1 (65.8%), LT (61.6%), STa (26.5%) and VT2e (16.4%). Six pathotypes accounted for 65.7% of all isolates investigated. Using possession of virulence factor genes as reference, O-serogrouping employing a selection of antisera representing common pig pathogenic serogroups and detection of hemolysis were evaluated as epidemiological markers for pathogenicity. Both criteria were associated with pathogenicity (P<0.001, for both), however, both methods also resulted in false classifications regarding pathogenicity for 11.9 and 13.2% of isolates, respectively. Detection of adhesion factor genes F4, F18 and intimin is suggested as an operational alternative when diagnosing PWD and ED.     

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.     

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.     

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.     

An investigation on the effect of transport and lairage on the faecal shedding prevalence of Escherichia coli O157 in cattle

The aim of the study was to investigate the effect of transport and lairage on the prevalence of Escherichia coli O157 faecal shedding and the subsequent contamination of beef carcasses. Individual rectal faecal samples were taken from two cohorts of cattle (109 and 59) at the farm before transport and at the abattoir post-transport and lairage. The entire outer and inner surfaces of the carcass of each animal were swabbed immediately following slaughter and dressing. The prevalence of E. coli O157 shedding in cattle sampled at farm, post-transport and lairage was 18% (20), 13% (14) and 12% (13) for cohort A and 1.7% (1), 1.7% (1) and 0 for cohort B, respectively. No E. coli O157 was recovered from the 168 dressed carcasses. In total, 98% (46 of 47) of the E. coli O157 isolates from cohort A were potentially pathogenic to man. Transport and lairage do not cause an increase in the prevalence of E. coli O157 faecal shedding in cattle. This study demonstrates that even positive cohorts of cattle may be slaughtered and processed to produce clean carcasses by following good hygienic practices.     

Characterization of pathogenic Escherichia coli isolated from humans in Austria: phenotypes, toxin gene types and epidemiology

One hundred and ten clinical Escherichia coli isolates of serovar O157 (n = 102) and O26 (n = 8) were characterized for the presence of putative virulence genes by PCR. All but one of these isolates contained the eae gene. The EHEC-hly gene could be detected in all E. coli O157 and in 50% of E. coli O26 isolates. Forty-five (40.9%) of the 110 E. coli were positive for both stx(1) and stx(2) genes, 2 (1.8%) isolates were positive for stx(1) and 57 isolates (51.8%) were positive for stx(2) only. Among the 102 stx(2) positive isolates, 14 (13.7%) E. coli O157 contained also the stx(2c) variant gene. No other stx(2) variant was identified. Six clinical isolates (five E. coli O157:H7 and one E. coli O26) did not contain stx genes. Ten non-pathogenic E. coli isolates which were amplified as controls didn't contain any stx and eae gene but two of the ten strains contained the EHEC-hly gene. By their growth on chromogenic media, all but two of 50 E. coli O157 could be differentiated from eight E. coli O26 and 10 non-pathogenic E. coli. Sixty-one of the O157:H7 isolates were further subjected to pulsed-field gel electrophoresis (PFGE) which identified 49 distinguishable patterns. In five cases where contact infection among family members was suspected, indistinguishable PFGE patterns confirmed the epidemiological relatedness of the isolates. Moreover, two PFGE clusters were identified which comprised five and three strains, respectively. These findings indicate the occurrence of both family and diffuse outbreaks of E. coli O157 infections in Austria during recent years and demonstrate the need for molecular subtyping of these pathogens.     

The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland.

E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.     

Prevalence of serogroups and virulence factors of Escherichia coli strains isolated from pigs with postweaning diarrhoea in eastern China

This study was undertaken to determine the present distribution of serogroups, hemolytic activity and virulence factors among Escherichia coli strains isolated from pigs with postweaning diarrhoea from eight provinces in eastern China. Two hundred and fifteen E. coli isolates were serogrouped with O-antisera, investigated for hemolytic activity, assessed for F4, F5, F6, F18 and F41 fimbrial antigens by monoclonal antibodies and detected for genes of enterotoxins and shiga-toxin-two-variant (Stx2e) by a multiplex polymerase chain reaction (PCR). Among these E. coli isolates, 140 were determined to be placed in serogroups, 52 were unable to be serogrouped and the rest 23 auto-agglutinated. These isolates distributed in 45 serogroups and 64.3% (90/140) belonged to 12 O serogroups: O8, O9, O11, O20, O32, O91, O93, O101, O107, O115, O116 and O131. Hemolytic activity was detected in 11.6% (25/215) of all isolates. Several uncommon O serogroups were discovered in this study. Agglutination tests showed that 50.2% (108/215) of these isolates were positive for one or more of the five fimbrial antigens. Seventy-two E. coli strains expressed single fimbria and 36 strains expressed two or more fimbriae. Among these 215 E. coli isolates, strains expressing F18, F4, F6, F6 + F18 or F5 + F41 occurred more frequently. PCR analysis showed that 60.5% (130/215) of the isolates only harboured the gene of estI (STI) while 6.0% (13/215) strains possessed the genes of stx2e, estI and estII and 5.6% (12/215) of strains had the genes of estI/estII. Of all these isolates, 107 (49.8%) were negative for the fimbrial antigens examined. The fimbria-negative isolates usually possessed genetic determinant of estI (78, 72.9%).     

Detection with nonradioactively labeled probes of the toxin genes of different E. coli pathotypes from swine]

We tested hemolytic E. coli from 86 pigs with edema disease or colidiarrhea. They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes. By slide-agglutination we detected 38 cases with E. coli O149:K88, 28 with E. coli O139:82B and 20 with E. coli O141. E. coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes. Edema disease E. coli O139:82B reacted with the SLTII probe. E. coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns. All the E. coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII. No strains isolated from pigs with edema disease possessed any of these enterotoxin genes. Gene probe technique confirmed the serological method as useful tool for diagnosing E. coli O149:K88 and O139:82B as ETEC or VTEC, respectively. On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E. coli of serogroup O141.     

Significance of fecal volatile fatty acids in shedding of Escherichia coli O157 from calves: experimental infection and preliminary use of a probiotic product

Cattle have been recognized as a principal reservoir of Escherichia coli O157:H7. This organism appears to be confined to the gastrointestinal tract and is shed in feces. A probiotic product containing lactic acid-producing Streptococcus bovis LCB6 and Lactobacillus gallinarum LCB 12 isolated from adult cattle was developed, and a preliminary experiment was conducted to evaluate its effect on the elimination of E. coli O157 from experimentally infected calves. Eight 4-month-old Holstein calves were orally challenged with E. coli O157 and the probiotic product was administered against four calves continued fecal shedding of E. coli O157 by the 7th day after infection. Fecal shedding of E. coli O157 was completely inhibited and re-shedding was not detected in any of the animals. Remarkable increase of VFAs, especially that of acetic acid in feces after the administration of probiotic bacteria correlated with the diminution of E. coli O157. Four calves that had spontaneously ceased fecal shedding of E. coli O157 by the 7th day exhibited a high concentration of VFAs in feces before and after experimental infection. Although our results are preliminary and obtained from calves under limited conditions, the possible application of probiotic product to reduce fecal shedding of E. coli O157 from cattle is suggested.     

Verocytotoxin-producing and attaching and effacing activity of Escherichia coli isolated from diseased farm livestock

Between May 2005 and June 2008, strategically selected isolates of Escherichia coli obtained from clinical submissions to Veterinary Laboratories Agency (VLA) regional laboratories in England and Wales were serogrouped and examined by PCR for verocytotoxin (VT) production and attaching and effacing (eae) genes, both of which are zoonotic determinants. VT-encoding genes were detected in 54 (5.3 per cent) of the 1022 isolates examined. Only one isolate (0.1 per cent) was identified as verocytotoxigenic E coli (VTEC) O157. Non-O157 VTECs were present in 4.7 per cent of isolates from cattle, compared with 7.9 per cent in pigs, 2.3 per cent in sheep and 6.7 per cent in goats. The predominant serogroup identified in cattle was O26 and the predominant serogroup in pigs was O2. Attaching and effacing activity was attributed to 69 (6.8 per cent) of all isolates.     

Dry-rolled or steam-flaked grain-based diets and fecal shedding of Escherichia coli O157 in feedlot cattle

Hindgut is a major colonization site for Escherichia coli O157 in cattle. In this study, diets were formulated to effect changes in hindgut fermentation to test our hypothesis that changes in the hindgut ecosystem could have an impact on fecal shedding of E. coli O157. Feedlot heifers (n = 347) were prescreened for the prevalence of E. coli O157 by fecal and rectoanal mucosal swab cultures. A subset of 40 heifers identified as being positive for fecal shedding of E. coli O157 was selected, housed in individual pens, and randomly allocated to 4 dietary treatments. Treatments were arranged as a 2 x 2 factorial, with factor 1 consisting of grain type (sorghum or wheat) and factor 2 being method of grain processing (steam-flaking or dry-rolling). Four transition diets, each fed for 4 d, were used to adapt the animals to final diets that contained 93% concentrate and 7% roughage. The grain fraction consisted of dry-rolled sorghum, steam-flaked sorghum, a mixture of dry-rolled wheat and steam-flaked corn, or a mixture of steam-flaked wheat and steam-flaked corn. Wheat diets contained 52% wheat and 31% steam-flaked corn (DM basis). Fecal and rectoanal mucosal swab samples were obtained 3 times a week to isolate (enrichment, immunomagenetic separation, and plating on selective medium) and identify (sorbitol negative, indole production, and agglutination test) E. coli O157. The data were analyzed as repeated measures of binomial response (positive or negative) on each sampling day. Method of processing (dry-rolled vs. steam-flaked), sampling day, and the grain type x day interaction were significant (P < 0.05), but not the method of processing x grain type interaction. The average prevalence of E. coli O157 from d 9 was greater (P < 0.001) in cattle fed steam-flaked grains (65%) compared with those fed dry-rolled grains (30%). Average prevalence in cattle fed sorghum (51%) or wheat (43%) were similar (P > 0.10) on most sampling days. Results from this study indicate that feeding dry-rolled grains compared with steam-flaked grains reduced fecal shedding of E. coli O157. Possibly, dry-rolling allowed more substrate to reach the hindgut where it was fermented, thus making the hindgut inhospitable to the survival of E. coli O157. Dietary intervention to influence hindgut fermentation offers a simple and practical mitigation strategy to reduce the prevalence of E. coli O157 in feedlot cattle.     

Polyclonal sandwich ELISAs to detect F18 fimbriated Escherichia coli in pigs in Northern Ireland.

F18 fimbriated Escherichia coli are a newly described cause of postweaning diarrhea in pigs. Polyclonal rabbit antisera were raised to the antigenic variants, F18ab and F18ac, of these fimbriae and were used to develop monospecific sandwich enzyme-linked immunosorbent assays (ELISAs). The ELISAs were standardized with type cultures characterized by polymerase chain reaction techniques (PCR) and then used to conduct a study of the prevalence of F18 fimbriated E. coli in pigs in Northern Ireland. A total of 176 isolates were tested by ELISA and PCR. Eight isolates were positive for F18 by ELISA, of which 2 were shown to be false positives by PCR and one was PCR positive but ELISA negative. Of the 6 confirmed ELISA positives, all produced VT2 toxin and 3 produced ST toxin. Four positives were from serogroups O138 and O139, previously associated with porcine diarrhea.     

Influence of processed grains on fecal pH, starch concentration, and shedding of Escherichia coli O157 in feedlot cattle

Manipulation of cattle diets has been proposed as a possible preharvest control measure for Escherichia coli O157. Altering hindgut fermentation through diet changes may be a means to reduce fecal shedding of E. coli O157. In Exp. 1, the objective was to determine whether fecal shedding of E. coli O157 was related to fecal starch concentration. Beginning on d 20, and every week thereafter until d 61, steers in 54 pens (6 to 7 steers per pen) were sampled (n = 122) by fecal collection and rectoanal mucosal swabs (RAMS) for E. coli O157 and fecal starch concentration determinations. Escherichia coli O157 prevalence was 3.3% in fecal samples, 4.1% as measured by RAMS, and 4.9% by fecal or RAMS samples. Steers positive for E. coli O157 contained 21% more (P < 0.05) fecal starch than steers that were negative for E. coli O157. In Exp. 2, we attempted to alter the concentration of starch escaping rumen fermentation by feeding finishing diets based on steam-flaked corn (SFC) and dry-rolled corn (DRC) to 30 heifers prescreened for being culture positive for fecal E. coli O157. Beginning on d 13, heifers were sampled (feces and RAMS) weekly to monitor fecal pH and starch concentration, and prevalence of E. coli O157. Prevalence of E. coli O157 remained above 30% for the first 13 d, but declined (P < 0.05) over the entire 7-wk period. Based on RAMS, the prevalence of E. coli O157 tended to be greater (P = 0.08) for heifers fed SFC than for those fed the DRC diet. After d 20, heifers fed DRC had greater (P < 0.05) fecal starch and lower (P < 0.05) fecal pH than heifers fed SFC. Fecal pH was negatively correlated (r = - 0.34; P < 0.05; n = 143) with fecal starch concentration. Fecal starch concentration and pH were not different (P > 0.05) for heifers that were positive or negative for E. coli O157. Our data suggest that fecal shedding of E. coli O157 was not related to fecal pH or starch concentration in cattle fed grain-based diets.     

Escherichia coli O157 in feedlot cattle feces and water in four major feeder-cattle states in the USA

The prevalence of Escherichia coli O157 was determined in 10662 fecal samples, 2130 water and 1132 water tank-sediment samples collected during the summer months in 2001 from 711 pens in 73 feedlots located in Kansas, Nebraska, Texas, or Oklahoma, USA. Overall, 10.2% of fecal samples were positive for E. coli O157, with 52% of the pens and 95.9% of the feedlots having at least one positive fecal sample. There were no differences among states or months in the fecal prevalences. Water or water tank-sediment was positive in 13.1% of the water tanks, and 60.3% of feedlots had at least one positive tank. Cattle were more likely to be shedding E. coli O157 in pens with positive water tanks, and water was more likely to be positive when E. coli O157 was detected in the sediment.     

Prevalence of genotypes for fimbriae and enterotoxins and of O serogroups in Escherichia coli isolated from diarrheic piglets in Korea.

Polymerase chain reaction for 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa, STb), and 1 heat-labile enterotoxin (LT) were performed on 400 Escherichia coli isolates to determine their genotype prevalence among enterotoxigenic E. coli isolates from preweaned pigs with diarrhea in the Republic of Korea. A total of 200 of the 400 E. coli isolates were also selected for characterization of the O serogroup. Of these 200 isolates, serogroup could be determined in 139 (69.5%) but not in 61 isolates (30.5%). Isolates of serogroup O101 were the most common, followed in descending order by 08, 020, 0162, 0141, and 0149. Ninety-seven (24.3%) of the 400 E. coli isolates carried genes for at least 1 of the entertoxins or fimbrial adhesins. Of these 97 isolates, 27 carried genes for at least 1 of the fimbrial adhesins and entertoxins. Sixty-six percent of the isolates that carried fimbrial adhesin genes carried genes for at least 1 of the enterotoxins, and 71% of the isolates that carried enterotoxin genes carried genes for at least 1 of the fimbrial adhesins. Genes for the F6 fimbriae were detected in 6% of the E. coli isolates, and F4+, F41+, and F5+ genes were detected in 4.3%, 3.3%, and 2% of the isolates, respectively. Genes for STa, STb, and LT were detected in 10%, 8.5%, and 4.3% of the isolates, respectively. The 6 major genotypes observed in this study (in decreasing order) were F6+, STb+, F41+, STa+STb+, F6+STa+, and STa+.     

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.     

Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.